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Chien-Chung Yang,1,2 Li-Der Hsiao,3 Chuen-Mao Yang3– 5 1Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital at Tao-Yuan, Kwei-San, Tao-Yuan 33302, Taiwan; 2School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 33302, Taiwan; 3Department of Pharmacology, College of Medicine, China Medical University, Taichung 40402, Taiwan; 4Program for Biotch Pharmaceutical Industry, China Medical University, Taichung 40402, Taiwan; 5Department of Post-Baccalaureate Veterinary Medicine, College of Medical and Health Science, Asia University, Wufeng, Taichung 41354, TaiwanCorrespondence: Chuen-Mao Yang Tel +886-4-22053366 (ext. 2229)Email chuenmao@mail.cmu.edu.twPurpose: Neuroinflammation, characterized by the increased expression of inflammatory proteins such as matrix metalloproteinases (MMPs), plays a critical role in neurodegenerative disorders. Lipopolysaccharide (LPS) has been shown to upregulate MMP-9 expression through the activation of various transcription factors, including activator protein 1 (AP-1) and forkhead box protein O1 (FoxO1). The flavonoid 3,5,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one (galangin) has been demonstrated to possess antioxidant and anti-inflammatory properties in various types of cells. Here, we investigated the mechanisms underlying the inhibitory effect of galangin on LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells).Methods: Pharmacological inhibitors and siRNAs were employed to explore the effects of galangin on LPS-challenged RBA-1 cells. Gelatin zymography, Western blotting, real-time PCR, and a luciferase reporter assay were used to detect MMP-9 activity, protein expression, mRNA levels, and promoter activity, respectively. The protein kinases involved in the LPS-induced MMP-9 expression were determined by Western blot. A chromatin immunoprecipitation (ChIP) assay was employed to evaluate the activity of c-Jun at the MMP-9 promoter.Results: Galangin treatment attenuated the LPS-mediated induction of MMP-9 protein and mRNA expression, as well as the activity at the MMP-9 promoter. In addition, galangin exerted its inhibitory effects on MMP-9 expression through suppressing the LPS-stimulated activation of proline-rich tyrosine kinase (Pyk2), platelet-derived growth factor receptor beta (PDGFRβ), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), and mitogen-activated protein kinases (MAPKs). Pretreatment with galangin attenuated the LPS-induced phosphorylation of c-Jun and FoxO1. LPS-induced cell migration was also suppressed by galangin pretreatment.Conclusion: Galangin attenuates the LPS-induced inflammatory responses, including the induction of MMP-9 expression and cell migration, via inhibiting Pyk2/PDGFRβ/PI3K/Akt/mTOR/JNK1/JNK2 and p44/p42 MAPK cascade-dependent AP-1 and FoxO1 activities. These results provide new insights into the mechanisms through which galangin mitigates LPS-induced inflammatory responses, and suggest novel strategies for the management of LPS-related brain diseases.Keywords: neuroinflammation, astrocytes, LPS, matrix metalloproteinase, protein kinases, Chinese herbal medicine

Chien-Chung Yang,1,2 Li-Der Hsiao,3 Hui-Ching Tseng,3 Ching-Ming Kuo,3 Chuen-Mao Yang3,4 1Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital at Tao-Yuan, Tao-Yuan 33302, Taiwan; 2School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Tao-Yuan 33302, Taiwan; 3Department of Pharmacology, College of Medicine, China Medical University, Taichung 40402, Taiwan; 4Department of Post-Baccalaureate Veterinary Medicine, College of Medical and Health Science, Asia University, Taichung 41354, TaiwanCorrespondence: Chuen-Mao Yang Tel +886 4-22053366 (Ext. 2229)Email chuenmao@mail.cmu.edu.twPurpose: Neuroinflammation plays a crucial role in neurodegenerative diseases. Matrix metalloproteinases (MMPs) are a landmark of neuroinflammation. Lipopolysaccharide (LPS) has been demonstrated to induce MMP-9 expression. The mechanisms underlying LPS-induced MMP-9 expression have not been completely elucidated in astrocytes. Nuclear factor-kappaB (NF-κB) is well known as one of the crucial transcription factors in MMP-9 induction. Moreover, reactive oxygen species (ROS) could be an important mediator of neuroinflammation. Here, we differentiated whether ROS and NF-κB contributed to LPS-mediated MMP-9 expression in rat brain astrocytes (RBA-1). Besides, pristimerin has been revealed to possess antioxidant and anti-inflammatory effects. We also evaluated the effects of pristimerin on LPS-induced inflammatory responses.Methods: RBA-1 cells were used for analyses. Pharmacological inhibitors and siRNAs were used to evaluate the signaling pathway. Western blotting and gelatin zymography were conducted to evaluate protein and MMP-9 expression, respectively. Real-time PCR was for mRNA expression. Wound healing assay was for cell migration. 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (H2DCF-DA) and dihydroethidium (DHE) staining were for ROS generation. Immunofluorescence staining was conducted to assess NF-κB p65. Promoter-reporter gene assay and chromatin immunoprecipitation (ChIP) assay were used to detect promoter activity and the association of nuclear proteins with the promoter.Results: Our results showed that the increased level of ROS generation was attenuated by edaravone (a ROS scavenger), apocynin (APO; an inhibitor of p47Phox), diphenyleneiodonium (DPI; an inhibitor of NOX), and pristimerin in RBA-1 cells exposed to LPS. Besides, pretreatment with APO, DPI, edaravone, Bay11-7082, and pristimerin also inhibited the phosphorylation, nuclear translocation, promoter binding activity of NF-κB p65 as well as upregulation of MMP-9 expression-mediated cell migration in RBA-1 cells challenged with LPS.Conclusion: These results suggested that LPS enhances the upregulation of MMP-9 through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)/ROS-dependent NF-κB activity. These results also provide new insights into the mechanisms by which pristimerin attenuates LPS-mediated MMP-9 expression and neuroinflammatory responses.Keywords: neuroinflammation, NOX, ROS, LPS, MMP-9, pristimerin, astrocytes

OBJECTIVE: To investigate the roles of MAPKs and NF-kappaB in tumor necrosis factor alpha (TNFalpha)-induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: Human RASFs were isolated from synovial tissue obtained from patients with RA who underwent knee or hip surgery. The involvement of MAPKs and NF-kappaB in TNFalpha-induced VCAM-1 expression was investigated using pharmacologic inhibitors and transfection with short hairpin RNA (shRNA) and measured using Western blot, reverse transcriptase-polymerase chain reaction, and gene promoter assay. NF-kappaB translocation was determined by Western blot and immunofluorescence staining. The functional activity of VCAM-1 was evaluated by lymphocyte adhesion assay. RESULTS: TNFalpha-induced VCAM-1 expression, phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK, and translocation of NF-kappaB were attenuated by the inhibitors of MEK-1/2 (U0126), p38 (SB202190), JNK (SP600125), and NF-kappaB (helenalin) or by transfection with their respective shRNA. TNFalpha-stimulated translocation of NF-kappaB into the nucleus and NF-kappaB promoter activity were blocked by Bay11-7082, but not by U0126, SB202190, or SP600125. VCAM-1 promoter activity was enhanced by TNFalpha in RASFs transfected with VCAM-1-Luc, and this promoter activity was inhibited by Bay11-7082, U0126, SB202190, and SP600125. Moreover, up-regulation of VCAM-1 increased the adhesion of lymphocytes to the RASF monolayer, and this adhesion was attenuated by pretreatment with helenalin, U0126, SP600125, or SB202190 prior to exposure to TNFalpha or by anti-VCAM-1 antibody before the addition of lymphocytes. CONCLUSION: In RASFs, TNFalpha-induced VCAM-1 expression is mediated through activation of the p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB pathways. These results provide new insights into the mechanisms underlying cytokine-initiated joint inflammation in RA and may inspire new targeted therapeutic approaches. [ABSTRACT FROM AUTHOR]

Mevastatin (MVS) is known for its anti-inflammatory effects, potentially achieved by upregulating heme oxygenase-1 (HO-1), an enzyme involved in cytoprotection against oxidative injury. Nonetheless, the specific processes by which MVS stimulates HO-1 expression in human cardiac fibroblasts (HCFs) are not yet fully understood. In this study, we found that MVS treatment increased HO-1 mRNA and protein levels in HCFs. This induction was inhibited by pretreatment with specific inhibitors of p38 MAPK, JNK1/2, and FoxO1, and by siRNAs targeting NOX2, p47 phox , p38, JNK1, FoxO1, Keap1, and Nrf2. MVS also triggered ROS generation and activated JNK1/2 and p38 MAPK, both attenuated by NADPH oxidase or ROS inhibitors. Additionally, MVS promoted the phosphorylation of FoxO1 and Nrf2, which was suppressed by p38 MAPK or JNK1/2 inhibitor. Furthermore, MVS inhibited TNF-α-induced NF-κB activation and vascular cell adhesion molecule-1 (VCAM-1) expression via the HO-1/CO pathway in HCFs. In summary, the induction of HO-1 expression in HCFs by MVS is mediated through two primary signaling pathways: NADPH oxidase/ROS/p38 MAPK, and JNK1/2/FoxO1 and Nrf2. This research illuminates the underlying processes through which MVS exerts its anti-inflammatory effects by modulating HO-1 in cardiac fibroblasts., (© 2024 Wiley Periodicals LLC.)

The use of manufactured silica nanoparticles (SiNPs) has become widespread in everyday life, household products, and various industrial applications. While the harmful effects of crystalline silica on the lungs, known as silicosis or chronic pulmonary diseases, are well understood, the impact of SiNPs on the airway is not fully explored. This study aimed to investigate the potential effects of SiNPs on human tracheal smooth muscle cells (HTSMCs). Our findings revealed that SiNPs induced the expression of cyclooxygenase-2 (COX-2) mRNA/protein and the production of prostaglandin E 2 (PGE 2 ) without causing cytotoxicity. This induction was transcription-dependent, as confirmed by cell viability assays and COX-2 luciferase reporter assays. Further analysis, including Western blot with pharmacological inhibitors and siRNA interference, showed the involvement of receptor tyrosine kinase (RTK) EGF receptor (EGFR), non-RTK Pyk2, protein kinase Cα (PKCα), and p42/p44 MAPK in the induction process. Notably, EGFR activation initiated cellular signaling that led to NF-κB p65 phosphorylation and translocation into the cell nucleus, where it bound and stimulated COX-2 gene transcription. The resulting COX-2 protein triggered PGE 2 production and secretion into the extracellular space. Our study demonstrated that SiNPs mediate COX-2 up-regulation and PGE 2 secretion in HTSMCs through the sequential activation of the EGFR/Pyk2/PKCα/p42/p44MAPKs-dependent NF-κB signaling pathway. Since PGE 2 can have both physiological bronchodilatory and anti-inflammatory effects, as well as pathological pro-inflammatory effects, the increased PGE 2 production in the airway might act as a protective compensatory mechanism and/or a contributing factor during airway exposure to SiNPs.

Bradykinin (BK) has been recognized as a stimulant for matrix metalloproteinase (MMP)-9 expression, contributing to neuroinflammation. Modulating the BK/MMP-9 pathway offers potential in the treatment of neuroinflammatory disorders. Rhamnetin (RNT), a flavonoid compound known for its antioxidant and anti-inflammatory effects, has shown promise. However, the specific mechanisms through which RNT inhibits BK-induced MMP-9 expression remain unclear. Therefore, this study aims to delve into the intricate mechanisms underlying this process. Here, we initially demonstrated that RNT effectively attenuated BK-induced MMP-9 expression and its associated cell migration in rat brain astrocyte-1 (RBA-1) cells. Further investigation revealed that BK-driven MMP-9 protein, mRNA, and promoter activity linked to cell migration relied on c-Src, Pyk2, EGFR, PDGFR, PI3K/Akt, JNK1/2, and c-Jun. This was validated by the inhibition of these effects through specific inhibitors, a finding substantiated by the introduction of siRNAs targeting these signaling molecules. Notably, the phosphorylated levels of these signaling components induced by BK were significantly reduced by their respective inhibitors and RNT, underscoring the inhibitory role of RNT in this process. These findings indicate that, in RBA-1 cells, RNT diminishes the heightened induction of MMP-9 triggered by BK through the inhibition of c-Src/Pyk2/PDGFR and EGFR/PI3K/Akt/JNK1/2-dependent AP-1 activation. This suggests that RNT holds promise as a potential therapeutic approach for addressing neuroinflammation in the brain.

In this study, we confirmed that thrombin significantly increases the production of COX-2 and PGE 2 in human tracheal smooth muscle cells (HTSMCs), leading to inflammation in the airways and lungs. These molecules are well-known contributors to various inflammatory diseases. Here, we investigated in detail the involved signaling pathways using specific inhibitors and small interfering RNAs (siRNAs). Our results demonstrated that inhibitors targeting proteins such as protein kinase C (PKC)δ, proline-rich tyrosine kinase 2 (Pyk2), c-Src, epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), or activator protein-1 (AP-1) effectively reduced thrombin-induced COX-2 and PGE 2 production. Additionally, transfection with siRNAs against PKCδ, Pyk2, c-Src, EGFR, protein kinase B (Akt), or c-Jun mitigated these responses. Furthermore, our observations revealed that thrombin stimulated the phosphorylation of key components of the signaling cascade, including PKCδ, Pyk2, c-Src, EGFR, Akt, and c-Jun. Thrombin activated COX-2 promoter activity through AP-1 activation, a process that was disrupted by a point-mutated AP-1 site within the COX-2 promoter. Finally, resveratrol (one of the most researched natural polyphenols) was found to effectively inhibit thrombin-induced COX-2 expression and PGE 2 release in HTSMCs through blocking the activation of Pyk2, c-Src, EGFR, Akt, and c-Jun. In summary, our findings demonstrate that thrombin-induced COX-2 and PGE 2 generation involves a PKCδ/Pyk2/c-Src/EGFR/PI3K/Akt-dependent AP-1 activation pathway. This study also suggests the potential use of resveratrol as an intervention for managing airway inflammation.

The risk of lung exposure to silica nanoparticles (SiNPs) and related lung inflammatory injury is increasing with the wide application of SiNPs in a variety of industries. A growing body of research has revealed that cyclooxygenase (COX)-2/prostaglandin E 2 (PGE 2 ) up-regulated by SiNP toxicity has a role during pulmonary inflammation. The detailed mechanisms underlying SiNP-induced COX-2 expression and PGE 2 synthesis remain unknown. The present study aims to dissect the molecular components involved in COX-2/PGE 2 up-regulated by SiNPs in human pulmonary alveolar epithelial cells (HPAEpiCs) which are one of the major targets while SiNPs are inhaled. In the present study, we demonstrated that SiNPs induced COX-2 expression and PGE 2 release, which were inhibited by pretreatment with a reactive oxygen species (ROS) scavenger (edaravone) or the inhibitors of proline-rich tyrosine kinase 2 (Pyk2, PF-431396), epidermal growth factor receptor (EGFR, AG1478), phosphatidylinositol 3-kinase (PI3K, LY294002), protein kinase B (Akt, Akt inhibitor VIII), p38 mitogen-activated protein kinase (MAPK) (p38 MAPK inhibitor VIII), c-Jun N-terminal kinases (JNK)1/2 (SP600125), Forkhead Box O1 (FoxO1, AS1842856), and activator protein 1 (AP-1, Tanshinone IIA). In addition, we also found that SiNPs induced ROS-dependent Pyk2, EGFR, Akt, p38 MAPK, and JNK1/2 activation in these cells. These signaling pathways induced by SiNPs could further cause c-Jun and FoxO1 activation and translocation from the cytosol to the nucleus. AP-1 and FoxO1 activation could increase COX-2 and PGE 2 levels induced by SiNPs. Finally, the COX-2/PGE 2 axis might promote the inflammatory responses in HPAEpiCs. In conclusion, we suggested that SiNPs induced COX-2 expression accompanied by PGE 2 synthesis mediated via ROS/Pyk2/EGFR/PI3K/Akt/p38 MAPK- and JNK1/2-dependent FoxO1 and AP-1 activation in HPAEpiCs.

Tumor necrosis factor (TNF)-α is involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. It is a crucial pro-inflammatory cytokine in many heart disorders, including chronic heart failure and ischemic heart disease, contributing to cardiac remodeling and dysfunction. The implication of TNF-α in inflammatory responses in the heart has been indicated to be mediated through the induction of C-C Motif Chemokine Ligand 20 (CCL20). However, the detailed mechanisms of TNF-α-induced CCL20 upregulation in human cardiac fibroblasts (HCFs) are not completely defined. We demonstrated that in HCFs, TNF-α induced CCL20 mRNA expression and promoter activity leading to an increase in the secretion of CCL20. TNF-α-mediated responses were attenuated by pretreatment with TNFR1 antibody, the inhibitor of epidermal growth factor receptor (EGFR) (AG1478), p38 mitogen-activated protein kinase (MAPK) (p38 inhibitor VIII, p38i VIII), c-Jun amino N-terminal kinase (JNK)1/2 (SP600125), nuclear factor kappaB (NF-κB) (helenalin), or forkhead box O (FoxO)1 (AS1841856) and transfection with siRNA of TNFR1, EGFR, p38α, JNK2, p65, or FoxO1. Moreover, TNF-α markedly induced EGFR, p38 MAPK, JNK1/2, FoxO1, and NF-κB p65 phosphorylation which was inhibited by their respective inhibitors in these cells. In addition, TNF-α-enhanced binding of FoxO1 or p65 to the CCL20 promoter was inhibited by p38i VIII, SP600125, and AS1841856, or helenalin, respectively. Accordingly, in HCFs, our findings are the first to clarify that TNF-α-induced CCL20 secretion is mediated through a TNFR1-dependent EGFR/p38 MAPK and JNK1/2/FoxO1 or NF-κB cascade. We demonstrated that TNFR1-derived EGFR transactivation is involved in the TNF-α-induced responses in these cells. Understanding the regulation of CCL20 expression by TNF-α on HCFs may provide a potential therapeutic strategy in cardiac inflammatory disorders.

The inflammation of the airway and lung could be triggered by upregulation cyclooxygenase (COX)-2 and prostaglandin E 2 (PGE 2 ) induced by various proinflammatory factors. COX-2 induction by thrombin has been shown to play a vital role in various inflammatory diseases. However, in human tracheal smooth muscle cells (HTSMCs), how thrombin enhanced the levels of COX-2/PGE 2 is not completely characterized. Thus, in this study, the levels of COX-2 expression and PGE 2 synthesis induced by thrombin were determined by Western blot, promoter-reporter assay, real-time PCR, and ELISA kit. The various signaling components involved in the thrombin-mediated responses were differentiated by transfection with siRNAs and selective pharmacological inhibitors. The role of NF- κ B was assessed by a chromatin immunoprecipitation (ChIP) assay, immunofluorescent staining, as well as Western blot. Our results verified that thrombin markedly triggered PGE 2 secretion via COX-2 upregulation which were diminished by the inhibitor of thrombin (PPACK), PAR1 (SCH79797), G i/o protein (GPA2), G q protein (GPA2A), PKC α (Gö6976), p38 MAPK (SB202190), JNK1/2 (SP600125), MEK1/2 (U0126), or NF- κ B (helenalin) and transfection with siRNA of PAR1, G q α , G i α , PKC α , JNK2, p38, p42, or p65. Moreover, thrombin induced PAR1-dependent PKC α phosphorylation in HTSMCs. We also observed that thrombin induced p38 MAPK, JNK1/2, and p42/p44 MAPK activation through a PAR1/PKC α pathway. Thrombin promoted phosphorylation of NF- κ B p65, leading to nuclear translocation and binding to the COX-2 promoter element to enhance promoter activity, which was reduced by Gö6976, SP600125, SB202190, or U0126. These findings supported that COX-2/PGE 2 expression triggered by thrombin was engaged in PAR1/G q or G i/o /PKC α /MAPK-dependent NF- κ B activation in HTSMCs., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Chien-Chung Yang et al.)

Lung inflammation is a pivotal event in the pathogenesis of acute lung injury. Heme oxygenase-1 (HO-1) is a key antioxidant enzyme that could be induced by kaempferol (KPR) and exerts anti-inflammatory effects. However, the molecular mechanisms of KPR-mediated HO-1 expression and its effects on inflammatory responses remain unknown in human pulmonary alveolar epithelial cells (HPAEpiCs). This study aimed to verify the relationship between HO-1 expression and KPR treatment in both in vitro and in vivo models. HO-1 expression was determined by real time-PCR, Western blotting, and promoter reporter analyses. The signaling components were investigated by using pharmacological inhibitors or specific siRNAs. Chromatin immunoprecipitation (ChIP) assay was performed to investigate the interaction between nuclear factor erythroid-2-related factor (Nrf2) and antioxidant response elements (ARE) binding site of HO-1 promoter. The effect of KPR on monocytes (THP-1) binding to HPAEpiCs challenged with lipopolysaccharides (LPS) was determined by adhesion assay. We found that KPR-induced HO-1 level attenuated the LPS-induced intercellular cell adhesion protein 1 (ICAM-1) expression in HPAEpiCs. KPR-induced HO-1 mRNA and protein expression also attenuated ICAM-1 expression in mice. Tin protoporphyrin (SnPP)IX reversed the inhibitory effects of KPR in HPAEpiCs. In addition, in HPAEpiCs, KPR-induced HO-1 expression was abolished by both pretreating with the inhibitor of NADPH oxidase (NOX, apocynin (APO)), reactive oxygen species (ROS) (N-acetyl-L-cysteine (NAC)), Src (Src kinase inhibitor II (Srci II)), Pyk2 (PF431396), protein kinase C (PKC)α (Gö6976), p38 mitogen-activated protein kinase (MAPK) inhibitor (p38i) VIII, or c-Jun N-terminal kinases (JNK)1/2 (SP600125) and transfection with their respective siRNAs. The transcription of the homx1 gene was enhanced by Nrf2 activated by JNK1/2 and p38α MAPK. The binding activity between Nrf2 and HO-1 promoter was attenuated by APO, NAC, Srci II, PF431396, or Gö6983. KPR-mediated NOX/ROS/c-Src/Pyk2/PKCα/p38α MAPK and JNK1/2 activate Nrf2 to bind with ARE on the HO-1 promoter and induce HO-1 expression, which further suppresses the LPS-mediated inflammation in HPAEpiCs. Thus, KPR exerts a potential strategy to protect against pulmonary inflammation via upregulation of the HO-1.

Heme oxygenase-1 (HO-1) has been shown to exert antioxidant, anti-inflammatory, and anti-apoptotic effects in various types of cells. Therefore, the induction of HO-1 is an excellent rationale for the development of protective drugs. 15-Deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) can modulate the expression of antioxidant defense proteins and be beneficial for neuroinflammation. Brain endothelial cells play an important role in the pathophysiology of brain disorders. Whether 15d-PGJ 2 can induce HO-1 expression and protect against the inflammatory responses in mouse brain microvascular endothelial (bEnd.3) cells remains unclear. Here, we reveal that 15d-PGJ 2 stimulated HO-1 protein and mRNA expression in a time- and concentration-dependent manner in bEnd.3 cells, which was attenuated by diphenyleneiodonium chloride (DPI) and MitoTempo. Thus, activation of NADPH oxidase (NOX)- and mitochondria-derived reactive oxygen species (ROS) mediated 15d-PGJ 2 -induced HO-1 expression. ROS generation could cause phosphorylation of protein kinase C (PKC)δ, leading to HO-1 expression, which was suppressed by Rottlerin (selective inhibitor PKCδ), DPI, and MitoTempo. We further demonstrated that phosphorylation of c-Jun N-terminal kinase (JNK)1/2 participated in 15d-PGJ 2 -upregulated HO-1 expression, which was blocked by SP600125 or Rottlerin. Moreover, 15d-PGJ 2 -induced HO-1 expression was mediated through the activation of c-Jun (a subunit of activator protein 1 (AP-1)) and specificity protein 1 (Sp1), leading to their interaction with the HO-1 promoter, revealed by chromatin immunoprecipitation assay, which was attenuated by SP600125, Mithramycin A, or Tanshinone II A. We further verified the anti-inflammatory effect of HO-1 expression. Our results showed that 15d-PGJ 2 -induced HO-1 could mitigate the lipopolysaccharide-triggered interleukin-6 expression and secretion, as measured by an ELISA assay kit. These results suggest that 15d-PGJ 2 -induced HO-1 expression is mediated through the activation of NOX- and mitochondria-derived ROS-dependent PKCδ/JNK1/2/Sp1 and the AP-1 signaling pathway and protects against inflammatory responses in bEnd.3 cells.

Recently, we found that 5,8-dihydroxy-4',7-dimethoxyflavone (DDF) upregulated the expression of heme oxygenase (HO)-1 via p38 mitogen-activated protein kinase/nuclear factor-erythroid factor 2-related factor 2 (MAPK/Nrf2) pathway in human cardiac fibroblasts (HCFs). However, the alternative processes by which DDF induces the upregulation of HO-1 expression are unknown. Activation of epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), and protein kinase C (PKC) α may initiate specificity protein (Sp)1 activity, which has been reported to induce expression of antioxidant molecules. Thus, we explored whether these components are engaged in DDF-induced HO-1 upregulation in HCFs. Western blotting, promoter-reporter analyses, and real-time polymerase chain reactions were adopted to measure HO-1 and vascular cell adhesion molecule (VCAM)-1 expressions in HCFs. Respective small interfering (si)RNAs and pharmacological inhibitors were employed to investigate the signaling components engaged in DDF-induced HO-1 upregulation. The chromatin immunoprecipitation assay was conducted to detect the binding interaction of Sp1 and antioxidant response elements (ARE) on the promoter of HO-1. An adhesion assay of THP-1 monocyte was undertaken to examine the functional effect of HO-1 on tumor necrosis factor (TNF)- α -induced VCAM-1 expression. DDF stimulated the EGFR/PKC α /PI3K/Akt pathway leading to activation of Sp1 in HCFs. The roles of these protein kinases in HO-1 induction were ensured by transfection with their respective siRNAs. Chromatin immunoprecipitation assays revealed the interaction between Sp1 and the binding site of proximal ARE on the HO-1 promoter, which was abolished by glutathione, AG1478, Gö6976, LY294002, or mithramycin A. HO-1 expression enhanced by DDF abolished the monocyte adherence to HCFs and VCAM-1 expression induced by TNF- α . Pretreatment with an inhibitor of HO-1: zinc protoporphyrin IX reversed these inhibitory effects of HO-1. We concluded that DDF-induced HO-1 expression was mediated via an EGFR/PKC α /PI3K/Akt-dependent Sp1 pathway and attenuated the responses of inflammation in HCFs., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Chien-Chung Yang et al.)

Human cardiac fibroblasts (HCFs) play key roles in normal physiological functions and pathological processes in the heart. Our recent study has found that, in HCFs, sphingosine 1-phosphate (S1P) can upregulate the expression of cyclooxygenase-2 (COX-2) leading to prostaglandin E 2 (PGE 2 ) generation mediated by S1P receptors/PKC α /MAPKs cascade-dependent activation of NF- κ B. Alternatively, G protein-coupled receptor- (GPCR-) mediated transactivation of receptor tyrosine kinases (RTKs) has been proved to induce inflammatory responses. However, whether GPCR-mediated transactivation of RTKs participated in the COX-2/PGE 2 system induced by S1P is still unclear in HCFs. We hypothesize that GPCR-mediated transactivation of RTKs-dependent signaling cascade is involved in S1P-induced responses. This study is aimed at exploring the comprehensive mechanisms of S1P-promoted COX-2/PGE 2 expression and apoptotic effects on HCFs. Here, we used pharmacological inhibitors and transfection with siRNA to evaluate whether matrix metalloprotease (MMP)2/9, heparin-binding- (HB-) epidermal growth factor (EGF), EGF receptor (EGFR), PI3K/Akt, MAPKs, and transcription factor AP-1 participated in the S1P-induced COX-2/PGE 2 system determined by Western blotting, real-time polymerase chain reaction (RT-PCR), chromatin immunoprecipitation (ChIP), and promoter-reporter assays in HCFs. Our results showed that S1PR1/3 activated by S1P coupled to G q - and G i -mediated MMP9 activity to stimulate EGFR/PI3K/Akt/MAPKs/AP-1-dependent activity of transcription to upregulate COX-2 accompanied with PGE 2 production, leading to stimulation of caspase-3 activity and apoptosis. Moreover, S1P-enhanced c-Jun bound to COX-2 promoters on its corresponding binding sites, which was attenuated by these inhibitors of protein kinases, determined by a ChIP assay. These results concluded that transactivation of MMP9/EGFR-mediated PI3K/Akt/MAPKs-dependent AP-1 activity was involved in the upregulation of the COX-2/PGE 2 system induced by S1P, in turn leading to apoptosis in HCFs., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Chien-Chung Yang et al.)

Excessive production of reactive oxygen species (ROS) by NADPH oxidase (Nox) resulted in inflammation. The negative regulator of ROS (NRROS) dampens ROS generation during inflammatory responses. 15-Deoxy-∆12,14-prostaglandin J 2 (15d-PGJ 2 ) exhibits neuroprotective effects on central nervous system (CNS). However, whether 15d-PGJ 2 -induced NRROS expression was unknown in rat brain astrocytes (RBA-1). NRROS expression was determined by Western blot, RT/real-time PCR, and promoter activity assays. The signaling components were investigated using pharmacological inhibitors or specific siRNAs. The interaction between transcription factors and the NRROS promoter was investigated by chromatin immunoprecipitation assay. Upregulation of NRROS on the hydrogen peroxide (H 2 O 2 )-mediated ROS generation and interleukin 6 (IL-6) secretion was measured. 15d-PGJ 2 -induced NRROS expression was mediated through PI3K/Akt-dependent activation of Sp1 and FoxO1 and established the essential promoter regions. We demonstrated that 15d-PGJ 2 activated PI3K/Akt and following by cooperation between phosphorylated nuclear FoxO1 and Sp1 to initiate the NRROS transcription. In addition, Nrf2 played a key role in NRROS expression induced by 15d-PGJ 2 which was mediated through its phosphorylation. Finally, the NRROS stable clones attenuated the H 2 O 2 -induced ROS generation and expression of IL-6 through suppressing the Nox-2 activity. These results suggested that 15d-PGJ 2 -induced NRROS expression is mediated through a PI3K/Akt-dependent FoxO1 and Sp1 phosphorylation, and Nrf2 cascade, which suppresses ROS generation through attenuating the p47 phox phosphorylation and gp91 phox formation and IL-6 expression in RBA-1 cells. These results confirmed the mechanisms underlying 15d-PGJ 2 -induced NRROS expression which might be a potential strategy for prevention and management of brain inflammatory and neurodegenerative diseases., (© 2021. The Author(s).)

Bradykinin (BK) has been shown to induce matrix metalloproteinase (MMP)-9 expression and participate in neuroinflammation. The BK/MMP-9 axis can be a target for managing neuroinflammation. Our previous reports have indicated that reactive oxygen species (ROS)-mediated nuclear factor-kappaB (NF-κB) activity is involved in BK-induced MMP-9 expression in rat brain astrocytes (RBA-1). Rhamnetin (RNT), a flavonoid compound, possesses antioxidant and anti-inflammatory effects. Thus, we proposed RNT could attenuate BK-induced response in RBA-1. This study aims to approach mechanisms underlying RNT regulating BK-stimulated MMP-9 expression, especially ROS and NF-κB. We used pharmacological inhibitors and siRNAs to dissect molecular mechanisms. Western blotting and gelatin zymography were used to evaluate protein and MMP-9 expression. Real-time PCR was used for gene expression. Wound healing assay was applied for cell migration. 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) were used for ROS generation and NOX activity, respectively. Promoter luciferase assay and chromatin immunoprecipitation (ChIP) assay were applied to detect gene transcription. Our results showed that RNT inhibits BK-induced MMP-9 protein and mRNA expression, promoter activity, and cell migration in RBA-1 cells. Besides, the levels of phospho-PKCδ, NOX activity, ROS, phospho-ERK1/2, phospho-p65, and NF-κB p65 binding to MMP-9 promoter were attenuated by RNT. In summary, RNT attenuates BK-enhanced MMP-9 upregulation through inhibiting PKCδ/NOX/ROS/ERK1/2-dependent NF-κB activity in RBA-1.

Purpose: Tumor necrosis factor-α (TNF-α) has been shown to exert as a pathogenic factor in cardiac fibrosis and heart failure which were associated with the up-regulation of cyclooxygenase (COX)-2/prostaglandin E 2 (PGE 2 ) axis. However, whether TNF-α-induced COX-2/PGE 2 upregulation mediated through ROS-dependent cascade remains elusive in human cardiac fibroblasts (HCFs). This study aims to address the underlying mechanisms of TNF-α-induced COX-2/PGE 2 expression., Methods: Here, we used TNF receptor neutralizing antibody (TNFR nAb), pharmacologic inhibitors, and siRNAs to dissect the involvement of signaling components examined by Western blot and ELISA in TNF-α-mediated responses in HCFs. MitoSOX Red was used to measure mitoROS generation. Isolation of subcellular fractions was performed to determine membrane translocation of PKCα. Promoter luciferase assay and chromatin immunoprecipitation (ChIP) assay were used to determine the role of transcription factor., Results: We found that TNF-α time- and concentration-dependently upregulated COX-2 protein and mRNA expression as well as PGE 2 synthesis which was attenuated by TNFR1 nAb, the inhibitor of mitochondrial ROS scavenger (MitoTEMPO), protein kinase C [(PKC)α, Gö6976], p38 MAPK [p38 inhibitor VIII, (p38i VIII)], JNK1/2 (SP600125), or forkhead box protein O1 [(FoxO1), AS1842856], and transfection with their respective siRNAs in HCFs. TNF-α-stimulated PKCα phosphorylation was inhibited by TNFR1 nAb, MitoTEMPO, or Gö6976. TNF-α stimulated phosphorylation of p38 MAPK and JNK1/2 was attenuated by TNFR1 nAb, MitoTEMPO, Gö6976, and their inhibitors p38i VIII and SP600125. Moreover, TNF-α-triggered FoxO1 phosphorylation was abolished by AS1842856, TNFR1 nAb, and its upstream inhibitors MitoTEMPO, Gö6976, p38i VIII, and SP600125. Phosphorylation of FoxO1 could enhance its interaction with the COX-2 promoter element revealed by ChIP assay, which was attenuated by AS1842856., Conclusion: Our results suggested that TNF-α-induced COX-2/PGE 2 upregulation is mediated through TNFR1-dependent MitoROS/PKCα/p38 MAPK and JNK1/2 cascade to activate FoxO1 binding with the COX-2 promoter in HCFs., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Yang et al.)

Carbon monoxide releasing molecule-3 ( CORM-3) has been shown to protect inflammatory diseases via the upregulation of heme oxygenases-1 (HO-1). However, in rat brain astrocytes (RBA-1), the mechanisms underlying CORM-3-induced HO-1 remain poorly defined. This study used western blot, real-time PCR, and promoter activity assays to determine the levels of HO-1 expression and 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA) and dihydroethidium (DHE) to measure reactive oxygen species (ROS). We found that CORM-3-induced HO-1 expression was mediated through ROS generation by Nox or mitochondria. The signaling components were differentiated by pharmacological inhibitors and small interfering RNA (siRNA). Subcellular fractions, immunofluorescent staining, and chromatin immunoprecipitation assay were used to evaluate the nuclear translocation and promoter binding activity of Nrf2 induced by CORM-3. The roles of mTOR and FoxO1 in CORM-3-stimulated responses are still unknown in RBA-1 cells. Our results demonstrated that transfection with siRNAs or pretreatment with pharmacological inhibitors attenuated the levels of HO-1 and phosphorylation of signaling components including Akt, mTOR, FoxO1, and Nrf2 stimulated by CORM-3. Moreover, pretreatment with N-acetyl-L-cysteine, diphenyleneiodonium chloride, apocynin, or rotenone blocked nuclear translocation and promoter binding activity of Nrf2 induced by CORM-3. The present study concluded that in RBA-1 cells, CORM-3-induced HO-1 expression is, at least partially, mediated through Nox and mitochondria/ROS-dependent PI3K/Akt/mTOR cascade to activate FoxO1 or ROS leading to activation of Nrf2 activity., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Chih-Chung Lin et al.)

Heme oxygenase-1 (HO-1) has been shown to exert as an antioxidant and anti-inflammatory enzyme in cardiovascular inflammatory diseases. Flavonoids have been demonstrated to display anti-inflammatory and antioxidant effects through the induction of HO-1. 5,8-Dihydroxy-4',7-dimethoxyflavone (DDF), one of the flavonoid compounds, is isolated from Reevesia formosana . Whether DDF induced HO-1 expression on human cardiac fibroblasts (HCFs) remained unknown. Here, we found that DDF time- and concentration-dependently induced HO-1 protein and mRNA expression, which was attenuated by pretreatment with reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC) in HCFs. DDF-enhanced ROS generation was attenuated by NAC, but not by either diphenyleneiodonium chloride (DPI, Nox inhibitor) or MitoTempol (mitochondrial ROS scavenger). Interestingly, pretreatment with glutathione (GSH) inhibited DDF-induced HO-1 expression. The ratio of GSH/GSSG was time-dependently decreased in DDF-treated HCFs. DDF-induced HO-1 expression was attenuated by an inhibitor of p38 MAPK (p38i VIII) or siRNA, but not by MEK1/2 (PD98059) or JNK1/2 (SP600125). DDF-stimulated p38 MAPK phosphorylation was inhibited by GSH or p38i VIII. Moreover, DDF-induced HO-1 expression was mediated through Nrf2 phosphorylation and translocation into the nucleus which was attenuated by NAC or p38 siRNA. DDF also stimulated antioxidant response element (ARE) promoter activity which was inhibited by NAC, GSH, or p38i VIII. Interaction between Nrf2 and the ARE-binding sites on the HO-1 promoter was revealed by chromatin immunoprecipitation assay, which was attenuated by NAC, GSH, or p38i VIII. We further evaluated the functional effect of HO-1 expression on the thrombin-induced fibrotic responses. Our result indicated that the induction of HO-1 by DDF can attenuate the thrombin-induced connective tissue growth factor expression. These results suggested that DDF-induced HO-1 expression is, at least, mediated through the activation of the ROS-dependent p38 MAPK/Nrf2 signaling pathway in HCFs. Thus, the upregulation of HO-1 by DDF could be a candidate for the treatment of heart fibrosis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Chien-Chung Yang et al.)

In the regions of tissue injuries and inflammatory diseases, sphingosine 1-phosphate (S1P), a proinflammatory mediator, is increased. S1P may induce the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E 2 (PGE 2 ) system in various types of cells to exacerbate heart inflammation. However, the detailed molecular mechanisms by which S1P induces COX-2 expression in human cardiac fibroblasts (HCFs) remain unknown. HCFs were incubated with S1P and analyzed by Western blotting, real time-Polymerase chain reaction (RT-PCR), and immunofluorescent staining. Our results indicated that S1P activated S1PR 1/3 -dependent transcriptional activity to induce COX-2 expression and PGE 2 production. S1P recruited and activated PTX-sensitive G i or -insensitive G q protein-coupled S1PR and then stimulated PKCα-dependent phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, leading to activating transcription factor NF-κB. Moreover, S1P-activated NF-κB was translocated into the nucleus and bound to its corresponding binding sites on COX-2 promoters determined by chromatin immunoprecipitation (ChIP) and promoter-reporter assays, thereby turning on COX-2 gene transcription associated with PGE 2 production in HCFs. These results concluded that in HCFs, activation of NF-κB by PKCα-mediated MAPK cascades was essential for S1P-induced up-regulation of the COX-2/PGE 2 system. Understanding the mechanisms of COX-2 expression and PGE 2 production regulated by the S1P/S1PRs system on cardiac fibroblasts may provide rationally therapeutic interventions for heart injury or inflammatory diseases., (Copyright © 2020 Yang, Hsiao, Su and Yang.)

Mevastatin (MVS) has been previously shown to induce heme oxygenase (HO)-1 expression through Nox/ROS-dependent PDGFRα/PI3K/Akt/Nrf2/ARE axis in human pulmonary alveolar epithelial cells (HPAEpiCs). However, alternative signaling pathways might involve in MVS-induced HO-1 expression. We found that tumor necrosis factor α (TNFα) induced vascular cell adhesion protein 1 (VCAM-1) expression and NF-κB p65 phosphorylation which were attenuated by pretreatment with MVS via up-regulation of HO-1, determined by Western blot and real-time qPCR. TNFα-induced VCAM-1 expression was attenuated by an NF-κB inhibitor, Bay117082. The inhibitory effects of MVS were reversed by tin protoporphyrin (SnPP)IX (an inhibitor of HO-1 activity). In addition, pretreatment with the inhibitor of pan-Protein kinase C (PKC) (GF109203X), PKCα (Gö6983), Pyk2 (PF431396), p38α MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA), and transfection with their respective siRNAs abolished MVS-induced HO-1 expression in HPAEpiCs. c-Jun (one of AP-1 subunits) was activated by PKCα, Pyk2, p38α MAPK, and JNK1/2, which turned on the transcription of the homx1 gene. The interaction between c-Jun and HO-1 promoter was confirmed by a chromatin immunoprecipitation (ChIP) assay, which was attenuated by these pharmacological inhibitors. These results suggested that MVS induces AP-1/HO-1 expression via PKCα/Pyk2/p38α MAPK- or JNK1/2-dependent c-Jun activation, which further binds with AP-1-binding site on HO-1 promoter and suppresses the TNFα-mediated inflammatory responses in HPAEpiCs. Thus, upregulation of the AP-1/HO-1 system by MVS exerts a potentially therapeutic strategy to protect against pulmonary inflammation., Competing Interests: The authors declare that they have no competing interests.

Background: Mevastatin (MVS), a 3-hydroxy-3-methylglutaryl coenzyme, a reductase (HMG-CoA) inhibitor, has anti-inflammatory effects potentially via up-regulation of heme oxygenase-1 (HO-1). However, the mechanisms underlying MVS-induced HO-1 expression remain largely unknown in human pulmonary alveolar epithelial cells (HPAEpiCs)., Methods: HO-1 and intercellular adhesion molecule (ICAM)-1 expression were determined using real-time PCR, Western blotting, and promoter reporter analyses. The signaling components were investigated using pharmacological inhibitors or specific small interfering RNA (siRNA)s. Interaction between Nrf2 and the antioxidant response element (ARE) binding site for the HO-1 promoter was determined by chromatin immunoprecipitation (ChIP) assay., Results: Upregulation of HO-1 by MVS attenuated the tumor necrosis factor (TNF)-α-stimulated ICAM-1 expression associated with THP-1 adhesion to HPAEpiCs. These inhibitory effects of HO-1 were reversed by tin protoporphyrin (SnPP)IX or by transfection with HO-1 siRNA. MVS-induced HO-1 expression was mediated via NADPH oxidase (Nox)-derived reactive oxygen species (ROS) generation. Activation of Nox2/ROS further stimulated the phosphorylation of p47 phox , proto-oncogene tyrosine-protein kinase (c-Src), platelet-derived growth factor receptor (PDFGR)α, protein kinase B (Akt), and Nrf2, which were inhibited by siRNAs. Pretreatment with pharmacological inhibitors, including diphenyleneiodonium (DPI), apocynin (APO), N-acetyl-L-cysteine (NAC), PP1, AG1296, or LY294002, reduced the MVS-activated Nrf2 nuclear-translocation binding to the ARE on the HO-1 promoter., Conclusions: MVS-induced HO-1 is, at least in part, mediated through a p47 phox /Nox2/ROS-dependent activation of c-Src/PDGFRα/PI3K/Akt-regulated Nrf2/ARE axis and suppresses the TNF-α-mediated inflammatory responses in HPAEpiCs.

Neuroinflammation is a landmark of neuroinflammatory and neurodegenerative diseases. Matrix metalloproteinase (MMP)-9, one member of MMPs, has been shown to contribute to the pathology of these brain diseases. Several experimental models have demonstrated that lipopolysaccharide (LPS) exerts a pathological role through Toll-like receptors (TLRs) in neuroinflammation and neurodegeneration. However, the mechanisms underlying LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1) are not completely understood. Here, we applied pharmacological inhibitors and siRNA transfection to assess the levels of MMP-9 protein, mRNA, and promoter activity, as well as protein kinase phosphorylation in RBA-1 cells triggered by LPS. We found that LPS-induced expression of pro-form MMP-9 and cell migration were mediated through TLR4, proto-oncogene tyrosine-protein kinase (c-Src), proline-rich tyrosine kinase 2 (Pyk2), platelet-derived growth factor receptor (PDGFR), phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), p38 mitogen-activated protein kinase (MAPK), and Jun amino-terminal kinase (JNK)1/2 signaling molecules in RBA-1 cells. In addition, LPS-stimulated binding of c-Jun to the MMP-9 promoter was confirmed by chromatin immunoprecipitation (ChIP) assay, which was blocked by pretreatment with c-Src inhibitor II, PF431396, AG1296, LY294002, Akt inhibitor VIII, p38 MAP kinase inhibitor VIII, SP600125, and tanshinone IIA. These results suggest that in RBA-1 cells, LPS activates a TLR4/c-Src/Pyk2/PDGFR/PI3K/Akt/p38 MAPK and JNK1/2 pathway, which in turn triggers activator protein 1 (AP-1) activation and ultimately induces MMP-9 expression and cell migration.

Lysophosphatidylcholine (LPC) may accumulate in the heart to cause fibrotic events, which is mediated through fibroblast activation and collagen accumulation. Here, we evaluated the mechanisms underlying LPC-mediated collagen induction via mitochondrial events in human cardiac fibroblasts (HCFs), coupling application of the pharmacologic cyclooxygenase-2 (COX-2) inhibitor, celecoxib, and genetic mutations in FOXO1 on the fibrosis pathway. In HCFs, LPC caused prostaglandin E 2 (PGE 2 )/PGE 2 receptor 4 (EP 4 )-dependent collagen induction via activation of transcriptional activity of forkhead box protein O1 (FoxO1) on COX-2 gene expression. These responses were mediated through LPC-induced generation of mitochondrial reactive oxygen species (mitoROS), as confirmed by ex vivo studies, which indicated that LPC increased COX-2 expression and oxidative stress. LPC-induced mitoROS mediated the activation of protein kinase C (PKC)α, which interacted with and phosphorylated dynamin-related protein 1 (Drp1) at Ser 616 , thereby increasing Drp1-mediated mitochondrial fission and mitochondrial depolarization. Furthermore, inhibition of PKCα and Drp1 reduced FoxO1-mediated phosphorylation at Ser 256 and nuclear accumulation, which suppressed COX-2/PGE 2 expression and collagen production. Moreover, pretreatment with celecoxib or COX-2 siRNA suppressed WT FoxO1; mutated Ser 256 -to-Asp 256 FoxO1-enhanced collagen induction, which was reversed by addition of PGE 2 Our results demonstrate that LPC-induced generation of mitoROS regulates PKCα-mediated Drp1-dependent mitochondrial fission and COX-2 expression via a PKCα/Drp1/FoxO1 cascade, leading to PGE 2 /EP 4 -mediated collagen induction. These findings provide new insights about the role of LPC in the pathway of fibrotic injury in HCFs., (Copyright © 2019 Tseng et al.)

The up-regulation of heme oxygenase-1 (HO-1) is mediated through nicotinamaide adenine dinucleotide phosphate (NADPH) oxidases (Nox) and reactive oxygen species (ROS) generation, which could provide cytoprotection against inflammation. However, the molecular mechanisms of carbon monoxide-releasing molecule (CORM)-2-induced HO-1 expression in human tracheal smooth muscle cells (HTSMCs) remain unknown. Here, we found that pretreatment with CORM-2 attenuated the lipopolysaccharide (LPS)-induced intercellular adhesion molecule (ICAM-1) expression and leukocyte count through the up-regulation of HO-1 in mice, which was revealed by immunohistochemistrical staining, Western blot, real-time PCR, and cell count. The inhibitory effects of HO-1 by CORM-2 were reversed by transfection with HO-1 siRNA. Next, Western blot, real-time PCR, and promoter activity assay were performed to examine the HO-1 induction in HTSMCs. We found that CORM-2 induced HO-1 expression via the activation of protein kinase C (PKC)α and proline-rich tyrosine kinase (Pyk2), which was mediated through Nox-derived ROS generation using pharmacological inhibitors or small interfering ribonucleic acids (siRNAs). CORM-2-induced HO-1 expression was mediated through Nox-(1, 2, 4) or p47 phox , which was confirmed by transfection with their own siRNAs. The Nox-derived ROS signals promoted the activities of extracellular signal-regulated kinase 1/2 (ERK1/2). Subsequently, c-Fos and c-Jun-activator protein-1 (AP-1) subunits-were up-regulated by activated ERK1/2, which turned on transcription of the HO-1 gene by regulating the HO-1 promoter. These results suggested that in HTSMCs, CORM-2 activates PKCα/Pyk2-dependent Nox/ROS/ERK1/2/AP-1, leading to HO-1 up-regulation, which suppresses the lipopolysaccharide (LPS)-induced airway inflammation.

Neuroinflammation is characterized by the elevated expression of various inflammatory proteins, including matrix metalloproteinases (MMPs), induced by various pro-inflammatory mediators, which play a critical role in neurodegenerative disorders. Interleukin-1β (IL-1β) has been shown to induce the upregulation of MMP-9 through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-reactive oxygen species (ROS)-dependent signaling pathways. N-(2-cyano-3,12-dioxo-28-noroleana-1,9(11)-dien-17-yl)-2-2-difluoropropanamide (RTA 408), a novel synthetic triterpenoid, has been shown to possess anti-oxidant and anti-inflammatory properties in various types of cells. Here, we evaluated the effects of RTA 408 on IL-1β-induced inflammatory responses by suppressing MMP-9 expression in a rat brain astrocyte (RBA-1) line. IL-1β-induced MMP-9 protein and mRNA expression, and promoter activity were attenuated by RTA 408. The increased level of ROS generation in RBA-1 cells exposed to IL-1β was attenuated by RTA 408, as determined by using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and CellROX. In addition, the inhibitory effects of RTA 408 on MMP-9 expression resulted from the suppression of the IL-1β-stimulated activation of Pyk2 (proline-rich tyrosine kinase), platelet-derived growth factor receptor β (PDGFRβ), Akt, ROS, and mitogen-activated protein kinases (MAPKs). Pretreatment with RTA 408 attenuated the IL-1β-induced c-Jun phosphorylation, mRNA expression, and promoter activity. IL-1β-stimulated nuclear factor-κB (NF-κB) p65 phosphorylation, translocation, and promoter activity were also attenuated by RTA 408. Furthermore, IL-1β-induced glial fibrillary acidic protein (GFAP) protein and mRNA expression, and cell migration were attenuated by pretreatment with RTA 408. These results provide new insights into the mechanisms by which RTA 408 attenuates IL-1β-mediated inflammatory responses and exerts beneficial effects for the management of brain diseases.

The upregulation of heme oxygenase-1 (HO-1) by the carbon monoxide-releasing molecule (CORM)-2 may be mediated through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases [Nox] and reactive oxygen species (ROS) generation, which could provide cytoprotection against various cellular injuries. However, the detailed mechanisms of CORM-2-induced HO-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain largely unknown. Therefore, we dissected the mechanisms underlying CORM-2-induced HO-1 expression in HPAEpiCs. We found that the administration of mice with CORM-2 attenuated the tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule-1 (ICAM-1) expression and leukocyte count as revealed by immunohistochemical staining, western blot, real-time polymerase chain reaction (PCR), and cell count. Furthermore, TNF-α-induced ICAM-1 expression associated with monocyte adhesion to HPAEpiCs was attenuated by infection with adenovirus (adv)-HO-1 or incubation with CORM-2. These inhibitory effects of HO-1 were reversed by pretreatment with hemoglobin (Hb). Moreover, CORM-2-induced HO-1 expression was mediated via the phosphorylation of p47 phox , c-Src, epidermal growth factor receptor (EGFR), Akt, and NF-E2-related factor 2 (Nrf2), which were inhibited by their pharmacological inhibitors, including diphenyleneiodonium (DPI) or apocynin (APO), ROS [N-acetyl-L-cysteine (NAC)], PP1, AG1478, PI3K (LY294002), or Akt (SH-5), and small interfering RNAs (siRNAs). CORM-2-enhanced Nrf2 expression, and anti-oxidant response element (ARE) promoter activity was also inhibited by these pharmacological inhibitors. The interaction between Nrf2 and AREs was confirmed with a chromatin immunoprecipitation (ChIP) assay. These findings suggest that CORM-2 increases the formation of the Nrf2 and AREs complex and binds with ARE-binding sites via Src, EGFR, and PI3K/Akt, which further induces HO-1 expression in HPAEpiCs. Thus, the HO-1/CO system might suppress TNF-α-mediated inflammatory responses and exert a potential therapeutic strategy in pulmonary diseases.

Galangin, a member of the flavonol compounds of the flavonoids, could exert anti-inflammatory effects in various cell types. It has been used for the treatment of arthritis, airway inflammation, stroke, and cognitive impairment. Thrombin, one of the regulators of matrix metalloproteinase (MMPs), has been known as a vital factor of physiological and pathological processes, including cell migration, the blood⁻brain barrier breakdown, brain edema formation, neuroinflammation, and neuronal death. MMP-9 especially may contribute to neurodegenerative diseases. However, the effect of galangin in combating thrombin-induced MMP-9 expression is not well understood in neurons. Therefore, we attempted to explore the molecular mechanisms by which galangin inhibited MMP-9 expression and cell migration induced by thrombin in SK-N-SH cells (a human neuroblastoma cell line). Gelatin zymography, western blot, real-time PCR, and cell migration assay were used to elucidate the inhibitory effects of galangin on the thrmbin-mediated responses. The results showed that galangin markedly attenuated the thrombin-stimulated phosphorylation of proto-oncogene tyrosine-protein kinase (c-Src), proline-rich tyrosine kinase 2 (Pyk2), protein kinase C (PKC)α/β/δ, protein kinase B (Akt), mammalian target of rapamycin (mTOR), p42/p44 mitogen-activated protein kinase (MAPK), Jun amino-terminal kinases (JNK)1/2, p38 MAPK, forkhead box protein O1 (FoxO1), p65, and c-Jun and suppressed MMP-9 expression and cell migration in SK-N-SH cells. Our results concluded that galangin blocked the thrombin-induced MMP-9 expression in SK-N-SH cells via inhibiting c-Src, Pyk2, PKCα/βII/δ, Akt, mTOR, p42/p44 MAPK, JNK1/2, p38 MAPK, FoxO1, c-Jun, and p65 phosphorylation and ultimately attenuated cell migration. Therefore, galangin may be a potential candidate for the management of brain inflammatory diseases.

Lysophosphatidylcholine (LysoPC) has been shown to induce the expression of inflammatory proteins, including cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6), associated with cardiac fibrosis. Here, we demonstrated that LysoPC-induced COX-2 and IL-6 expression was inhibited by silencing NADPH oxidase 1, 2, 4, 5; p65; and FoxO1 in human cardiac fibroblasts (HCFs). LysoPC-induced IL-6 expression was attenuated by a COX-2 inhibitor. LysoPC-induced responses were mediated via the NADPH oxidase-derived reactive oxygen species-dependent JNK1/2 phosphorylation pathway, leading to NF-κB and FoxO1 activation. In addition, we demonstrated that both FoxO1 and p65 regulated COX-2 promoter activity stimulated by LysoPC. Overexpression of wild-type FoxO1 and S256D FoxO1 enhanced COX-2 promoter activity and protein expression in HCFs. These results were confirmed by ex vivo studies, where LysoPC-induced COX-2 and IL-6 expression was attenuated by the inhibitors of NADPH oxidase, NF-κB, and FoxO1. Our findings demonstrate that LysoPC-induced COX-2 expression is mediated via NADPH oxidase-derived reactive oxygen species generation linked to the JNK1/2-dependent pathway leading to FoxO1 and NF-κB activation in HCFs. LysoPC-induced COX-2-dependent IL-6 expression provided novel insights into the therapeutic targets of the cardiac fibrotic responses.

Staphylococcus aureus ( S. aureus ) is a very common Gram-positive bacterium. It is widely distributed in air, soil, and water. S. aureus often causes septicemia and pneumonia in patients. In addition, it is considered to play a key role in mediating cell adhesion molecules upregulation. Resveratrol is a natural antioxidant with diverse biological effects, including the modulation of immune function, anti-inflammation, and cancer chemoprevention. In this study, we proved that S. aureus -upregulated vascular cell adhesion molecule-1 (VCAM-1) expression in human lung epithelial cells (HPAEpiCs) was inhibited by resveratrol. We also observed that resveratrol downregulated S. aureus -enhanced leukocyte count in bronchoalveolar lavage (BAL) fluid in mice. In HPAEpiCs, S. aureus stimulated c-Src, PDGFR, p38 MAPK, or JNK1/2 phosphorylation, which was inhibited by resveratrol. S. aureus induced the adhesion of THP-1 cells (a human monocytic cell line) to HPAEpiCs, which was also reduced by resveratrol. Finally, we found that S. aureus induced c-Src/PDGFR/p38 MAPK and JNK1/2-dependent c-Jun and ATF2 activation and in vivo binding of c-Jun and ATF2 to the VCAM-1 promoter, which were inhibited by resveratrol. Thus, resveratrol functions as a suppressor of S. aureus -induced inflammatory signaling, not only by inhibiting VCAM-1 expression but also by diminishing c-Src, PDGFR, JNK1/2, p38 MAPK, and AP-1 activation in HPAEpiCs.

Background and Purpose: Haem oxygenase-1 (HO-1) is induced by thiazolidinediones including rosiglitazone and exerts anti-inflammatory effects in various models. However, the molecular mechanisms underlying rosiglitazone-induced HO-1 expression remain largely unknown in human pulmonary alveolar epithelial cells (HPAEpiCs)., Experimental Approach: HO-1 expression was determined by real time-PCR, Western blotting and promoter reporter analyses. Signalling pathways were investigated using pharmacological inhibitors or specific siRNAs. Interactions between nuclear factor erythroid-2-related factor (Nrf2) and antioxidant response elements (ARE) binding site of the HO-1 promoter were investigated with chromatin immunoprecipitation assays., Key Results: Up-regulation of HO-1 in HPAEpiCs or in mice by rosiglitazone blunted ICAM-1 expression and monocyte adhesion to HPAEpiCs challenged with LPS. Rosiglitazone-induced HO-1 expression was significantly attenuated by NADPH oxidase (NOX) inhibitors (apocynin and diphenyleneiodonium) or ROS scavenger (N-acetyl cysteine). The involvement of NOX activity and ROS generation in rosiglitazone-induced HO-1 expression was confirmed by transfection with p47 phox or NOX2 siRNA. Moreover, pretreatment with the inhibitors of c-Src (c-Srci II), proline-rich tyrosine kinase 2 (Pyk2) (PF431396), Akt (Akti VIII) or PPARγ (GW9662) and transfection with siRNA of c-Src, Pyk2, Akt or PPARγ abolished the rosiglitazone-induced HO-1 expression in HPAEpiCs. Subsequently, Nrf2 was activated by phosphorylation of c-Src, Pyk2 and Akt, which turned on transcription of HO-1 gene by binding to AREs binding site and enhancing ARE promoter activity., Conclusions and Implications: Rosiglitazone induces HO-1 expression via either NOX/ROS/c-Src/Pyk2/Akt-dependent Nrf2 activation or PPARγ in HPAEpiCs and suppresses LPS-mediated inflammatory responses, suggesting that PPARγ agonists may be useful for protection against pulmonary inflammation., (© 2018 The British Pharmacological Society.)

Arachidonic acid (AA) is a major product of phospholipid hydrolysis catalyzed by phospholipase A 2 during neurodegenerative diseases. AA exerts as a second messenger to regulate various signaling components which may be involved in different pathophysiological processes. Astrocytes are the main types of CNS resident cells which maintain and support the physiological function of brain. AA has been shown to induce ROS generation through activation of NADPH oxidases (Noxs) which may play a key role in the expression of heme oxygenase-1 (HO-1). Therefore, this study was designed to investigate the mechanisms underlying AA-induced HO-1 expression in rat brain astrocytes (RBA-1). We found that AA induced HO-1 protein and mRNA expression and promoter activity in RBA-1, which was mediated through the synthesis of 15-deoxy-Δ12,14-prostaglandin D 2 -activated peroxisome proliferator-activated receptor-γ (PPARγ) receptors. This note was confirmed by transfection with PPARγ small interfering RNAs (siRNA) which attenuated the AA-mediated responses. AA-induced HO-1 expression was mediated through Nox/ROS generation, which was inhibited by Nox inhibitors (diphenyleneiodonium and apocynin) and ROS scavengers (N-acetyl cysteine). Moreover, AA-induced HO-1 expression was mediated through phosphorylation of Src, Pyk2, platelet-derived growth factor, PI3K/Akt, and ERK1/2 which were inhibited by the pharmacological inhibitors including PP1, PF431396, AG1296, LY294002, and U0126 or by transfection with respective siRNAs. AA-enhanced Nrf2 expression and HO-1 promoter activity was inhibited by transfection with Nrf2 siRNA or by these pharmacological inhibitors. Furthermore, chromatin immunoprecipitation assay confirmed that Nrf2 and PPARγ were associated with the proximal antioxidant response element (ARE)-binding site on HO-1 promoter, suggesting that Nrf2/PPARγ are key transcription factors modulating HO-1 expression. AA-induced ARE promoter activity was also reduced by these pharmacological inhibitors. These findings suggested that AA increases formation of Nrf2 and PPARγ complex and binding with ARE1 binding site through Src, Pyk2, PI3K/Akt, and ERK1/2, which further induced HO-1 expression in RBA-1 cells.

HO-1 (heme oxygenase-1), an antioxidant enzyme, induced by rosiglitazone (PPAR ligands) can be a potential treatment of inflammation. However, the mechanisms of rosiglitazone-induced HO-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain largely unknown. In this study, we found that upregulation of HO-1 in vitro or in vivo by rosiglitazone attenuated VCAM-1 gene expression and monocyte adhesion to HPAEpiCs challenged with lipopolysaccharide (LPS). The inhibitory effects of rosiglitazone on LPS-mediated responses were reversed by transfection with HO-1 siRNA. LPS-induced VCAM-1 expression was mediated through NF-κB activation which was attenuated by rosiglitazone via suppressing p65 activation and translocation into the nucleus. Moreover, pretreatment with the inhibitor of PKCs (H7), PKCα (Gö6976), AMPKα (Compound C), p38 MAPKα (p38i VIII), SIRT1 (Sirtinol), or PPARγ (T0070907) and transfection with siRNA of PKCα, AMPKα, p38 MAPKα, SIRT1, or PPARγ abolished the rosiglitazone-induced HO-1 expression in HPAEpiCs. Further studies indicated that rosiglitazone stimulated SIRT1 deacetylase leading to PGC1α translocation from the cytosol into the nucleus, promoting fragmentation of NCoR and phosphorylation of PPARγ. Subsequently, PPARγ was activated by phosphorylation of PKCα, AMPKα, p38 MAPKα, and SIRT1, which turned on transcription of HO-1 gene by binding to PPAR response element (PPRE) and enhancing PPARγ promoter activity. These results suggested that rosiglitazone-induced HO-1 expression is mediated through PKCα/AMPKα/p38 MAPKα/SIRT1-dependent deacetylation of Ac-PGC1α and fragmentation of NCoR/PPARγ activation in HPAEpiCs. Up-regulation of HO-1 protected against the inflammatory responses triggered by LPS, at least in part, through attenuation of NF-κB., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background and Purpose: Haem oxygenase-1 (HO-1) could provide cytoprotection against various inflammatory diseases. However, the mechanisms underlying the protective effect of CO-releasing molecule-2 (CORM-2)-induced HO-1 expression against TNF-α-induced inflammatory responses in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unknown., Experimental Approach: CORM-2-induced HO-1 protein and mRNA expression, and signalling pathways were determined by Western blot and real-time PCR, coupled with respective pharmacological inhibitors or transfection with siRNAs. The effect of CORM-2 on TNF-α-induced increase in leukocyte counts in BAL fluid and VCAM-1 expression in lung was determined by cell counting and Western blot analysis., Key Results: CORM-2 attenuated the TNF-α-induced pulmonary haematoma, VCAM-1 expression and increase in leukocytes through an up-regulation of HO-1 in mice; this effect of CORM-2 was reversed by the HO-1 inhibitor zinc protoporphyrin IX. Furthermore, CORM-2 increased HO-1 protein and mRNA expression as well as the phosphorylation of PYK2, PKCα and ERK1/2 (p44/p42 MAPK) in HPAEpiCs; these effects were attenuated by their respective pharmacological inhibitors or transfection with siRNAs. Inhibition of PKCα by Gö6976 or Gö6983 attenuated CORM-2-induced stimulation of PKCα and ERK1/2 phosphorylation but had no effect on PYK2 phosphorylation. Moreover, inhibition of PYK2 by PF431396 reduced the phosphorylation of all three protein kinases. Finally, PYK2/PKCα/ERK1/2-mediated stimulation of activator protein 1 was shown to play a key role in CORM-2-induced HO-1 expression via an up-regulation of c-Fos mRNA., Conclusions and Implications: CORM-2 activates a PYK2/PKCα/ERK1/2/AP-1 pathway leading to HO-1 expression in HPAEpiCs. This HO-1/CO system might have potential as a therapeutic target in pulmonary inflammation., (© 2017 The British Pharmacological Society.)

Neurodegenerative disorders and brain damage are initiated by excessive production of reactive oxygen species (ROS), which leads to tissue injury, cellular death and inflammation. In cellular anti-oxidant systems, heme oxygenase-1 (HO-1) is an oxidative-sensor protein induced by ROS generation or carbon monoxide (CO) release. CO releasing molecules (CORMs), including CORM-3, exert anti-oxidant and anti-inflammatory effects. However, the molecular mechanisms of CORM-3-induced HO-1 expression and protection against interleukin (IL)-1β-induced inflammatory responses have not been fully elucidated in rat brain astrocytes (RBA-1). To study the regulation of CORM-3-induced HO-1 expression, signaling pathways, promoter activity, mRNA and protein expression were assessed following treatment with pharmacological inhibitors and gene-specific siRNA knockdown. We found that CORM-3 mediated HO-1 induction via transcritional and translational processes. Furthermore, CORM-3-induced HO-1 expression was mediated by phosphorylation of several protein kinases, such as c-Src, Pyk2, protein kinase Cα (PKCα) and p42/p44 mitogen-activated protein kinase (MAPK), which were inhibited by respective pharmacological inhibitors or by gene-specific knockdown with siRNA transfections. Next, we found that CORM-3 sequentially activated the c-Src/Pyk2/PKCα/p42/p44 MAPK pathway, thereby up-regulating mRNA for the activator protein (AP)-1 components c-Jun and c-Fos; these effects were attenuated by an AP-1 inhibitor (Tanshinone IIA; TSIIA) and other relevant inhibitors. Moreover, CORM-3-induced upregulation of HO-1 attenuated the IL-1β-induced cell migration and matrix metallopeptidase-9 mRNA expression in RBA-1 cells. These effects were reversed by an matrix metalloproteinase (MMP)2/9 inhibitor or by transfection with HO-1 siRNA.

Neuroinflammation is a hallmark of neurodegenerative disorders in the central nerve system (CNS). Thrombin has been known as one of the factors in pathological processes including migration, blood-brain barrier breakdown, brain edema formation, neuroinflammation, and neuronal death. Thrombin has been shown to be a regulator of matrix metalloproteinase (MMPs) expression leading to cell migration. Among MMPs, the elevated expression of MMP-9 has been observed in patients with brain diseases, which may contribute to the pathology of neuroinflammatory and neurodegenerative diseases. However, the mechanisms underlying thrombin-induced MMP-9 expression in SK-N-SH cells were not completely understood. Here, we used gelatin zymography, Western blot, real-time PCR, promoter activity assay, and cell migration assay to demonstrate that thrombin induced the expression of pro-form MMP-9 protein and messenger RNA (mRNA), and promoter activity in SK-N-SH cells, which were attenuated by pretreatment with the pharmacological inhibitor of protease-activated receptor-1 (PAR-1, SCH79797), Gi-coupled receptor (GPA2), c-Src (PP1), Pyk2 (PF431396), EGFR (AG1478), PI3K (LY294002), Akt (SH-5), MEK1/2 (U0126), or AP-1 (TanshinoneIIA) and transfection with small interfering RNA (siRNA) of PAR-1, Gi, c-Src, Pyk2, EGFR, Akt, p44, p42, or c-Jun. Moreover, thrombin-stimulated c-Src, Pyk2, EGFR, Akt, p42/p44 MAPK, or c-Jun phosphorylation was attenuated by their respective inhibitor of PP1, PF431396, AG1478, SH-5, U0126, or TanshinoneIIA. Finally, pretreatment with these inhibitors also blocked thrombin-induced SK-N-SH cell migration. Our results concluded that thrombin binding to PAR-1 receptor activated Gi-protein/c-Src/Pyk2/EGFR/PI3K/Akt/p42/p44 MAPK cascade, which in turn elicited AP-1 activation and ultimately evoked MMP-9 expression and cell migration in SK-N-SH cells.

Bradykinin (BK) induces inflammation in rheumatoid arthritis (RA). Resveratrol is a potent activator of Sirt1 which could modulate inflammation through deacetylating histones of transcription factors. Here, we investigated the mechanisms underlying BK-induced COX-2 expression which is modulated by resveratrol/Sirt1 in human rheumatoid arthritis synovial fibroblasts (RASFs). We found that BK-induced COX-2 protein and mRNA expression associated with PGE 2 synthesis, and promoter activity was mediated through B2R receptors, which were attenuated by selective B2R antagonist Hoe140 or transfection with B2R siRNA. BK-induced responses were mediated through PKCμ, MAPKs, AP-1 and NF-κB which were inhibited by their respective inhibitors or siRNAs. Up-regulation of Sirt1 by resveratrol suppressed the BK-induced COX-2/PGE 2 production through inhibiting the interaction of AP-1 and NF-κB with COX-2 promoter in RASFs. BK-induced COX-2/PGE 2 expression is mediated through a B2R-PKCμ-dependent MAPKs, AP-1, and NF-κB cascade. Resveratrol inhibited the phosphorylation and acetylation of p65, c-Jun, and Fos and reduced the binding to the COX-2 promoter, thereby attenuated the COX-2 expression. Therefore, resveratrol may be a promising therapeutic intervention for treatment of inflammatory arthritis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tumor necrosis factor-α (TNF-α) triggers activation of cytosolic phospholipase A 2 (cPLA 2 ) and then enhancing the synthesis of prostaglandin (PG) in inflammatory diseases. However, the detailed mechanisms of TNF-α induced cPLA 2 expression were not fully defined in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that TNF-α-stimulated increases in cPLA 2 mRNA (5.2 folds) and protein (3.9 folds) expression, promoter activity (4.3 folds), and PGE 2 secretion (4.7 folds) in HPAEpiCs, determined by Western blot, real-time PCR, promoter activity assay and PGE 2 ELISA kit. These TNF-α-mediated responses were abrogated by the inhibitors of NADPH oxidase [apocynin (APO) and diphenyleneiodonium chloride (DPI)], ROS [N-acetyl cysteine, (NAC)], NF-κB (Bay11-7082) and transfection with siRNA of ASK1, p47 phox , TRAF2, NIK, IKKα, IKKβ, or p65. TNF-α markedly stimulated NADPH oxidase activation and ROS including superoxide and hydrogen peroxide production which were inhibited by pretreatment with a TNFR1 neutralizing antibody, APO, DPI or transfection with siRNA of TRAF2, ASK1, or p47 phox . In addition, TNF-α also stimulated p47 phox phosphorylation and translocation in a time-dependent manner. On the other hand, TNF-α induced TNFR1, TRAF2, ASK1, and p47 phox complex formation in HPAEpiCs, which were attenuated by a TNF-α neutralizing antibody. We found that pretreatment with NAC, DPI, or APO also attenuated the TNF-α-stimulated IKKα/β and NF-κB p65 phosphorylation, NF-κB (p65) translocation, and NF-κB promoter activity in HPAEpiCs. Finally, we observed that TNF-α-stimulated NADPH oxidase activation and ROS generation activates NF-κB through the NIK/IKKα/β pathway. Taken together, our results demonstrated that in HPAEpiCs, up-regulation of cPLA 2 by TNF-α is, at least in part, mediated through the cooperation of TNFR1, TRAF2, ASK1, and NADPH oxidase leading to ROS generation and ultimately activates NF-κB pathway.

Thrombin has been known to activate inflammatory genes including matrix metalloproteinases (MMPs). The elevated expression of MMP-9 has been observed in patients with neuroinflammatory diseases and may contribute to the pathology of brain diseases. However, the mechanisms underlying thrombin-induced MMP-9 expression in SK-N-SH cells remain unknown. The effects of thrombin on MMP-9 expression were examined in SK-N-SH cells by gelatin zymography, Western blot, real-time PCR, promoter activity assay, and cell migration assay. The detailed mechanisms were analyzed by using pharmacological inhibitors and small intefering RNA (siRNA) transfection. Here, we demonstrated that thrombin induced the expression of proform MMP-9 and migration of SK-N-SH cells, which were attenuated by pretreatment with the inhibitor of thrombin (PPACK), Gq (GPA2A), PC-PLC (D609), PI-PLC (ET-18-OCH 3 ), nonselective protien kinase C (PKC, GF109203X), PKCα/βII (Gö6983), PKCδ (Rottlerin), p38 mitogen-activated protein kinases (MAPK) (SB202190), JNK1/2 (SP600125), or NF-κB (Bay11-7082 or Helenalin) and transfection with siRNA of Gq, PKCα, PKCβ, PKCδ, p38, JNK1/2, IKKα, IKKβ, or p65. Moreover, thrombin-stimulated PKCα/βII, PKCδ, p38 MAPK, JNK1/2, or p65 phosphorylation was abrogated by their respective inhibitor of PPACK, GPA2A, D609, ET-18-OCH 3 , Gö6983, Rottlerin, SB202190, SP600125, Bay11-7082, or Helenalin. Pretreatment with these inhibitors or transfection with MMP-9 siRNA also blocked thrombin-induced SK-N-SH cell migration. Our results show that thrombin stimulates a Gq/PLC/PKCs/p38 MAPK and JNK1/2 cascade, which in turn triggers NF-κB activation and ultimately induces MMP-9 expression and cell migration in SK-N-SH cells.

Upregulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Lipopolysaccharide (LPS) has been shown to play a key role in inflammation via adhesion molecule induction and then causes lung injury. However, the mechanisms underlying LPS-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. We showed that LPS induced ICAM-1 expression in HPAEpiCs, revealed by Western blotting, RT-PCR, real-time PCR, and promoter assay. Pretreatment with the inhibitor of c-Src (protein phosphatase-1, PP1), reactive oxygen species (ROS) (Edaravone), NADPH oxidase (apocynin and diphenyleneiodonium chloride), EGFR (AG1478), PDGFR (AG1296), phosphatidylinositol-3-kinase (PI3K) (LY294002), MEK1/2 (U0126), or NF-κB (Bay11-7082) and transfection with siRNAs of c-Src, EGFR, PDGFR, Akt, p47(phox), Nox2, Nox4, p42, and p65 markedly reduced LPS-induced ICAM-1 expression and monocyte adherence to HPAEpiCs challenged with LPS. In addition, we established that LPS stimulated phosphorylation of c-Src, EGFR, PDGFR, Akt, or p65, which was inhibited by pretreatment with their respective inhibitors. LPS induced Toll-like receptor 4 (TLR4), MyD88, TNF receptor-associated factor 6 (TRAF6), c-Src, p47(phox), and Rac1 complex formation 2, which was attenuated by transfection with c-Src or TRAF6 siRNA. Furthermore, LPS markedly enhanced NADPH oxidase activation and intracellular ROS generation, which were inhibited by PP1. We established that LPS induced p42/p44 MAPK activation via a c-Src/NADPH oxidase/ROS/EGFR, PDGFR/PI3K/Akt-dependent pathway in these cells. Finally, we observed that LPS significantly enhanced NF-κB and IκBα phosphorylation, NF-κB translocation, and NF-κB promoter activity, which were inhibited by PP1, Edaravone, apocynin, diphenyleneiodonium chloride, AG1478, AG1296, LY294002, or U0126. These results demonstrated that LPS induces p42/p44 MAPK activation mediated through the TLR4/MyD88/TRAF6/c-Src/NADPH oxidase/ROS/EGFR, PDGFR/PI3K/Akt pathway, which in turn initiates the activation of NF-κB and ultimately induces ICAM-1 expression in HPAEpiCs., (Copyright © 2016 the American Physiological Society.)

The intercellular adhesion molecule-1 (ICAM-1) expression is frequently correlated with the lung inflammation. In lung injury, sphingosine-1-phosphate (S1P, bioactive sphingolipid metabolite), participate gene regulation of adhesion molecule in inflammation progression and aggravate tissue damage. To investigate the transduction mechanisms of the S1P in pulmonary epithelium, we demonstrated that exposure of HPAEpiCs (human pulmonary alveolar epithelial cells) to S1P significantly induces ICAM-1 expression leading to increase monocyte adhesion on the surface of HPAEpiCs. These phenomena were effectively attenuated by pretreatments with series of inhibitors such as Rottlerin (PKCδ), PF431396 (PYK2), diphenyleneiodonium chloride (DPI), apocynin (NADPH oxidase), Edaravone (ROS), and Bay11-7082 (NF-κB). Consistently, knockdown with siRNA transfection of PKCδ, PYK2, p47 (phox) , and p65 exhibited the same results. Pretreatment with both Gq-coupled receptor antagonist (GPA2A) and Gi/o-coupled receptor antagonist (GPA2) also blocked the upregulation of ICAM-1 protein and mRNA induced by S1P. We observed that S1P induced PYK2 activation via a Gq-coupled receptor/PKCδ-dependent pathway. In addition, S1P induced NADPH oxidase activation and intracellular ROS generation, which were also reduced by Rottlerin or PF431396. We demonstrated that S1P induced NF-κB p65 phosphorylation and nuclear translocation in HPAEpiCs. Activated NF-κB was blocked by Rottlerin, PF431396, APO, DPI, or Edaravone. Besides, the results of monocyte adhesion assay indicated that S1P-induced ICAM-1 expression on HPAEpiCs can enhance the monocyte attachments. In the S1P-treated mice, we found that the levels of ICAM-1 protein and mRNA in the lung fractions, the pulmonary hematoma and leukocyte count in bronchoalveolar lavage fluid were enhanced through a PKCδ/PYK2/NADPH oxidase/ROS/NF-κB signaling pathway. We concluded that S1P-accelerated lung damage is due to the ICAM-1 induction associated with leukocyte recruitment.

The inflammation-dependent adhesion molecule expressions are characterized in cardiovascular diseases and myocardial tissue infiltrations. Several pro-inflammatory cytokines are elevated in the acute myocardial injury and infarction. Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, is raised in the injury tissues and inflammatory regions and involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. In fibroblasts, TNF-α-triggered expression of vascular cell adhesion molecule (VCAM)-1 aggravated the heart inflammation. However, the mechanisms underlying TNF-α-mediated VCAM-1 expression in cardiac fibroblasts remain unclear. Here, the primary cultured human cardiac fibroblasts (HCFs) were used to investigate the effects of TNF-α on VCAM-1 expression. The molecular evidence, including protein, mRNA, and promoter analyses, indicated that TNF-α-induced VCAM-1 gene expression is mediated through the TNFR-dependent manner. Activation of TNF-α/TNFR system triggered PKCα-dependent NADPH oxidase (Nox)/reactive oxygen species (ROS) signal linking to MAPK cascades, and then led to activation of the transcription factor, AP-1. Moreover, the results of mRNA and promoter assay demonstrated that c-Jun/AP-1 phosphorylated by TNF-α turns on VCAM-1 gene expression. Subsequently, up-regulated VCAM-1 on the cell surface of TNF-α-challenged HCFs increased the number of monocytes adhering to these cells. These results indicated that in HCFs, activation of AP-1 by PKCα-dependent Nox/ROS/MAPKs cascades is required for TNF-α-induced VCAM-1 expression. To clarify the mechanisms of TNF-α-induced VCAM-1 expression in HCFs may provide therapeutic strategies for heart injury and inflammatory diseases.

Carbon monoxide (CO) is one of the cytoprotective byproducts of heme oxygenase (HO)-1 and exerts anti-inflammatory action in various models. However, the detailed mechanisms underlying CO-induced HO-1 expression in primary human cardiomyocytes remain largely unidentified. We used primary left ventricle myocytes as a model and applied CO releasing molecule (CORM)-2 to investigate the relationship of CO and HO-1 expression. We herein used Western blot, real-time PCR, promoter activity and EIA to investigate the role of HO-1 expression protecting against thrombin-mediated responses. We found that thrombin-induced COX-2 expression, PGE2 release and cardiomyocyte hypertrophy markers (increase in ANF/BNP, α-actin expression and cell surface area) was attenuated by pretreatment with CORM-2 which was partially reversed by hemoglobin (Hb) or ZnPP (an inhibitor of HO-1 activity), suggesting that HO-1/CO system may be of clinical importance to ameliorate heart failure through inhibition of inflammatory responses. CORM-2-induced HO-1 protein expression, mRNA and promoter was attenuated by pretreatment with the inhibitors of Pyk2 (PF431396), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), p38 (SB202530), JNK1/2 (SP600125), FoxO1 (AS1842856) and Sp1 (mithramycin A). The involvement of these signaling components was further confirmed by transfection with respective siRNAs, consistent with those of pharmacological inhibitors. These results suggested that CORM-2-induced HO-1 expression is mediated through a Pyk2/PDGFR/PI3K/Akt/FoxO1/Sp1-dependent manner and exerts a cytoprotective effect in human cardiomyocytes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Purpose: Sphingosine 1-phosphate (S1P), an important inflammatory mediator, has been shown to regulate COX-2 production and promote various cellular responses such as cell migration. Mevastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA), effectively inhibits inflammatory responses. However, the mechanisms underlying S1P-evoked COX-2-dependent cell migration, which is modulated by mevastatin in human tracheal smooth muscle cells (HTSMCs) remain unclear., Experimental Approach: The expression of COX-2 was determined by Western blotting, real time-PCR and promoter analyses. The signalling molecules were investigated by pretreatment with respective pharmacological inhibitors or transfection with siRNAs. The interaction between COX-2 promoter and transcription factors was determined by chromatin immunoprecipitation assay. Finally, the effect of mevastatin on HTSMC migration and leukocyte counts in BAL fluid and COX-2 expression induced by S1P was determined by a cell migration assay, cell counting and Western blot., Key Results: S1P stimulated mTOR activation through the Nox2/ROS and PI3K/Akt pathways, which can further stimulate FoxO1 phosphorylation and translocation to the cytosol. We also found that S1P induced CREB activation and translocation via an mTOR-independent signalling pathway. Finally, we showed that pretreatment with mevastatin markedly reduced S1P-induced cell migration and COX-2/PGE2 production via a PPARγ-dependent signalling pathway., Conclusions and Implications: Mevastatin attenuates the S1P-induced increased expression of COX-2 and cell migration via the regulation of FoxO1 and CREB phosphorylation and translocation by PPARγ in HTSMCs. Mevastatin could be beneficial for prevention of airway inflammation in the future., (© 2015 The British Pharmacological Society.)

Carbon monoxide (CO), a reaction product of the cytoprotective heme oxygenase (HO)-1, displays an anti-inflammatory effect in various cellular injuries, but the precise mechanisms of HO-1 expression remain unknown. We used the transition metal carbonyl compound carbon monoxide-releasing molecule-2 (CORM-2) that acts as carbon monoxide donor. The effects of CORM-2 on expression of HO-1 in human tracheal smooth muscle cells (HTSMCs) were determined by Western blot, real-time PCR, and promoter activity assay. In HTSMCs, CORM-2 activated Nrf2 through the activation of a c-Src/EGFR/PI3K/Akt-dependent pathway, resulting in HO-1 expression. We showed that CORM-2-induced HO-1 protein and mRNA levels were inhibited by the inhibitor of c-Src (PP1 or SU6656), EGFR (AG1478), PI3K (LY294002), Akt (SH-5), JNK1/2 (SP600125), or p38 MAPK (SB202190) and transfection with siRNA of c-Src, EGFR, Akt, p38, JNK2, or Nrf2 in HTSMCs. We also showed that CORM-2 stimulated c-Src, EGFR, Akt, p38 MAPK, and JNK1/2 phosphorylation. CORM-2 also enhanced Nrf2 translocation from the cytosol to the nucleus and antioxidant response element (ARE) promoter activity. Moreover, CORM-2 mediated p38 MAPK and JNK1/2 activation via a c-Src/EGFR/PI3K/Akt pathway, which further enhanced Nrf2 activation and translocation. Finally, we observed that CORM-2 induced in vivo binding of Nrf2 to the HO-1 promoter. CORM-2 activates the c-Src/EGFR/PI3K/Akt/JNK1/2 and p38 MAPK pathways, which in turn trigger Nrf2 activation and ultimately induces HO-1 expression in HTSMCs. Thus, the HO-1/CO system might be potential therapeutics in airway diseases., (© 2015 Wiley Periodicals, Inc.)

The elevated level of endothelin-1 (ET-1) has been detected in the bronchoalveolar lavage of patients with severe asthma, acute lung injury, acute respiratory distress syndrome, and sepsis. ET-1 may affect vessel tone together with lung physiology and pathology. Vascular cell adhesion molecule-1 (VCAM-1) is one kind of adhesion molecules participating in the process of polymorphonuclear leukocyte transmigration and regulating the occurrence and amplification of tissue inflammation. However, the molecular mechanisms underlying ET-1-mediated expression of VCAM-1 on human tracheal smooth muscle cells (HTSMCs) were largely unknown. Here we reported that ET-1 stimulated expression of VCAM-1 gene on HTSMCs, which was blocked by pretreatment with the inhibitors of ET receptors, Src, matrix metalloproteinases (MMPs), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), phosphatidylinositol 3-kinase (PI3K), AKT, MEK1/2, and p300, suggesting the participation of these signaling components in ET-1-regulated HTSMC responses. Furthermore, transfection with small-interfering RNA (siRNA) of Src, AKT, p42 mitogen-activated protein kinase (MAPK), or p300 downregulated the respective proteins and significantly attenuated ET-1-induced VCAM-1 expression. ET-1 also stimulated phosphorylation of Src, EGFR, PDGFR, AKT, p42/p44 MAPK, and Elk-1 and acetylation of histone H4 on HTSMCs. Immunoprecipitation assay showed the association between Elk-1 and p300 in the nucleus. Adhesion assay revealed that the adhesion of THP-1 to HTSMCs challenged with ET-1 was increased, which was attenuated by the inhibitors of ET receptors, Src, MMPs, EGFR, PDGFR, PI3K, AKT, p42/p44 MAPK, and p300. Taken together, these data suggested that ET-1 promotes occurrence and amplification of pathology-related airway inflammation via enhancing VCAM-1 expression in an ET receptor/Src/MMP/EGFR, PDGFR/PI3K/AKT/p42/p44 MAPK/Elk-1/p300 pathway in HTSMCs., (Copyright © 2015 the American Physiological Society.)

Upregulation of heme oxygenase 1 (HO-1) by carbon monoxide (CO) delivered by CO-releasing molecules (CORMs) may be utilized as a therapeutic intervention for neurodegenerative diseases. This study was to delineate the two putative anti-oxidant response elements (AREs) in modulating HO-1 gene by participating with its promoter elements in rat brain astrocytes (RBA-1). CORM-2-induced HO-1 expression was mediated through superoxide, p38 mitogen-activated protein kinase(MAPK), extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), protein tyrosine kinase 2 (Pyk2), platelet-derived growth factor receptor (PDGFR), and phosphatidylinositol 3'-kinase (PI3K/Akt), revealed by the pharmacological inhibitors or knockdown of these signaling molecules. CORM-2-enhanced HO-1 promoter activity was inhibited by co-transfection with small interfering RNA (siRNA) of c-Jun, specificity protein 1 (Sp1), or nuclear factor-erythroid 2-related factor 2 (Nrf2). Immunoprecipitation assay showed that CORM-2 increased the association of nuclear Nrf2 with Sp1 and c-Jun. Furthermore, chromatin immunoprecipitation (ChIP) assay confirmed that Nrf2, Sp1, and c-Jun are associated with the proximal ARE binding site on HO-1 promoter, suggesting that Nrf2/Sp1/c-Jun cooperations are key transcription factors modulating HO-1 expression. Mechanistically, CORM-2-induced ARE promoter activity was reduced by the inhibitors of reactive oxygen species (ROS), p38 MAPK, Pyk2, MAPK/ERK kinases 1 and 2 (MEK1/2), PDGFR, and PI3K/Akt or the siRNAs of c-Jun, SP1, and Nrf2. These findings suggested that CORM-2 increases formation of c-Jun, Sp1, and Nrf2 complex and binding with ARE1 binding site, which is mediated through both ROS/p38 MAPK and Pyk2-dependent PDGFR/PI3K/Akt/Erk1/2 pathways, resulting in HO-1 expression in RBA-1 cells.

Background: Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine and elevated in the regions of tissue injury and inflammatory diseases. The deleterious effects of TNF-α on fibroblasts may aggravate heart inflammation mediated through the up-regulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1). However, the mechanisms underlying TNF-α-induced VCAM-1 expression in cardiac fibroblasts remain unknown. This study aimed to investigate the roles of TNF-α in VCAM-1 expression and its effects on human cardiac fibroblasts (HCFs)., Results: The primary culture HCFs were used in this study. The results obtained with Western blotting, real time-quantitative PCR, and promoter activity analyses showed that TNF-α-induced VCAM-1 expression was mediated through TNF receptor (TNFR) 1-dependent gene up-regulation. Activation of TNFR1 by TNF-α transactivated c-Src-dependent EGF receptor (EGFR) linking to PI3K/Akt cascade, and then led to transcriptional activity of NF-κB. Moreover, the results of promoter reporter assay demonstrated that the phosphorylated p65 NF-κB turned on VCAM-1 gene expression. Subsequently, up-regulation of VCAM-1 promoted monocytes adhesion to HCFs challenged with TNF-α determined by cell adhesion assay., Conclusions: Taken together, these results indicate that in HCFs, activation of NF-κB by c-Src-mediated transactivation of EGFR/PI3K/Akt cascade is required for TNF-α-induced VCAM-1 expression. Finally, increased VCAM-1 enhances monocytes adhering to HCFs challenged with TNF-α. Understanding the mechanisms of VCAM-1 up-regulated by TNF-α on HCFs may provide rationally therapeutic interventions for heart injury or inflammatory diseases.

Thrombin and COX-2 regulating cardiac hypertrophy are via various signaling cascades. Several transcriptional factors including CREB involve in COX-2 expression. However, the interplay among thrombin, CREB, and COX-2 in primary human neonatal ventricular cardiomyocytes remains unclear. In this study, thrombin-induced COX-2 promoter activity, mRNA and protein expression, and PGE2 synthesis were attenuated by pretreatment with the inhibitors of c-Src (PP1), Pyk2 (PF431396), EGFR (AG1478), PI3K/Akt (LY294002/SH-5), and p300 (GR343), or transfection with siRNAs of c-Src, Pyk2, EGFR, p110, Akt, CREB, and p300. Moreover, thrombin-stimulated phosphorylation of c-Src, Pyk2, EGFR, Akt, CREB and p300 was attenuated by their respective inhibitors. These results indicate that thrombin-induced COX-2 expression is mediated through PAR-1/c-Src/Pyk2/EGFR/PI3K/Akt linking to CREB and p300 cascades. Functionally, thrombin-induced hypertrophy and ANF/BNP release were, at least in part, mediated through a PAR-1/COX-2-dependent pathway. We uncover the importance of COX-2 regarding human cardiomyocyte hypertrophy that will provide a therapeutic intervention in cardiovascular diseases., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)