Search

Background: Cancer and its therapies can impact fertility in various ways, and therefore a growing number of cancer survivors face fertility as a significant concern. The cytotoxic alkylating agent cyclophosphamide (CP) is commonly used as an antineoplastic agent; unfortunately, its use is significantly associated with male infertility and damage to the reproductive system., Aim: The present study aimed to assess the possible beneficial effects of Irbesartan (IRB) in a rat model of CP-induced testicular toxicity., Main Methods: The effects of treatment were assessed by measuring peroxisome proliferator-activated receptor gamma (PPAR-γ) expression via qRT-PCR, the immunohistochemical (IHC) assessment of apoptotic markers, NOD-like receptor protein 3 (NLRP3), and nuclear factor-κB (NF-κB), determination of the count and viability of epididymal sperm, oxidative stress markers via biochemical analysis, serum testosterone, caspase-1, and interleukin-18 (IL-18) levels via ELISA, histopathological assessment, and fibrosis by Masson's trichrome (MT) stain., Key Findings: There was a significant increase in malondialdehyde (MDA), caspase-1, and IL-18 contents, NF-κB, NLRP3, Bcl-2-associated X protein (Bax), caspase-3, and MT staining in testicular tissue after CP administration compared to the normal control group. Whereas reduced glutathione (GSH), superoxide dismutase (SOD), PPAR-γ expression, B-cell lymphoma-2 (Bcl-2) staining, serum testosterone, and the count and viability of epididymal sperm were decreased compared to the normal control group. The IRB treatment has reversed CP-induced testicular toxicity., Significance: It is possible to conclude that IRB revealed a significant testicular protective effect against CP via antioxidant, anti-apoptotic, and anti-inflammatory effects., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Diabetic keratopathy (DK) is an important diabetic complication at the ocular surface. Chronic low-grade inflammation mediated by the NLRP3 inflammasome promotes pathogenesis of diabetes and its complications. However, the effect of the NLRP3 inflammasome on DK pathogenesis remains elusive. Wild-type (WT) and Nlrp3 knockout (KO) C57 mice were used to establish a type I diabetes model by intraperitoneal injection of streptozotocin. The effect of the NLRP3 inflammasome on diabetic corneal wound healing and never regeneration was examined by a corneal epithelial abrasion model. Western blot, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and pharmacological treatment were performed to investigate the regulatory mechanism of advanced glycation end products (AGEs) on NLRP3 inflammasome activation and corneal wound healing in vivo . The cultured mouse corneal epithelial cells (TKE2) were used to evaluate the effect and mechanism of AGEs on NLRP3 inflammasome activation in vitro . We revealed that NLRP3 inflammasome-mediated inflammation and pyroptosis contributed to DK pathogenesis. Under physiological conditions, the NLRP3 inflammasome was required for corneal wound healing and nerve regeneration. However, under a diabetic scenario, sustained activation of the NLRP3 inflammasome resulted in postponed corneal wound healing and impaired nerve regeneration. Mechanistically, the accumulated AGEs promoted hyperactivation of the NLRP3 inflammasome through ROS production. Moreover, genetically and pharmacologically blocking the AGEs/ROS/NLRP3 inflammasome axis significantly expedited diabetic corneal epithelial wound closure and nerve regeneration. Our results revealed that AGEs-induced hyperactivation of the NLRP3 inflammasome resulted in delayed diabetic corneal wound healing and impaired nerve regeneration, which further highlighted the NLRP3 inflammasome as a promising target for DK treatment., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Intrauterine inflammation affects fetal development of the nervous system and may cause prenatal brain injury in offspring. Previously, neural stem cells have been extensively used as a therapeutic choice for nervous system diseases. Recently, the therapeutic ability of conditioned medium, harvested from cultured stem cells, has captured the attention of researchers in the field. Our study aimed to compare the therapeutic effect of neural stem cells (NSCs) or NSC-conditioned medium (NSC-CM) after prenatal brain injury. The animal model was induced by intraperitoneal injection of lipopolysaccharide into the pregnant mice and NSCs or NSC-CM were transplanted into the lateral ventricle of embryos in treatment groups. Inflammation and apoptosis were evaluated postpartum in offspring via measuring the expression of NLRP3 gene and protein, the expression and the activity of caspase-3, and the expression of pro-inflammatory cytokines by real-time PCR, immunohistochemistry, western blotting, ELISA, and colorimetric assay kit. A rotarod test was performed for motor function evaluation. Data showed that although NSC-CM fought against the inflammation and apoptosis and improved the motor function, NSCs acted more efficiently. In conclusion, the results of our study contend that NSCs have a better therapeutic effect than CM in prenatal brain injury., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) seriously threatens human's health. Researches have shown a close correlation between long non-coding RNAs (lncRNAs) and PD. However, the biological function of lncRNA homeobox transcript antisense RNA (HOTAIR) in PD remains largely unknown. In this study, we established PD models in vivo and in vitro by using 1-methyl-4-phenyl-2, 3, 6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP + ) to assess the role of HOTAIR in pyroptotic cell death and neuronal damage. RNA immunoprecipitation (RIP) and dual luciferase reporter assay were used to verify the interaction between miR-326 and HOTAIR or ELAV like RNA binding protein 1 (ELAVL1). LncRNA HOTAIR was upregulated in PD mice and MPP + induced SH-SY5Y cells. Additionally, knockdown of HOTAIR notably attenuated the symptom of PD in vivo. Downregulation of HOTAIR could obviously promoted cell viability and suppressed NLR family pyrin domain containing 3 (NLRP3) mediated pyroptotic cell death of SH-SY5Y cells in the presence of MPP + . Further, lncRNA HOTAIR positively regulated ELAVL1 expression by targeting miR-326, and downregulation of HOTAIR or ELAVL1 notably suppressed promotive effects of miR-326 inhibitor on MPP + induced pyroptosis via activation of NLRP3 inflammasome. Collectively, HOTAIR silencing significantly inhibits neuronal damage through repressing NLRP3 mediated pyroptosis activation via regulation of miR-326/ELAVL1 axis in PD, which may contribute to a better understanding of PD pathogenesis and provide new treatment strategies for this disease., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Chaperonin 60.1 (Cpn60.1) is a protein derived from Mycobacterium tuberculosis that has been shown, along with its peptide fragment IRL201104, to have beneficial effects in models of allergic inflammation. To further investigate the anti-inflammatory properties of Cpn60.1 and IRL201104, we have investigated these molecules in a model of nonallergic lung inflammation. Mice were treated with Cpn60.1 (0.5-5,000 ng/kg) or IRL201104 (0.00025-2.5 ng/kg), immediately before intranasal instillation of bacterial lipopolysaccharide (LPS). Cytokine levels and cell numbers in mouse bronchoalveolar lavage (BAL) fluid were measured 4 h after LPS administration. In some experiments, mice were depleted of lung-resident phagocytes. Cells from BAL fluid were analyzed for inflammasome function. Human umbilical vein endothelial cells (HUVECs) were analyzed for adhesion molecule expression. Human neutrophils were analyzed for integrin expression, chemotaxis, and cell polarization. Cpn60.1 and IRL201104 significantly inhibited neutrophil migration into the airways, independently of route of administration. This effect of the peptide was absent in TLR4 and annexin A1 knockout mice. Intravital microscopy revealed that IRL201104 reduced leukocyte adhesion and migration into inflamed tissues. However, IRL201104 did not significantly affect adhesion molecule expression in HUVECs or integrin expression, chemotaxis, or polarization of human neutrophils at the studied concentrations. In phagocyte-depleted animals, the anti-inflammatory effect of IRL201104 was not significant. IRL201104 significantly reduced IL-1β and NLRP3 expression and increased A20 expression in BAL cells. This study shows that Cpn60.1 and IRL201104 potently inhibit LPS-induced neutrophil infiltration in mouse lungs by a mechanism dependent on tissue-resident phagocytes and to a much lesser extent, the proresolving factor annexin A1.

Microbiota can exert immunomodulatory effects by short-chain fatty acids (SCFA) in experimental models of graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT). Therefore we aimed to analyze the expression of SCFAs sensing G-protein coupled receptor GPR109A and GPR43 by quantitative PCR in 338 gastrointestinal (GI) biopsies obtained from 199 adult patients undergoing allo-SCT and assessed the interaction of GPR with FOXP3 expression and regulatory T cell infiltrates. GPR expression was strongly upregulated in patients with stage II-IV GvHD (p=0.000 for GPR109A, p=0.01 for GPR43) and at the onset of GvHD (p 0.000 for GPR109A, p=0.006 for GPR43) and correlated strongly with FOXP3 and NLRP3 expression. The use of broad-spectrum antibiotics (Abx) drastically suppressed GPR expression as well as FOXP3 expression in patients' gut biopsies (p=0.000 for GPRs, FOXP3 mRNA and FOXP3+ cellular infiltrates). Logistic regression analysis revealed treatment with Abx as an independent factor associated with GPR and FOXP3 loss. The upregulation of GPRs was evident only in the absence of Abx (p=0.001 for GPR109A, p=0.014 for GPR43) at GvHD onset. Thus, GPR expression seems to be upregulated in the presence of commensal bacteria and associates with infiltration of FOXP3+ T regs, suggesting a protective, regenerative immunomodulatory response. However, Abx, which has been shown to induce dysbiosis, interferes with this protective response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ghimire, Weber, Hippe, Meedt, Hoepting, Kattner, Hiergeist, Gessner, Matos, Ghimire, Wolff, Edinger, Hoffmann, Poeck, Herr and Holler.)

Hypoxia inducible factor 1 alpha (HIF-1α) is involved in regulating the biological functions of neuronal death after traumatic brain injury (TBI), and attaches importance in the inflammatory response, but its potential mechanism is still unknown. Our study aimed to explore the regulatory mechanism between HIF-1α and NLRP3 inflammasome after TBI. Male mice underwent controlled cortical impact (CCI) or sham-operated procedures. Brain water content and blood-brain barrier permeability were measured at the indicated time after TBI. The behavioral performance, ELISA, immunofluorescence, and western blot analysis were used to determine whether HIF-1α specifically targeted TBI-induced pyroptosis. We discovered that TBI-induced brain injury caused by external mechanical forces is characterized by edema and blood-brain barrier disorder, and the release of IL-1β, IL-18, and LDH and upregulation of HIF-1α expression, reaching the peak on the third day post-TBI. In addition, HIF-1α accumulated NLRP3 inflammasome-mediated pyroptosis and activation of microglia. The protein expressions of NLRP3, GSDMD, GSDMD-N, pro-caspase 1, and cleaved caspase 1 were markedly increased in the injured cortex, which were restored to normal levels by the interference of HIF-1α. The inactivation of HIF-1α conferred neuroprotection and alleviated brain injury after TBI. HIF-1α was implicated in TBI-induced brain injury, aggravated NLRP3 inflammasome -mediated pyroptosis, and the activation of microglia, which provided a potential target for treating TBI., (Copyright © 2021. Published by Elsevier B.V.)

Diabetes mellitus (DM) induces damage to the ocular surface, which leads to vision decline. In the current study, we investigated whether N-acetylcysteine (NAC) plays a protective role in diabetes-induced ocular surface damage. The diabetic mice model was treated with 0.3% NAC topically. Corneal epithelial integrity, tear volume and corneal sensitivity were examined by sodium fluorescein staining, phenol red cotton thread and esthesiometer respectively. The level of reactive oxygen species (ROS) was measured with 2',7-dichlorofluorescein diacetate. The expression of NLRP3, IL-1β and caspase-1 were evaluated by RT-PCR, western blot and immunostaining. The level of SOD1 was assessed by RT-PCR. We found that the expression of NLRP3, IL-1β and caspase-1 were elevated in diabetic cornea and conjunctiva. Treatment with NAC improved corneal epithelial integrity, increased tear production and corneal sensitivity in diabetic mice. Moreover, NAC markedly attenuated ROS accumulation and decreased NLRP3, IL-1β and caspase-1 levels in diabetic cornea and conjunctiva. These results suggest that NAC improves ocular surface damage in STZ-induced diabetic mice, which may be related to the inhibition of the ROS/NLRP3/Caspase-1/IL-1β signaling pathway., (Copyright © 2021. Published by Elsevier Ltd.)

Vitamin D (VD) deficiency delays corneal wound healing in those with diabetes, which cannot be rescued with supplemental diet. Here, we employed topical calcitriol application to evaluate its efficiency in corneal wound healing and reinnervation in diabetic mice. Type 1 diabetic mice were topically administrated calcitriol, or subconjunctivally injected with NLRP3 antagonist MCC950 or IL-1β blocking antibody after epithelial debridement. Serum VD levels, corneal epithelial defect, corneal sensation and nerve density, NLRP3 inflammasome activation, neutrophil infiltration, macrophage phenotypes, and gene expressions were examined. Compared with those of normal mice, diabetic mice showed reduced serum VD levels. Topical calcitriol application promoted corneal wound healing and nerve regeneration, as well as sensation recovery in diabetic mice. Moreover, calcitriol ameliorated neutrophil infiltration and promoted the M1-to-M2 macrophage transition, accompanied by suppressed overactivation of the NLRP3 inflammasome. Treatment with NLRP3 antagonist or IL-1β blockage demonstrated similar improvements as those of topical calcitriol application. Additionally, calcitriol administration upregulated desmosomal and hemidesmosomal gene expression in the diabetic cornea. In conclusion, topical calcitriol application promotes corneal wound healing and reinnervation during diabetes, which may be related to the suppression of the overactivation of NLRP3 inflammasome., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Immune system hypersensitivity is believed to contribute to mental frailty in the elderly. Solid evidence indicates NOD-like receptor pyrin domain containing-3 (NLRP3)-inflammasome activation intimately connects aging-associated chronic inflammation (inflammaging) to senile cognitive decline. Thioredoxin interacting protein (TXNIP), an inducible protein involved in oxidative stress, is essential for NLRP3 inflammasome activity. This study aims to find whether TXNIP/NLRP3 inflammasome pathway is involved in senile dementia. According to our studies on sex-matched mice, TXNIP was significantly upregulated in aged animals, paralleled by the NLRP3-inflammasome over-activity leading to enhanced caspase-1 cleavage and IL-1β maturation, in both sexes. This was closely associated with depletion of the anti-aging and cognition enhancing protein klotho, in aged males. Txnip knockout reversed age-related NLRP3-hyperactivity and enhanced thioredoxin (TRX) levels. Further, TXNIP inhibition along with verapamil replicated TXNIP/NLRP3-inflammasome downregulation in aged animals, with FOXO-1 and mTOR upregulation. These alterations concurred with substantial improvements in both cognitive and sensorimotor abilities. Together, these findings substantiate the pivotal role of TXNIP to drive inflammaging in parallel with klotho depletion and functional decline, and delineate thioredoxin system as a potential target to decelerate senile dementia., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

L-arginine (Arg), a semi-essential amino acid, has recently been shown to attenuate inflammatory response during cardiovascular disease. NLRP3 inflammasome serves a central role in amplification of cellular inflammation. In this study, we aimed to confirm the modulatory effect of Arg on NLRP3 inflammasome and the underlying mechanisms in vascular endothelial cells (ECs). Arg suppressed NLRP3 inflammasome activation in ECs stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Moreover, treatment with Arg increased the expression of the deacetylase sirtuin 1 (SIRT1) in ECs. Importantly, knockdown of SIRT1 abolished the inhibitory potential of Arg on the activation of NLRP3 inflammasome. Further study indicated that Arg also alleviated LPS plus ATP-induced the generation of reactive oxygen species (ROS) in ECs. In addition, Arg may regulate NLRP3 inflammasome activation partly through suppression of ROS production. In combination, we speculate that Arg exerts an inhibitory effect on the activation of NLRP3 inflammasome in ECs, which may be partly mediated by SIRT1 and ROS., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Objectives: Stroke is an important cause of death and disability. Recent evidence suggests that post-stroke inflammation is an important factor in stroke pathology and a root cause of its lasting consequences. Phenothiazine drugs, like chlorpromazine and promethazine (C + P), induce hypothermia and have been shown to play a major role in neuroprotection. In the present study, we investigated this neuroprotective mechanism by assessing the anti-inflammatory effect of these drugs. Methods: Adult Sprague-Dawley rats underwent 2 h of middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion, with or without C + P (8 mg/kg). Infarct volumes, neurological deficits, along with mRNA and protein quantities of receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), NLRPyrin domain containing 3 (NLRP3), and interleukin-1β (IL-1β) were assessed, as well as the infiltration of neutrophils and macrophages. Results: C + P induced hypothermia that significantly reduced RIP1, RIP3, NLRP3 and IL-1β expression, infarction, and immune cell infiltration, while C + P treatment with temperature control at 37°C induced lesser effect. Conclusion: These findings suggest that the anti-inflammatory effect of C + P may be dependent on drug-induced hypothermia and regulation of the NLRP3 inflammasome via the RIP1/RIP3 complex. Future investigations are needed regarding C + P as potential treatment of ischemic stroke.

Diabetic retinopathy (DR) has been considered to involve mitochondrial alterations and be related to the nucleotide-binding oligomerization domain-like receptors 3 (NLRP3) inflammasome activation. The voltage-dependent anion channel 1 (VDAC1) protein is one of the key proteins that regulates the metabolic and energetic functions of the mitochondria. To explore the involvement of VDAC1 in mitophagy regulation of NLRP3 inflammasome activation under high-glucose (HG) conditions, this study examined expressions of VDAC1, mitochondrial function and mitophagy-related proteins, and NLRP3 inflammasome-related proteins in human retinal capillary endothelial cells (HRCECs) cultured with 30 mM of glucose in the presence or absence of mitophagy inhibitor (Mdivi-1) using Western blot. Mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were detected using flow cytometry. GFP-tagged pAdTrack-VDAC1 adenovirus was used to overexpress VDAC1. Cell biological behaviors, including proliferation, migration, tubule formation, and apoptosis, were also observed. Our results showed that when compared to the normal glucose and high mannitol groups, increased amounts of mitochondrial fragments, reduced mitochondrial membrane potential, increased expression of mitochondrial fission protein Drp 1, decreased expression of mitochondrial fusion protein Mfn 2, accumulation of mtROS, and activation of the NLRP3 inflammasome were observed in the HG group. Meanwhile, HG markedly reduced the protein expressions of PINK1, Parkin and VDAC1. Inhibition of mitophagy reduced PINK1 expression, enhanced NLRP3 expression, but failed to alter VDAC1. VDAC1 overexpression promoted PINK1 expression, inhibited NLRP3 activation and changed the cell biological behaviors under HG conditions. These findings demonstrate that VDAC1-mediated mitophagy plays a crucial role in regulating NLRP3 inflammasome activation in retinal capillary endothelial cells under HG conditions, suggesting that VDAC1 may be a potential target for preventing or treating DR., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Neuroinflammation is a major driver of age-related brain degeneration and concomitant functional impairment. In patients with Alzheimer's disease, the most common form of age-related dementia, factors that enhance neuroinflammation may exacerbate disease progression, in part by impairing the glymphatic system responsible for clearance of pathogenic beta-amyloid. Inflammatory bowel diseases (IBDs) induce neuroinflammation and exacerbate cognitive impairment in the elderly. The NACHT-LRR and pyrin (PYD) domain-containing protein 3 (NLRP3) inflammasome has been implicated in neuroinflammation. Therefore, we examined if the NLRP3 inflammasome contributes to glymphatic dysfunction and cognitive impairment in an aging mouse model of IBD., Methods: Sixteen-month-old C57BL/6J and NLRP3 knockout (KO) mice received 1% wt/vol dextran sodium sulfate (DSS) in drinking water to model IBD. Colitis induction was confirmed by histopathology. Exploratory behavior was examined in the open field, associative memory by the novel-object recognition and Morris water maze tests, glymphatic clearance by in vivo two-photon imaging, and neuroinflammation by immunofluorescence and western blotting detection of inflammatory markers., Results: Administration of DSS induced colitis, impaired spatial and recognition memory, activated microglia, and increased A1-like astrocyte numbers. In addition, DSS treatment impaired glymphatic clearance, aggravated amyloid plaque accumulation, and induced neuronal loss in the cortex and hippocampus. These neurodegenerative responses were associated with increased NLRP3 inflammasome expression and accumulation of gut-derived T lymphocytes along meningeal lymphatic vessels. Conversely, NLRP3 depletion protected against cognitive dysfunction, neuroinflammation, and neurological damage induced by DSS., Conclusions: Colitis can exacerbate age-related neuropathology, while suppression of NLRP3 inflammasome activity may protect against these deleterious effects of colitis.

This study aimed to explore the effects of N-acetylserotonin (NAS) on the expression of interleukin-1β (IL-1β) in the retina of retinal ischemia-reperfusion injury (RIRI) rats via the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB)/nod-like receptor pyrin domain containing 3 (NLRP3) signaling pathway. In this study, adult male Sprague Dawley rats were randomly divided into the sham, RIRI, RIRI + NAS and RIRI + TAK-242 + NAS groups. The rats in the RIRI + NAS and RIRI + TAK-242 + NAS groups were intraperitoneally injected with NAS 30 min before and after modeling. TAK-242, a selective TLR4 inhibitor, was administered by intraperitoneal injection in RIRI + TAK-242 + NAS group. The RIRI rat model was established by elevating the intraocular pressure to 110 mmHg for 60 min. The retinal structure and edema were assessed by H&E staining. The expression levels of TLR4, phosphorylated NF-κB (p-NF-κB), NLRP3, cleaved Caspase-1, and IL-1β in the retina of each group were detected using immunohistochemistry and Western blot. The correlations of the differences of TLR4 + and cleaved Caspase-1 + with IL-1β + cells (between the NAS and the RIRI groups) were analyzed, using linear regression in the RIRI + NAS group. Results showed that thinner retina, more RGCs, and less TLR4 + , p-NF-κB + , NLRP3 + , cleaved Caspase-1 + , and IL-1β + cells in the retina were observed in the RIRI + NAS and RIRI + TAK-242 + NAS groups compared with the RIRI group 12 h after RIRI (all P < 0.01). Western blot analysis results showed that the expression of IL-1β in the RIRI + NAS group began to increase 6 h after RIRI, and it reached a high level 12 h after RIRI, and then decreased. And it was lower at each time point in the RIRI + NAS group than in the RIRI group, and there existed significant difference (all P < 0.01). Besides, the expression levels of TLR4, p-NF-κB, NLRP3, and cleaved Caspase-1 proteins in the RIRI + NAS and RIRI + TAK-242 + NAS groups decreased 12 h after RIRI compared with those in the RIRI group (all P < 0.01). The difference in IL-1β + cells was significantly correlated with those of TLR4 + and cleaved Caspase-1 + cells in the RIRI + NAS group (r 2  = 0.9054 or 0.7431, P < 0.01). In conclusion, NAS could attenuate the expression of IL-1β by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway, reduce the retina edema, and promote the survival of RGCs, thereby alleviating the retinal injury and exert its neuroprotective effect., (Copyright © 2021. Published by Elsevier Ltd.)

Background: The functional characteristics of NLRP3 in the pathogenesis of coxsackievirus B3- (CVB3-) induced viral myocarditis (VMC) have not been fully elucidated, and the targeted therapeutic effect of NLRP3 or its related pathway in VMC has not been reported., Method: In this work, the change patterns of NLRP3- and Th17-related factors were detected during the pathological process of CVB3-induced VMC in Balb/c mice. The correlation between NLRP3 and Th17 cells during the VMC process was analyzed by Spearman test. The coculture system of spleen CD4 + T and bone marrow CD11c + DC cells was set to explore the orchestration of NLRP3 and Th17 in the pathological development of VMC in vitro. Anti-IL-1 β antibody or NLRP3 -/- Balb/c were used to block the NLRP3 pathway indirectly and directly to analyze the NLRP3-targeting therapeutic value., Results: The change patterns of NLRP3- and Th17-related molecules in the whole pathological process of mouse CVB3-induced VMC were described. Through Spearman correlation analysis, it was confirmed that there was a close correlation between NLRP3 and Th17 cells in the whole pathological process of VMC. And the interaction mode between NLRP3 and Th17 was preliminarily explored in the cell experiment in vitro. Under the intervention of an anti-IL-1 β antibody or NLRP3 knockout, the survival rate of the intervention group was significantly improved, the degree of myocardial inflammation and fibrosis was significantly alleviated, and the content of myocardial IL-17 and spleen Th17 was also significantly decreased., Conclusion: Our findings demonstrated a key role of the NLRP3 inflammasome and its close relationship with Th17 in the pathological progression of CVB3-induced VMC and suggested a possible positive feedback-like mutual regulation mechanism between the NLRP3 inflammasome and Th17 in vitro and in the early stage of CVB3 infection. Taking NLRP3 as a new starting point, it provides a new target and idea for the prevention and treatment of CVB3-induced VMC., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Jifei Chen et al.)

Aim: Spinal cord injury (SCI) is a serious disabling injury worldwide, and the excessive inflammatory response it causes plays an important role in secondary injury. Regulating the inflammatory response can be a potential therapeutic strategy for improving the prognosis of SCI. Zinc has been demonstrated to have a neuroprotective effect in experimental spinal cord injury models. In this study, we aimed to explore the neuroprotective effect of zinc through the suppression of the NLRP3 inflammasome., Method: Allen's method was used to establish an SCI model in C57BL/6J mice. The Basso Mouse Scale (BMS), Nissl staining were employed to confirm the protective effect of zinc on neuronal survival and functional recovery in vivo. Western blotting (WB), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression levels of NLRP3 inflammasome and autophagy-related proteins. Transmission electron microscopy (TEM) was used to confirm the occurrence of zinc-induced autophagy. In vitro, lipopolysaccharide (LPS) and ATP polarized BV2 cells to a proinflammatory phenotype. 3-Methyladenine (3-MA) and bafilomycin A1 (BafA1) were chosen to explore the relationship between the NLRP3 inflammasome and autophagy. A coimmunoprecipitation assay was used to detect the ubiquitination of the NLRP3 protein., Results: Our data showed that zinc significantly promoted motor function recovery after SCI. In vivo, zinc treatment inhibited the protein expression level of NLRP3 while increasing the level of autophagy. These effects were fully validated by the polarization of BV2 cells to a proinflammatory phenotype. The results showed that when 3-MA and BafA1 were applied, the promotion of autophagy by zinc was blocked and that the inhibitory effect of zinc on NLRP3 was reversed. Furthermore, co-IP confirmed that the promotion of autophagy by zinc also activated the protein expression of ubiquitin and suppressed high levels of NLRP3., Conclusion: Zinc provides neuroprotection by regulating NLRP3 inflammasome through autophagy and ubiquitination after SCI., (© 2020 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.)

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis and overall survival. Clinical investigations show that chronic stress is commonly present in the course of AML and associated with adverse outcome. However, the underlying molecular mechanisms are elusive. In the present study, a chronic restraint stress mouse model was established to evaluate the effect of stress on AML. We found that mice under chronic stress exhibited significantly increased liver and spleen infiltration of leukemic cells and poorer overall survival. This was accompanied by elevated cellular NLR family pyrin domain containing 3 (NLRP3) and interleukin-1β (IL-1β) in the liver or bone marrow, and secreted IL-1β in the plasma, indicating the activation of inflammasomes under chronic restraint stress. High mobility group box 1 (HMGB1) expression was markedly increased in newly diagnosed AML patients, but reduced in complete remission AML patients. The expression level of HMGB1 was positively correlated with NLRP3 mRNA in AML patients. Knockdown of HMGB1 significantly decreased NLRP3 and IL-1β expression in AML cell lines, and secreted IL-1β in supernatant of AML cell culture, while HMGB1 stimulation caused contrary effects. These results implied that HMGB1 could be involved in the regulation of inflammasome activation in AML development. Mice model showed that chronic restraint stress-facilitated proliferation and infiltration of AML cells were largely abrogated by knocking down HMGB1. Knockdown of HMGB1 also ameliorated overall survival and remarkably neutralized NLRP3 and IL-1β expression under chronic restraint stress. These findings provide evidences that chronic stress promotes AML progression via HMGB1/NLRP3/IL-1β dependent mechanism, suggesting that HMGB1 is a potential therapeutic target for AML. KEY MESSAGES: • Chronic restraint stress promoted acute myeloid leukemia (AML) progression and mediated NLRP3 inflammasome activation in xenograft mice. • HMGB1 mediated NLRP3 inflammasome activation in AML cells. • Knockdown of HMGB1 inhibited AML progression under chronic stress in vivo.

Background: S100 calcium binding protein A9 (S100A9) is a pro-inflammatory alarmin associated with several inflammation-related diseases. However, the role of S100A9 in lung injury in sepsis has not been fully investigated. Therefore, the present study aimed to determine the role of S100A9 in a lipopolysaccharide (LPS)-induced lung injury murine model and its underlying molecular mechanisms., Methods: LPS was utilized to induce sepsis and lung injury in C57BL/6 or NOD-like receptor family pyrin domain containing 3 (NLRP3) -/- mice. To investigate the effects of S100A9 blockade, mice were treated with a specific inhibitor of S100A9. Subsequently, lung injury and inflammation were evaluated by histology and enzyme‑linked immunosorbent assay (ELISA), respectively. Furthermore, western blot analysis and RT-qPCR were carried out to investigate the molecular mechanisms underlying the effects of S100A9., Results: S100A9 was upregulated in the lung tissues of LPS-treated mice. However, inhibition of S100A9 alleviated LPS-induced lung injury. Additionally, S100A9 blockade also attenuated the inflammatory responses and apoptosis in the lungs of LPS-challenged mice. Furthermore, the increased expression of NLRP3 was also suppressed by S100A9 blockade, while S100A9 blockade had no effect on NLRP3 -/- mice. In vitro, S100A9 downregulation mitigated LPS-induced inflammation. Interestingly, these effects were blunted by NLRP3 overexpression., Conclusion: The results of the current study suggested that inhibition of S100A9 could protect against LPS-induced lung injury via inhibiting the NLRP3 pathway. Therefore, S100A9 blockade could be considered as a novel therapeutic strategy for lung injury in sepsis.

This study aimed to determine the mechanism underlying the regulation of gout by the HOX transcript antisense RNA (HOTAIR) long non-coding RNA (lncRNA). The expression levels of HOTAIR, miR-20b, and Nlrp3 were estimated by qRT-PCR and western blotting. The methylation level of HOTAIR was detected by methylation-specific PCR. The recruitment of DNA methyltransferase 1 (DNMT1) to the lncRNA HOTAIR promoter was confirmed by a ChIP assay. RNA immunoprecipitation and RNA pull-down assays were used to confirm the interaction between HOTAIR and miR-20b. LncRNA HOTAIR and Nlrp3 expression was upregulated, and that of miR-20b was downregulated in synovial fluid mononuclear cells (SFMCs) collected from patients with gouty arthritis and monosodium urate (MSU)-stimulated THP-1 cells. Interleukin (IL)-1β level increased substantially upon stimulation by MSU crystals. The methylation percentage of HOTAIR was reduced in SFMCs from patients with gouty arthritis and MSU-stimulated THP-1 cells. DNMT1 expression was downregulated in MSU-stimulated THP-1 cells, and DNMT1 knockdown increased lncRNA HOTAIR expression. In addition, the interaction of HOTAIR with miR-20b was confirmed. HOTAIR knockdown suppressed Nlrp3 expression and the secretion of inflammatory cytokines via miR-20b regulation. Finally, in vivo experiments showed that HOTAIR knockdown alleviated ankle swelling in a mouse model of gouty arthritis. These findings suggest that lncRNA HOTAIR knockdown suppresses inflammatory cytokine secretion by upregulating miR-20b and downregulating NLRP3, thereby alleviating ankle swelling in gouty arthritis.

Long non-coding RNA (lncRNA) plays a crucial role in multiple disorders, while the role of it in Parkinson's disease (PD) is still unclear. Here, the increased lncRNA NEAT1 was discovered in MPP + -induced SH-SY5Y cells. Then, we proved that NEAT1 decreasing suppressed MPP + -induced neuronal apoptosis, upregulation of α-syn and activation of NLRP3 inflammasome. Rescue experiments shown that the inhibition of NEAT1 decreasing to MPP + -induced activation of NLRP3 inflammasome and subsequent neuronal apoptosis can be reversed by overexpressed α-syn. Subsequently, we indicated the interaction between NEAT1 and miR-1301-3p, as well as between NEAT1 and miR-5047. Interesting, we found that NEAT1 decreasing repressed the expression of GJB1, a downstream target of miR-1301-3p and miR-5047, through promoting miR-1301-3p rather than miR-5047 expression. Finally, we transfected miR-1301-3p inhibitor to MPP + -induced SH-SY5Y cells following si-NEAT1, and found that downregulation of NEAT1 repressed α-syn-mediated the activation of NLRP3 inflammasome through regulating miR-1301-3p/GJB1 signaling pathway. Overall, our data demonstrated that NEAT1 decreasing effectively suppressed MPP + -induced neuronal apoptosis. Mechanismly, downregulation of NEAT1 repressed α-syn-induced activation of NLRP3 inflammasome via inhibiting the expression of GJB1 by targeting miR-1301-3p. Our study supported a new and reliable evidence for lncRNA NEAT1 as a potential target for PD treatment.

The inflammatory process plays a crucial role in frailty syndrome, which can appear in middle age and is associated with a poor health outcome. Consequently, gerontologists recommend screening inflammatory biomarkers in middle-aged adults to detect frailty and, therefore, prevent chronic diseases and mortality. External factors could be a risk factor for frailty because they can generate and extend the inflammatory process. For these reasons, we analysed the effect of long-term contact lens wear on mRNA level of genes linked to inflammation (IL-6, NLRP3, NK1R, CD73, MUC16 and TRPV1 genes) in conjunctival cells of middle-aged individuals, by quantitative PCR. Middle-aged contact lens wearers presented a significant increase of NLRP3 and MUC16 mRNA level as well as a decrease of CD73 mRNA level, in comparison with non-contact lens wearers. Additionally, we checked for a potential correlation between these transcript levels and clinical changes of the participants' ocular surface. Unlike molecular analysis, clinical examination fails to detect inflammation in contact lens wearers. These data suggest that long-term contact lens wear could trigger an inflammatory response in middle age orchestrated by NLRP3 inflammasome and modulated by CD73 and MUC16 proteins. Further studies are needed to confirm our gene expression findings at the protein level as well as to investigate the potential role of long-term CL wear in the onset of ocular frailty., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In hyperlipidemia, pyroptosis in endothelial cells (ECs) induces atherosclerosis via the toll-like receptor 4 (TLR4) pathway. We evaluated the effects of Ecklonia cava extract (ECE) and pyrogallol-phloroglucinol-6,6-bieckol (PPB) on pyroptosis of ECs and vascular smooth muscle cells (VSMCs), which leads to attenuation of these cells and dysfunction of the aorta in high-fat-diet (HFD)-fed mice and in palmitate-treated ECs and VSMCs. The expression of TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which induce formation of NOD-LRR-and pyrin domain-containing protein 3 (NLRP3) inflammasomes, were increased by HFD and were decreased by ECE and PPB. The TLR4/NF-κB pathway was upregulated in palmitate-treated ECs and VSMCs and was decreased by ECE and PPB. The expressions of NLRP3/apoptosis-associated speck like protein containing a caspase recruitment domain, caspase-1, interleukin (IL)-1β, and IL-18 were increased by HFD and were decreased by ECE and PPB. Pyroptotic cells were increased by HFD and decreased by ECE and PPB. The expressions of the adhesion molecules, intercellular adhesion molecule and vascular cell adhesion molecule, and endothelin-1 were increased by HFD and were decreased by ECE and PPB. ECE and PPB decreased pyroptosis in the ECs and VSMCs, which was induced by HFD in the mouse aorta, and attenuated EC and VSMC dysfunction, an initiation factor of atherosclerosis.

Neuroinflammation is one of the most common etiology in various neurological disorders and responsible for multi-array neurotoxic manifestations such as neurodegeneration, neurotransmitters alteration and cognitive dysfunction. NR (Nerolidol) is a natural bioactive molecule which possesses significant antioxidant and anti-inflammatory potential, but suffers from glitches of low solubility, low bioavailability and fast hepatic metabolism. In the current study, we fabricated nano-engineered lipid carrier of nerolidol (NR-NLC) for its effective delivery into the brain and explored its effect on neuroinflammation, neurotransmitters level and on dysfunctional behavioral attributes induced by CYC (cyclophosphamide). The binding affinity of nerolidol with NLRP3 and TLR-4 was performed which showed stong interaction between them. NR-NLC was prepared by the ultrasonication methods and particle size was determined by Zeta-sizer. Swiss Albino mice were divided into 5 groups (n = 6), assessed for behavioral dysfunction, and sacrificed on the fifteenth day following cyclophosphamide treatment. Brains were then removed and used for biochemical, histopathological, immunohistochemical and fluorescence microscopic analysis. Biochemical analysis showed increased levels of MDA, TNF-α, IL-6, IL-1β, acetylcholine esterase, BDNF, 5-HT and dopamine, and reduced levels of SOD, CAT, GSH, IL-10, along with significant behavioral dysfunction in cyclophosphamide-treated animals. Significant neuronal damage was also observed in the histological study. Immunohistochemical analysis demonstrated increased expression of NLRP3 and caspase-1. Fluorescence microscopic analysis showed significant availability of NR-NLC in the hippocampus and cortex region. In contrast, treatment with NR-NLC effectively mitigated the aforementioned neurotoxic manifestation as compared to NR suspension. Our results showed potent neuroprotective effect of NR-NLC via modulation of oxidative stress, NLRP3 inflammasome, caspase-1 and neurotransmitter status., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Although the role of inflammation in epilepsy pathogenesis has been extensively investigated, the inflammasome complex, a key component of neuroinflammation, has been understudied in epilepsy patients., Methods: To better understand the involvement of this system in epilepsy, levels of inflammasome complex components (NLRP1, NLRP3, CASP1, ASC), end-products of inflammasome complex activity [IL-1β, IL-18, nitric oxide synthase (NOS) isoforms] and other inflammatory factors (NFκB, IL-6, TNF-α) were measured in peripheral blood of patients with focal epilepsy of unknown cause (FEoUC) (n = 47), mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (n = 35) and healthy controls using real time qPCR and/or ELISA., Results: Inflammasome complex associated factors were either downregulated or unchanged in epilepsy patients. Likewise, flow cytometry studies failed to show an increase in ratios of NLRP3-expressing CD3+ and CD14+ peripheral blood mononuclear cells (PBMC) in epileptic patients. Anti-neuronal antibody positive epilepsy patients showed increased NLRP1 and neuronal NOS mRNA expression levels, whereas patients under poly-therapy showed reduced serum inflammasome levels. FEoUC patients demonstrated increased PBMC NFκB mRNA expression levels and serum IL-1β and IL-6 levels. Both MTLE-HS and FEoUC patients displayed higher ratios of NFκB-expressing CD14+ PBMC than healthy controls., Conclusions: Although previous clinical studies have implicated increased inflammasome complex expression levels in epilepsy, our results indicate suppressed inflammasome complex activity in the peripheral blood of focal epilepsy patients. Alternatively, the IL-6-NFκB signaling pathway, appears to be activated in focal epilepsy, suggesting that factors of this pathway might be targeted for future theranostic applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Nod-like receptor protein 3 (NLRP3) inflammasome is a multi-protein complex composed of NLRP3, pro-caspase-1, and apoptosis-associated speck-like protein that contains a caspase recruitment domain (ASC). After NLRP3 priming by lipopolysaccharide (LPS), the ligand of toll-like receptor 4 (TLR4), activation of the NLRP3 inflammasome triggers caspase-1 maturation, leading to pyroptosis and release of interleukin-1beta (IL-1beta). Expression of TLR4 modulates LPS-triggered inflammatory cascades as well as the NLRP3 signaling. L-type calcium channel antagonists are widely used as anti-hypertensive drugs and also exert anti-inflammatory effects through inhibiting release of cytokines including IL-1beta. However, few studies reveal effects of L-type calcium channel antagonists on the NLRP3 inflammasome. In this study, we investigated the effects of nicardipine and verapamil, both L-type calcium channel antagonists, on the NLRP3 inflammasome using differentiated THP-1 cells. Pyroptosis or levels of IL-1beta and caspase-1 were assayed by flow cytometry or enzyme-linked immunosorbent assay, respectively. ASC oligomerization was assayed by immunofluorescence microscopy. Expression of NLRP3 or TLR4 was assayed by polymerase chain reaction and immunoblotting. Nuclear factor-kappaB (NF-kappaB) pathway was also studied. Our results showed that pyroptosis and IL-1beta release were attenuated by nicardipine, but not verapamil. Nicardipine also mitigated caspase-1 activation, inhibited ASC oligomerization, and reduced NLRP3 expression. Furthermore, nicardipine downregulated phosphorylation or nuclear translocation of NF-kappaB p65, consistent with the inhibitory effect of nicardipine on LPS-induced TLR4 expression. In conclusion, nicardipine exerted anti-inflammatory effects through inhibiting NLRP3 inflammasome pathway. Nicardipine may mitigate NLRP3 priming via inhibiting NF-kappaB activation, mediated by suppressing LPS-induced TLR4 expression.

Release of titanium ions (Ti ions) frequently occurs around dental implants and, as a consequence, higher content of Ti is typically present in peri-implantitis tissue. Unlike chronic periodontitis, Ti ions may play a role in the development of peri-implantitis. Inflammasomes are multiprotein signal transduction complexes, involved in inflammation and immune response, which lead to the secretion of mature cytokines associated with the progression of peri-implantitis. It is well known that T lymphocytes dominate the immune response in peri-implantitis, but whether Ti ions can impact the assembly of functional inflammasomes in T cells still remains unclear. Here, we observed that the mRNA expression of NLRP3 and CASP1, as well as the secretion of IL-1β, increased after 6-h incubation of Jurkat T cells with PHA and Ti ions. Moreover, measurement by confocal microscopy and flow cytometry assay indicates that Ti ions can promote the production of ROS, while NLRP3 expression and IL-1β secretion are reduced after treatment of Jurkat cells with NAC (ROS scavenger). Taken together, we presently show that Ti ions can activate NLRP3 inflammasome and then promote IL-β secretion in vitro, where ROS may play a mechanistic role in this activation process.

Background: An association between neuroinflammation and age-related neurologic disorders has been established but the molecular mechanisms and cell types involved have not been thoroughly characterized. Activity of the proinflammatory NLRP3 inflammasome is implicated in Alzheimer's and Parkinson's disease and our recent studies in patients suggest that dopaminergic neurons within the degenerating mesencephalon express NLRP3 throughout the progression of PD. Here, we directly test the impact of enhanced inflammasome activity in mesencephalic neurons by characterizing motor function, tissue integrity, and neuroinflammation in aging mice harboring hyperactivating mutations within the endogenous murine Nlrp3 locus, enabled only in cells expressing the dopaminergic neuron-specific Slc6a3 promoter., Methods: We compared mice harboring inducible alleles encoding the cryopyrin-associated periodic syndrome activating mutations Nlrp3 A350V and Nlrp3 L351P inserted into the endogenous mouse Nlrp3 locus. Tissue specific expression was driven by breeding these animals with mice expressing Cre recombinase under the control of the dopaminergic neuron-specific Slc6a3 promoter. The experimental mice, designed to express hyperactive NLRP3 only when the endogenous mouse Nlrp3 promotor is active in dopaminergic neurons, were analyzed throughout 18 months of aging using longitudinal motor function assessments. Biochemical and histologic analyses of mesencephalic tissues were conducted in 1- and 18-month-old animals., Results: We observed progressive and significant deficits in motor function in animals expressing Nlrp3 L351P , compared with animals expressing Nlrp3 WT and Nlrp3 A350V . Age-dependent neuroinflammatory changes in the mesencephalon were noted in all animals. Analysis of GFAP-immunoreactive astrocytes in the substantia nigra revealed a significant increase in astrocyte number in animals expressing Nlrp3 L351P compared with Nlrp3 WT and Nlrp3 A350V . Further analysis of Nlrp3 L351P striatal tissues indicated genotype specific gliosis, elevated Il1b expression, and both morphologic and gene expression indicators of proinflammatory A1 astrocytes., Conclusions: Dopaminergic neurons have the potential to accumulate NLRP3 inflammasome activators with age, including reactive oxygen species, dopamine metabolites, and misfolded proteins. Results indicate the Nlrp3 locus is active in dopaminergic neurons in aging mice, and that the hyperactive Nlrp3 L351P allele can drive neuroinflammatory changes in association with progressive behavioral deficits. Findings suggest neuronal NLRP3 inflammasome activity may contribute to neuroinflammation observed during normal aging and the progression of neurologic disorders.

Spinal cord injury (SCI) is the destruction of spinal cord motor and sensory resulted from an attack on the spinal cord, which can cause significant physiological damage. The inflammasome is a multiprotein oligomer resulting in inflammation; the NLRP3 inflammasome composed of NLRP3, apoptosis-associated speck-like protein (ASC), procaspase-1, and cleavage of procaspase-1 into caspase-1 initiates the inflammatory response. Subventricular Zone (SVZ) is the origin of neural stem/progenitor cells (NS/PCs) in the adult brain. Extracellular vesicles (EVs) are tiny lipid membrane bilayer vesicles secreted by different types of cells playing an important role in cell-cell communications. The aim of this study was to investigate the effect of intrathecal transplantation of EVs on the NLRP3 inflammasome formation in SCI rats. Male wistar rats were divided into three groups as following: laminectotomy group, SCI group, and EVs group. EVs was isolated from SVZ, and characterized by western blot and DLS, and then injected into the SCI rats. Real-time PCR and western blot were carried out for gene expression and protein level of NLRP3, ASC, and Caspase-1. H&E and cresyl violet staining were performed for histological analyses, as well as BBB test for motor function. The results indicated high level in mRNA and protein level in SCI group in comparison with laminectomy (p < 0.001), and injection of EVs showed a significant reduction in the mRNA and protein levels in EVs group compared to SCI (p < 0.001). H&E and cresyl violet staining showed recovery in neural cells of spinal cord tissue in EVs group in comparison with SCI group. BBB test showed the promotion of motor function in EVs group compared to SCI in 14 days (p < 0.05). We concluded that the injection of EVs could recover the motor function in rats with SCI and rescue the neural cells of spinal cord tissue by suppressing the formation of the NLRP3 inflammasome complex.

Objective: To explore the protective effect of curcumin on lidocaine-insulted PC12 cells., Materials and Methods: We first treated PC12 cells with different doses of lidocaine, and then treated the cells with curcumin or Nod-like receptor pyrin domain3 (NLRP3) inhibitor (MCC950). Subsequently, the cell viability, apoptosis, reactive oxygen species (ROS) production and NLRP3 inflammasome were detected by cell counting kit-8 (CCK8), Annexin V/PI staining, FCM and Western blot analysis, respectively, and the level of IL-1β in PC12 cells was determined by an enzyme-linked immunosorbent assay (ELISA) kit., Results: Lidocaine inhibited the viability of PC12 cells, and it induced cell apoptosis, promoted ROS release and activated NLRP3 inflammasome in PC12 cells, but its effects were reversed by the treatment of curcumin. Moreover, NLRP3 over-expression also induced cytotoxicity in PC12 cells, which was also rescued by the treatment of curcumin., Conclusions: Our study indicates that curcumin exerts protective effect against lidocaine-induced cytotoxicity on PC12 cells by suppressing the activity of NLRP3 inflammasome, which provides new ideas on screening natural product for neurological damage therapy.

Background: Neutrophilic subtype asthma occurs in approximately 15-25% of the asthma cases and is associated with severe airflow obstruction, corticosteroid resistance. MicroRNA plays a vital role in regulating many immune processes, but how miRNA circuits coordinate airway inflammation during neutrophilic asthma is unclear., Methods: To investigate the molecular mechanism of miR-223 in regulation of neutrophilic airway inflammation, miR-223 knockout mice were used to the OVA/CFA-induced neutrophilic asthma or treated with NLRP3 inhibitor and IL-1β receptor antagonist. Based on the results obtained, wide-type mice were subsequently treated with miR-223 agomirs or negative control agomirs, and the effects on airway inflammation were assessed using morphometric techniques, quantitative RT-PCR, western blot, ELISA and other molecular approaches., Results: The expression of miR-223 was upregulated in lung tissues of experimental mice model. Furthermore, miR-223 -/- mice led to aggravated neutrophilic airway inflammation with heightened histopathological, inflammatory cells and cytokines readouts. Moreover, miR-223 -/- mice also presented with enhanced NLRP3 inflammasome level with elevated IL-1β. Blocking NLRP3 or IL-1β diminished this phenotype. Finally, overexpression of miR-223 via treatment with miR-223 agomirs attenuated airway inflammation, NLRP3 levels and IL-1β release., Conclusions: The findings of this study revealed a crucial role for miR-223 in regulating the immunoinflammatory responses by depressing the NLRP3/ IL-1β axis in neutrophilic asthma.

Acrylamide (ACR), a potential neurotoxin, is present in diet and drinking water. Dietary exposure contributes to cognitive impairment, but relevant mechanism information is limited. Neuroinflammation plays important roles in neurodegenerative disorders. This study aimed to explore whether chronic acrylamide exposure induced neuronal lesions, microglial activation, NLRP3 inflammasome-mediated neuroinflammation and cognitive impairment. For this purpose, 36 Sprague-Dawley (SD) rats were randomly divided into three groups (n = 12/group) and maintained on treated drinking water providing dosages of 0, 0.5, or 5 mg/kg/day ACR for 12 months. Chronic exposure to ACR caused gait abnormality and cognitive dysfunction, which was associated with neuronal lesions, decrease in synapse associated proteins including synapsin I (SYN1), synaptophysin (SYP) and postsynaptic density protein 95 (PSD95), neurogenesis suppression as shown by reduced brain derived neurotrophic factor (BDNF) and doublecortin (DCX) in the hippocampus and frontal cortex. ACR stimulated glial proliferation and microglial activation by increasing GFAP + , Iba-1 + , Iba-1 + CD68 + positive cells. ACR markedly upregulated the protein levels of NLRP3 inflammasome constituents NLRP3, caspase-1 and increased pro-IL-1β and IL-1β. ACR elevated the protein P62 to suppress NLPR3 inflammasome cleavage. Inflammatory cytokines including TNF-α, IL-6 and Cox-2 were also significantly increased after NF-κB pathway activation, which aggravated neuronal lesions and caused memory deficits. This work helped to propose the possible mechanism of chronic exposure of ACR-induced neurotoxicity., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: High prevalence of obstructive sleep apnea (OSA) in the pulmonary hypertension (PH) population suggests that chronic intermittent hypoxia (CIH) is an important pathogenic factor of PH. However, the exact mechanism of CIH induced PH is not clear. One of the molecules that plays a key role in regulating pulmonary artery function under hypoxic conditions is superoxide dismutase 2 (SOD 2 )., Methods: Our study utilized heterozygous SOD 2 -/+ mice firstly in CIH model to explore the exact role of SOD 2 in CIH causing PH. Expression of SOD2 was analyzed in CIH model. Echocardiography and pulmonary hypertension were measured in wild type (WT) and SOD2 -/+ mice under normal air or CIH condition. Hematoxylin-Eosin (H&E) staining and masson staining were carried out to evaluate pulmonary vascular muscularization and remodeling. Micro-PET scanning of in vivo 99m Tc-labelled- MAG3-anti-CD11b was applied to assess CD11b in quantification and localization. Level of nod-like receptor pyrin domain containing 3 (NLRP3) was analyzed by real time PCR and immunohistochemistry (IHC)., Results: Results showed that SOD 2 was down-regulated in OSA/CIH model. Deficiency of SOD2 aggravated CIH induced pulmonary hypertension and pulmonary vascular hypertrophy. CD11b + cells, especially monocytic myeloid cell line-Ly6C + Ly6G - cells, were increased in the lung, bone marrow and the blood under CIH condition, and down-regulated SOD2 activated NLRP3 in CD11b + cells. SOD 2 -deficient-CD11b + myeloid cells promoted the apoptosis resistance and over-proliferation of human pulmonary artery smooth muscle cells (PASMCs) via up-regulating NLRP3., Conclusion: CIH induced down-regulating of SOD2 increased pulmonary hypertension and vascular muscularization. It could be one of the mechanism of CIH leading to PH.

This investigation evaluated the neuroprotective effect of melodinhenine B in a cerebral ischemia/reperfusion (I/R)-induced neuronal injury rat model. The effect of melodinhenine B was determined by evaluating the neurological deficit score, cerebral infarcted area, and blood-brain barrier (BBB) permeability. Moreover, the level of inflammatory cytokines and expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ), interleukin-1β (IL-1β), NLRP3, zonula occludens-1 (ZO-1), and occluding proteins were estimated by Western blotting. Histopathological changes and immunohistochemical analysis were performed to estimate the effect of melodinhenine B on neuronal injury. The neurological deficit score, percentage of infarcted area, and BBB permeability were improved in the melodinhenine B-treated group of rats. Treatment with melodinhenine B attenuated the altered expression of NF-κβ, IL-1β, NLRP3, ZO-1, and occluding proteins in the brain tissue of I/R-induced neuronal injury rats. The inflammatory cytokine levels were reduced in the melodinhenine B-treated group. Histopathologically, melodinhenine B reversed the pathological changes in the brain tissues of I/R-induced neuronal injury rats. In conclusion, melodinhenine B protects against neuronal injury in cerebral ischemia-reperfusion injury rats by regulating the inflammasomes.

Purpose : Our previous study has identified that plasma levels of S100A12 are closely associated with presence and severity of diabetic retinopathy (DR). In this work, we explored whether S100A12 can contribute to retinal microglial activation and inflammatory changes of DR via a microRNA-dependent mechanism. Material and Methods : Streptozotocin (STZ)-induced DR model was developed. Retinal microglia of rats were activated through intravitreal injection of S100A12. Differential expression of miRNAs on retinal microglia treated with S100A12 or DMEM/F-12 alone was determined using microarray analysis. Luciferase reporter assays were performed, which explored the regulation of a putative target of miR-30a. Results : S100A12 was increased approximately fivefold in the retina of 16-week diabetic rats compared with nondiabetic retinas. Furthermore, the levels of NLRP3, ASC, caspase-1, IL-1β, and IL-18 were significantly increased in the retina of rats treated with intravitreal injection of S100A12. Moreover, S100A12 induced an increased expression of NLRP3, ASC, caspase-1, IL-1β, and IL-18 in a dose- and time-dependent manner in retinal microglia. S100A12 was a proinflammatory trigger in diabetes-induced retinal microglial activation by activating NLRP3 in vivo and in vitro. In addition, S100A12 induced retinal microglial activation via a miR-30a-dependent mechanism. Mechanistically, S100A12 inhibited miR-30a expression, which was controlled by HDAC, and miR-30a downregulated NLRP3 expression by directly targeting its 3'-UTR. Conclusions : S100A12 plays an important role in the pathogenesis of DR by activating retinal microglia via a miR-30a-dependent mechanism.

Dexmedetomidine (Dex) is an anesthetic widely used in lumbar discectomy, but its effect on chondrocytes remains unclear. Dex is speculated to promote cartilage degeneration by activating α-2 adrenergic receptor. However, the antioxidative and anti-inflammatory effects of Dex implied the potential chondrocyte protective effect under stress conditions. The present study aimed to determine the effect of Dex on chondrocytes under non-stress and stress conditions. Chondrocytes were isolated from human annulus fibrosus (AF) tissues and oxidative stress was induced by treatment with 1 mM hydrogen peroxide (H 2 O 2 ). Chondrocytes were treated with Dex alone or in combination with H 2 O 2 Treatment with Dex alone decreased mRNA expression of COL2A1 and increased that of MMP-3 and MMP-13 , thus contributing to cartilage degeneration. However, Dex prevented H 2 O 2 -induced death and degeneration of chondrocytes partly by enhancing antioxidant capacity. Mechanistically, Dex attenuated H 2 O 2 -mediated activation of NF-κB and NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), both of which play key roles in inflammation and inflammatory damage. Dex inactivated NLRP3 through the suppression of NF-κB and JNK signals. Co-treatment with Dex and H 2 O 2 increased protein level of XIAP (X-linked inhibitor-of-apoptosis, an anti-apoptosis protein), compared with H 2 O 2 treatment alone. H 2 O 2 treatment increased the expression of neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) that is a ubiquitin ligase targeting XIAP. However, Dex decreased the amount of NEDD4 adhering to XIAP, thus protecting XIAP protein from NEDD4-mediated ubiquitination and degradation. Given that surgery inevitably causes oxidative stress and inflammation, the protective effect of Dex on chondrocytes during oxidative stress is noteworthy and warrants further study., (© 2019 The Author(s).)

Purpose: Visual (retinoid) cycle anomalies induce aberrant build-up of all-trans retinal (atRAL) in the retinal pigment epithelium (RPE), which is a cause of RPE atrophy in Stargardt disease type 1 and age-related macular degeneration. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation is implicated in the etiology of age-related macular degeneration. Here, we elucidated the relationship between NLRP3 inflammasome activation and atRAL-induced death of RPE cells., Methods: Cellular toxicities were assessed by MTS or MTT assays. Expression levels of mRNAs and proteins were determined by quantitative reverse transcription-polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. Fluorescence microscopy was used to examine intracellular signals. Ultrastructural features of organelles were examined by transmission electron microscope., Results: Abnormal accumulation of atRAL was associated with a significant increase in the proportion of human ARPE-19 cells exhibiting features of apoptosis and Caspase-3/gasdermin E (GSDME)-mediated pyroptosis. These cells also exhibited elevated expression of NLRP3, ASC, cleaved Caspase-1/poly ADP-ribose polymerase (PARP)/Caspase-3/GSDME, interleukin-1β (IL-1β), and IL-18, as well as NLRP3 inflammasome-related genes (IL1B and IL18). After exposure of human ARPE-19 cells to excess atRAL, reactive oxygen species (ROS) (including mitochondrial ROS) and cathepsins released from lysosomes transmitted signals leading to NLRP3 inflammasome activation. Suppressing the production of ROS, NLRP3 inflammasome, Caspase-1, cathepsin B, or cathepsin D protected ARPE-19 cells against atRAL-associated cytotoxicity. Damage to mitochondria, lysosomes, and endoplasmic reticulum in atRAL-exposed ARPE-19 cells was partially alleviated by treatment with MCC950, a selective NLRP3 inflammasome inhibitor., Conclusions: Aberrant build-up of atRAL promotes the death of RPE cells via NLRP3 inflammasome activation.

Evodiamine (EVO), an extraction from the traditional Chinese medicine Evodia rutaecarpa, has been reported to possess anti-inflammatory, anti-tumor and other pharmacological activities. However, the effectiveness of EVO to relieve dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) has not been evaluated. In this study, the protective effects and mechanisms of EVO on DSS-induced UC mice were investigated. The results indicated that treatment with EVO ameliorated DSS-induced UC mice body weight loss, disease activity index (DAI), colon length shortening, colonic pathological damage, and myeloperoxidase (MPO) activity. The production of TNF-α, IL-1β and IL-6 was also significantly inhibited by EVO. Further mechanistic results showed that EVO restrained the inflammation by regulating NF-κB signal and NLRP3 inflammasome. Furthermore, results also showed that EVO contributed to the tight junction (TJ) architecture integrity by modulating the expression of zonula occludens-1 (ZO-1) and occludin during colitis. Surprisingly, treatment with EVO reduced the concentration of plasmatic lipopolysaccharide (LPS) and re-balanced the levels of Escherichia coli and Lactobacillus. These findings suggested that EVO may have a potential protective effect on DSS-induced colitis and may be useful for the prevention and treatment of UC., (Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background The arteriovenous fistula ( AVF ) is the preferred hemodialysis access for patients with chronic kidney disease. Chronic kidney disease can increase neointima formation, which greatly contributes to AVF failure by an unknown mechanism. Our study aimed to determine the role of nucleotide-binding oligomerization domain-like receptor protein 3 ( NLRP 3) in neointima formation induced by experimental AVF s in the presence of chronic kidney disease. Methods and Results From our findings, NLRP 3 was upregulated in the intimal lesions of AVF s in both uremic mice and patients. Smooth muscle-specific knockout NLRP 3 mice exhibited markedly decreased neointima formation in the outflow vein of AVF s. Compared with primary vascular smooth muscle cells isolated from control mice, those isolated from smooth muscle-specific knockout NLRP 3 mice showed compromised proliferation, migration, phenotypic switching, and a weakened ability to activate mononuclear macrophages. To identify how NLRP 3 functions, several small-molecule inhibitors were used. The results showed that NLRP 3 regulates smooth muscle cell proliferation and migration through Smad2/3 phosphorylation rather than through caspase-1/interleukin-1 signaling. Unexpectedly, the selective NLRP 3-inflammasome inhibitor MCC 950 also repressed Smad2/3 phosphorylation and relieved chronic kidney disease-promoted AVF failure independent of macrophages. Conclusions Our findings suggest that NLRP 3 in vascular smooth muscle cells may play a crucial role in uremia-associated AVF failure and may be a promising therapeutic target for the treatment of AVF failure.

Acrolein is a common environmental pollutant that has been linked to cardiovascular diseases, such as atherosclerosis (AS). Increasing evidence demonstrates that acrolein impairs the cardiovascular system by targeting vascular endothelial cells, but the underlying mechanisms haven't been completely elucidated. In human umbilical vein endothelial cells (HUVECs), we observed that acrolein treatment induced cell reactive oxygen species (ROS) generation, autophagy, pyroptosis and reduced cell migration. In addition, exposure to acrolein resulted in NLRP3 inflammasome activation as evidenced by cleavage of caspase-1 and downstream mature interleukin (IL)-1β and IL-18 secretion. Knockdown of NLRP3 by small interfering RNA remarkably suppressed acrolein-induced pyroptosis and increased cell migration. Moreover, the scavenging ROS relieved the autophagy, NLRP3 inflammasome activation and pyroptosis. Furthermore, the role of autophagy in the acrolein-medicated pyroptosis and cell migration was investigated. In our study, 3-methyladenine (3-MA), an autophagy inhibitor, aggravated NLRP3 inflammasome activation, pyroptosis and decreased cell migration, rapamycin (Rapa), an autophagy inducer, alleviated aforementioned phenomenon under acrolein stress. Besides, we found damaged mitochondrion accentuated NLRP3 inflammasome and pyroptosis in acrolein-treated cells. In conclusion, it is possible that acrolein induced cell pyroptosis and suppressed cell migration via ROS-dependent autophagy. What's more, NLRP3 inflammasome activation plays a key role in this process., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background This study aimed at investigating whether NLRP 3 (the Nod like receptor family, pyrin domain-containing 3 protein) inflammasome activation induced HMGB 1 (high mobility group box-1 protein) secretion and foam cell formation in human vascular smooth muscle cells ( VSMC s) and atherosclerosis in ApoE -/- mice. Methods and Results VSMC s or ApoE -/- mice were treated with lipopolysaccharides ( LPS ) and/or ATP or LPS and high-fat diet to induce NLRP 3 inflammasome activation. HMGB 1 distribution and foam cell formation in VSMC s were characterized. Liver X receptor α and ATP -binding cassette transporter expression were determined. The impact of NLRP 3 or receptor for advanced glycation end product silencing, ZYVAD - FMK (caspase-1 inhibitor), glycyrrhizin ( HMGB 1 inhibitor) or receptor for advanced glycation end product antagonist peptide on HMGB 1 secretion, foam cell formation, liver X receptor α and ATP -binding cassette transporter expression was examined. Expression level of HMGB 1 in human atherosclerosis obliterans arterial tissues was characterized. Our results found that NLRP 3 inflammasome activation promoted foam cell formation and HMGB 1 secretion in VSMC s. Extracellular HMGB 1 was a key signal molecule in inflammasome activation-mediated foam cell formation. Furthermore, inflammasome activation-induced HMGB 1 activity and foam cell formation were achieved by receptor for advanced glycation end product/liver X receptor α / ATP -binding cassette transporter glycyrrhizin. Experiments in vivo found glycyrrhizin significantly attenuated the LPS /high-fat diet-induced atherosclerosis and serum HMGB 1 levels in mice. Finally, levels of HMGB 1 and NLRP 3 were increased in tunica media adjacent to intima of atherosclerosis obliteran arteries. Conclusions Our results revealed that HMGB1 is a key downstream signal molecule of NLRP 3 inflammasome activation and plays an important role in VSMC s foam cell formation and atherogenesis by downregulating liver X receptor α and ATP -binding cassette transporter expression through receptor for advanced glycation end product.

Glioblastoma(GBM) is most common brain tumor in adults. Currently standard treatments have limited effect to increase the survival, because there are still largely unclear mechanisms in glioblastoma development. miR-223 was involved in various types of cancer, however, the function of miR-223-3p in GBM was still unclear. In our study, real-time PCR was performed to exam the expression level of miR-223-3p and NLRP3 (Nucleotide-binding oligomerization domain(NOD)-like receptor family PYRIN domain containing-3) in GBM tissues. Following that, mimic or inhibitor of miR-223-3p were used to modulate miR-223-3p expression in GBM cell lines respectively. Then, we analyzed cell proliferation and migration by cell counting kit and transwell assay. Further, western blot was performed to detect several inflammation-associated cytokines level in GBM cell lines. We found that miR-223-3p was decreased but NLRP3 was increased in GBM tissues. Treatment with miR-223-3p mimic inhibits cell proliferation and migration via decreasing several inflammation-associated cytokines, including interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), IL-8 and IL-18. Importantly, these effects induced by miR-223-3p could be attenuated by NLRP3 overexpression, which was considered as one of target genes of miR-223-3p. In conclusion, these results indicated that miR-223-3p might act as a suppressor and a potential therapy target of GBM., (Copyright © 2018. Published by Elsevier GmbH.)

Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biologic functions, the contribution of endothelial TXNIP has not been well defined. To investigate the endothelial function of TXNIP, we generated a TXNIP knockout mouse on the Cdh5-cre background (TXNIP fl/fl cdh5 cre ). Control (TXNIP fl/fl ) and TXNIP fl/fl cdh5 cre mice were fed a high protein-low carbohydrate (HP-LC) diet for 3 mo to induce metabolic stress. We found that TXNIP fl/fl and TXNIP fl/fl cdh5 cre mice on an HP-LC diet displayed impaired glucose tolerance and dyslipidemia concretizing the metabolic stress induced. We evaluated the impact of this metabolic stress on mice with reduced endothelial TXNIP expression with regard to arterial structure and function. TXNIP fl/fl cdh5 cre mice on an HP-LC diet exhibited less endothelial dysfunction than littermate mice on an HP-LC diet. These mice were protected from decreased aortic medial cell content, impaired aortic distensibility, and increased plasminogen activator inhibitor 1 secretion. This protective effect came with lower oxidative stress and lower inflammation, with a reduced NLRP3 inflammasome expression, leading to a decrease in cleaved IL-1β. We also show the major role of TXNIP in inflammation with a knockdown model, using a TXNIP-specific, small interfering RNA included in a lipoplex. These findings demonstrate a key role for endothelial TXNIP in arterial impairments induced by metabolic stress, making endothelial TXNIP a potential therapeutic target.-Bedarida, T., Domingues, A., Baron, S., Ferreira, C., Vibert, F., Cottart, C.-H., Paul, J.-L., Escriou, V., Bigey, P., Gaussem, P., Leguillier, T., Nivet-Antoine, V. Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

Necroptosis is a manner of caspase-independent cell death,which accounts for delayed ischemic cerebral injury, and can be used as a novel tool to expand the treatment time window in ischemic cerebral injury. Q-VD-OPH, a novel pan caspase inhibitor, has been identified as an inducer of necroptosis. In this study, we determined the optimal dose of Q-VD-OPH, which induces necroptosis in rats by the middle cerebral artery occlusion, followed by reperfusion. Furthermore, we report that the NLRP3 inflammasome is involved in necroptosis, with levels of NLRP3 inflammasome proteins as well as inflammatory cytokines, such as IL-1β, being elevated. We also demonstrated that NLRP3 was not only expressed in microglia and vascular endothelial cell, but also in neurons when necroptosis is induced with Q-VD-OPH. Inhibition of NLRP3 by glyburide strongly suppressed the expression of NLRP3 inflammasome proteins and IL-1β, and markedly reduced brain tissue damage. Our findings provide evidence that pretreatment with Q-VD-OPH suppresses apoptosis and induces necroptosis in the cerebral ischemia-reperfusion model. We also identified that the NLRP3 inflammasome plays an important role in neuronal necroptosis, and that NLRP3 inflammasome deficiency reduces brain tissue damage after cerebral ischemia-reperfusion injury in rats.

It has been proved that hyperuricemia is associated with non-alcoholic fatty liver disease (NAFLD). The xanthine oxidase (XO) inhibitor, febuxostat, decreases free fatty acids-induced fat accumulation in HFDT-fed mice. Here, it is shown that febuxostat attenuates fat accumulation and reactive oxygen species (ROS) in HepG2 cells. It was further found that the underlying mechanism is related to the reduction in expression of NLRP3/caspase-1/IL-18/IL-1beta and improved insulin resistance (IR). This finding highlights the possible molecular pathways involving NLRP3 activation for management of ROS and insulin IR. In conclusion, febuxostat may be a promising potential treatment for patients with NAFLD.

Background: MicroRNAs (miRNAs) serve as important regulators of inflammatory and immune responses and are implicated in several immune disorders including gouty arthritis. The expression of miR-146a is upregulated in the peripheral blood mononuclear cells of patients with inter-critical gout when compared to normouricemic and hyperuricemic controls and those patients with acute gout flares. However, the role of miR-146a in the development of gout remains unknown. Here, we used miR-146a knockout (KO) mice to test miR-146a function in a monosodium urate (MSU)-induced gouty arthritis model., Methods: The footpad or ankle joint of miR-146a KO and wild-type (WT) mice were injected with an MSU suspension to induce acute gouty arthritis. Bone marrow-derived macrophages (BMDMs) were stimulated with MSU and the gene expression of miR-146a; interleukin 1 beta (IL-1β); tumor necrosis factor-α (TNF-α); and the NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome was evaluated. TNF-α and IL-1β protein levels in BMDMs were assessed by fluorescence-activated cell sorting and western blot analyses. Gene and protein levels of TNF receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase (IRAK1), the targets of miR-146a, were also measured., Results: Significantly increased paw swelling and index and ankle joint swelling were observed in miR-146a KO mice compared to WT controls after MSU treatment. MiR-146a expression in BMDMs from WT mice was dramatically upregulated at 4 h following MSU stimulation. Additionally, the expression of IL-1β, TNF-α, and NALP3 was higher in BMDMs from miR-146a KO mice after exposure to MSU crystals compared to those from WT mice. Consistent with the observed gene expression, the IL-1β and TNF-α proteins were upregulated in miR-146a KO mice. Additionally quantitative RT-PCR and western blot demonstrated that TRAF6 and IRAK1 were dramatically upregulated in BMDMs from miR-146 KO mice compared to those from WT mice., Conclusions: Collectively, these observations suggest that miR-146a provides negative feedback regulation of gouty arthritis development and lack of miR-146a enhances gouty arthritis via upregulation of TRAK6, IRAK-1, and the NALP3 inflammasome function.

Atherosclerosis (AS) is an inflammatory disease linked to endothelial dysfunction. Melatonin is reported to possess substantial anti-inflammatory properties, which has proven to be effective in AS. Emerging literature suggests that pyroptosis plays a critical role during AS progression. However, whether pyroptosis contributes to endothelial dysfunction and the underlying molecular mechanisms remained unexploited. This study was designed to investigate the antipyroptotic effects of melatonin in atherosclerotic endothelium and to elucidate the potential mechanisms. In this study, high-fat diet (HFD)-treated ApoE -/- mice were used as an atherosclerotic animal model. We found intragastric administration of melatonin for 12 weeks markedly reduced the atherosclerotic plaque in aorta. Meanwhile, melatonin also attenuated the expression of pyroptosis-related genes, including NLRP3, ASC, cleaved caspase1, NF-κB/GSDMD, GSDMD N-termini, IL-1β, and IL-18 in aortic endothelium of melatonin-treated animals. Consistent antipyroptotic effects were also observed in ox-LDL-treated human aortic endothelial cells (HAECs). We found that lncRNA MEG3 enhanced pyroptosis in HAECs. Moreover, MEG3 acted as an endogenous sponge by sequence complementarity to suppress the function of miR-223 and to increase NLRP3 expression and enhance endothelial cell pyroptosis. Furthermore, knockdown of miR-223 blocked the antipyroptotic actions of melatonin in ox-LDL-treated HAECs. Together, our results suggest that melatonin prevents endothelial cell pyroptosis via MEG3/miR-223/NLRP3 axis in atherosclerosis, and therefore, melatonin replacement might be considered a new strategy for protecting endothelium against pyroptosis, thereby for the treatment of atherosclerosis associated with pyroptosis., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Whether approaches to chronically increase VEGF-A in the heart may have beneficial effects and prevent the development of heart failure, in part by improving cardiac perfusion, or whether this increase could have detrimental effects on cardiac performance in the aging heart, has not been tested yet. In this study, a genetic mouse model with a chronic increase in VEGF-A in the heart is shown to have increased cardiac angiogenesis and develop cardiac hypertrophy with enhanced basal cardiac performance with age progression. However, in aged hearts, this increase in VEGF-A was associated with higher expression of fetal cardiac genes and reduced cardiac performance after β-agonistic stress, features consistent with pathologic cardiac hypertrophy. Expression of Nod-like receptor protein (NLRP)-3 was increased in the hearts of the mice, and its genetic inactivation prevented increased fetal cardiac gene expression and partially rescued the impaired cardiac performance after β-agonistic stimulation in aged hearts without reducing cardiac angiogenesis or hypertrophy. Thus, although a chronic increase in cardiac VEGF-A may improve cardiac perfusion, long-term upregulation of VEGF-A leads to reduced cardiac performance under stress, an effect that can be partially inhibited by NLRP3 inactivation. Targeting NLRP3 shifts the VEGF-A-induced cardiac hypertrophy from a pathologic toward a more physiologic hypertrophy.-Marneros, A. G. Effects of chronically increased VEGF-A on the aging heart.

Inflammasome-mediated production of mature IL-1β and IL-18 cytokines represents an important innate immune response against infecting pathogens. Helicobacter pylori, one of the most successful and persistent human pathogens, induces severe inflammation leading to gastritis and more serious gastric diseases. H. pylori modulates different immune responses for its survival and inflammasome signaling is manipulated by the cag pathogenicity island ( cagPAI), urease and VacA cytotoxin. Here we report that H. pylori regulates NLRP3 expression, an inflammasome forming regulator, in infected THP-1 monocytes. This response was independent of the major H. pylori pathogenicity-associated factors CagA, VacA, Cgt, FlaA and cagPAI. Two NLRP3 expression controlling factors, the NLRP3 mRNA targeting microRNA hsa-miR-223-3p and cytokine IL-10, were found to work in tandem for its regulation. H. pylori infection also induced copious amount of pro-IL-1β in THP-1 monocytes/macrophages but secreted a very low amount of mature IL-1β. Moreover, secreted IL-10 correlated with the down-regulation of nigericin-induced NLRP3 inflammasome activation of LPS-primed THP-1 monocytes and human PBMCs from volunteers. However, H. pylori-treated PBMCs secreted significantly more mature IL-1β throughout the infection period, which suggests a different mode of activation. Taken together, this study demonstrates targeting of inflammasome-forming NLRP3, an important innate immunity component, and crucial manipulation of pro- and anti-inflammatory cytokines in H. pylori infection.

Autoimmune hepatitis is a chronic inflammatory disease in the liver with potential to the development of liver fibrosis. Recent evidences suggest that bone marrow derived mesenchymal stem cells (BMSCs) may exert its therapeutic activity through exosomes. Moreover, miR-223 is highly expressed in BMSCs and plays an important role in autoimmune diseases. Therefore, in this study, hepatoprotective role of BMSCs and miR-223 was investigated in both mice and hepatocytes. Liver antigen S100 was used to establish autoimmune hepatitis model in mice while LPS and ATP were used to establish cell injury model in hepatocyte. Before the experiments, BMSCs were infected with pre-miR-223 and transfected with miR-223 inhibitor respectively. Exosomes from bone marrow stem cells were isolated by ultracentrifugation. Liver injury was evaluated by serum levels of ALT and AST as well as liver histology. Inflammation and cell death were examined by inflammatory cytokines and lactase dehydrogenase respectively. Both BMSCs-exo and BMSCs-exo miR-223(+) significantly reversed either S100 or LPS/ATP induced injury in mice and hepatocytes. Meanwhile, the expressions of cytokines, NLRP3 and caspase-1 were also downregulated by BMSCs-exo and BMSCs-exo miR-223(+) at both protein and mRNA levels in mice and hepatocytes. Moreover, BMSCs-exo miR-223(-) reverses the effects of BMSCs-exo and BMSCs-exo miR-223(+) in mouse AIH and in hepatocytes. In conclusion, bone marrow stem cell derived exosomes can protect liver injury in an experimental model of autoimmune hepatitis and the mechanism could be related to exosomal miR-223 regulation of NLRP3 and caspase-1., (Copyright © 2017 Elsevier Ltd. All rights reserved.)