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Cyclic nucleotides cAMP and cGMP are important regulators of immune cell functions. Phosphodiesterases (PDEs) hydrolyze cAMP and/or cGMP and, thus, play crucial roles in cyclic nucleotide homeostasis. Abnormal alterations of PDE expression have been implicated in several diseases. To understand the function of PDEs in macrophages, we screened for all PDE genes in both peritoneal and alveolar macrophages from C57BL/6J mice and found that PDE4B and PDE10A are highly induced by LPS. A number of PDE4 inhibitors have been used clinically for the treatment of inflammatory lung diseases. However, the role of PDE10A in inflammation is still poorly understood. We therefore investigated the role of PDE10A in macrophage inflammatory response in vitro and acute lung inflammation in vivo. We found that LPS induces a sustained PDE10A expression in macrophages, which is different from a transient induction by PDE4B. PDE10A inhibition blocked LPS-induced MCP-1 expression, but not TNF-α, whereas PDE4B inhibition blocked LPS-induced TNF-α expression, but not MCP-1. In addition, PDE10A inhibition or deficiency decreased LPS-induced HIF-1α protein expression and subsequently suppressed MCP-1 expression. In vivo, PDE10A expression was also elevated in lung tissue after LPS exposure. Global PDE10A knockout or systemic administration of the PDE10A inhibitor TP-10 in mice significantly suppressed inflammatory molecule levels in the lung tissue and bronchoalveolar lavage fluid as well as inflammatory cell infiltration. These findings show that PDE10A plays a critical role in lung inflammation by promoting the activation of resident macrophages and infiltration of neutrophils.

Otitis media (OM) is the most common bacterial infection in children. It remains a major health problem and a substantial socioeconomic burden. Streptococcus pneumoniae (S. pneumoniae) is one of the most common bacterial pathogens causing OM. Innate inflammatory response plays a critical role in host defense against bacterial pathogens. However, if excessive, it has a detrimental impact on the middle ear, leading to middle ear inflammation, a hallmark of OM. Currently, there has been limited success in developing effective therapeutic agents to suppress inflammation without serious side effects. In this study, we show that vinpocetine, an antistroke drug, suppressed S. pneumoniae–induced inflammatory response in cultured middle ear epithelial cells as well as in the middle ear of mice. Interestingly, vinpocetine inhibited S. pneumoniae–induced inflammation via upregulating a key negative regulator cylindromatosis (CYLD). Moreover, CYLD suppressed S. pneumoniae–induced inflammation via inhibiting the activation of ERK. Importantly, the postinfection administration of vinpocetine markedly inhibited middle ear inflammation induced by S. pneumoniae in a well-established mouse OM model. These studies provide insights into the molecular mechanisms underlying the tight regulation of inflammation via inhibition of ERK by CYLD and identified vinpocetine as a potential therapeutic agent for suppressing the inflammatory response in the pathogenesis of OM via upregulating negative regulator CYLD expression.

Background: Atherosclerosis is the predominant cause of cardiovascular diseases. Existing studies suggest that the development of atherosclerosis is closely related to inflammation and immunity, but whether there are differences and similarities between atherosclerosis occurring at different sites is still unknown. We elucidated the pathological characteristics of peripheral vascular diseases by using bioinformatic analyses on immune cells and inflammation-related gene expression in atherosclerotic arteries and plaques. Methods: Eight data sets regarding atherosclerosis were downloaded from the Gene Expression Omnibus database. Human immune genes were obtained from the IMMPORT website. The samples were scored and divided into highand low-immune groups. Then the samples were analysed using weighted gene co-expression network analysis, while the modules were analysed using functional enrichment. The protein–protein interaction network was constructed using the STRING and Cytoscape databases. The hub immune genes were screened, and the correlation between hub immune genes and immune cells was analysed. Results: Immune cells and their functions were significantly different during atherosclerosis development. The infiltration proportion of immune cells was approximately similar in samples from different sources of patients with carotid atherosclerosis. However, the sensitivity of lower extremity atherosclerosis samples to immune cells is lower than that of carotid atherosclerosis samples.The samples from the plaque and artery were mainly infiltrated by macrophages, T cells and mast cells. After immune cells were assessed, resting NK cells, activated mast cells and M0 macrophages were found to be key immune cells in atherosclerosis and plaque formation. In addition, CCL4, TLR2, IL1B and PTPRC were considered to be immune marker genes in atherosclerosis development. Conclusion: Bioinformatic data analysis confirms the essential role of immune cells in cardiovascular diseases, and also indicates some differences of immune and inflammation characteristics of atherosclerosis between carotid and lower extremity arteries., Oxidative medicine and cellular longevity, 2022, art. no. 5491038; 2022

Chronic low back pain (CLBP) has been proved to be the dominating cause of disability in patients with lumbar degenerative diseases. Of the various etiological factors, intervertebral disc degeneration (IVDD) has been the dominating cause. In the past few decades, the role and changes of nerve systems, especially the peripheral sensory fibers and their neurotransmitters, in the induction and progression of IVDD have attracted growing concerns. The expression of many neuropeptides, such as SP, NPY, and CGRP, in the nociceptive pathways is increased during the progression of IVDD and responsible for the discogenic pain. Here, the role of CGRP in the progression of IVDD was firstly investigated both in vitro and in vivo. Firstly, we confirmed that human degenerated intervertebral disc tissue exhibited elevated expression of CGRP and its receptor. Secondly, in vitro experiments suggested that CGRP could inhibit the proliferation and induce apoptosis in human nucleus pulposus (NP) cells, as well as promote inflammation and degenerated phenotypes through activating NF-κB and MAPK signaling pathways. Thirdly, CGRP receptor antagonist, Rimegepant, can ameliorate the adverse effects of CGRP imposed on NP cells, which were confirmed in vitro and in vivo. Our results will bring about a brand-new insight into the roles of neuromodulation in IVDD and related therapeutic attempts.

Scope: Gut microbiome-derived metabolites are the major mediators of diet-induced host-microbial interactions. Aryl hydrocarbon receptor (AHR) plays a crucial role in glucose, lipid, and cholesterol metabolism in the liver. In this study, we aimed to investigate the role of indole-3-acetic acid (IAA) and AHR in sulforaphane (SFN) alleviates hepatic steatosis in mice fed on a high-fat diet (HFD).Methods and Results: The HFD-fed male C57BL/6 mice were intervened with SFN for 6 weeks. HFD-mice showed classical pathophysiological characteristics of hepatic steatosis. The results showed that SFN significantly reduced body weight, liver inflammation and hepatic steatosis in HFD-fed mice. SFN reduced hepatic lipogenesis by activating AHR/SREBP-1C pathway, which was confirmed in HepG2 cell experiments. Moreover, SFN increased hepatic antioxidant activity by modulating Nrf-2/NQO1 expression. SFN increased serum and liver IAA level in HFD mice. Notably, SFN manipulated the gut microbiota, resulting in reducing Deferribacteres and proportions of the phylum Firmicutes/Bacteroidetes and increasing the abundance of specific bacteria that produce IAA. Furthermore, SFN upregulated Ahr expression and decreased the expression of inflammatory cytokines in Raw264.7 cells.Conclusions: SFN ameliorated hepatic steatosis not only by modulating lipid metabolism via AHR/SREBP-1C pathway but regulating IAA and gut microbiota in HFD-induced NAFLD mice.

The sphingomyelin synthase 2 (SMS2) is a potential target for pharmacological intervention in atherosclerosis. However, so far, few selective SMS2 inhibitors and their pharmacological activities were reported. In this study, a class of 2-benzyloxybenzamides were discovered as novel SMS2 inhibitors through scaffold hopping and structural optimization. Among them, Ly93 as one of the most potent inhibitors exhibited IC50 values of 91 nM and 133.9 μM against purified SMS2 and SMS1 respectively. The selectivity ratio of Ly93 was more than 1400-fold for purified SMS2 over SMS1. The in vitro studies indicated that Ly93 not only dose-dependently diminished apoB secretion from Huh7 cells, but also significantly reduced the SMS activity and increased cholesterol efflux from macrophages. Meanwhile, Ly93 inhibited the secretion of LPS-mediated pro-inflammatory cytokine and chemokine in macrophages. The pharmacokinetic profiles of Ly93 performed on C57BL/6J mice demonstrated that Ly93 was orally efficacious. As a potent selective SMS2 inhibitor, Ly93 significantly decreased the plasma SM levels of C57BL/6J mice. Furthermore, Ly93 was capable of dose-dependently attenuating the atherosclerotic lesions in the root and the entire aorta as well as macrophage content in lesions, in apolipoprotein E gene knockout mice treated with Ly93. In conclusion, we discovered a novel selective SMS2 inhibitor Ly93 and demonstrated its anti-atherosclerotic activities in vivo. The preliminary molecular mechanism-of-action studies revealed its function in lipid homeostasis and inflammation process, which indicated that the selective inhibition of SMS2 would be a promising treatment for atherosclerosis.

Objectives: An association between angiogenesis/inflammation status and tumor has been reported in various types of cancer. This study sought to assess the role of peripheral blood VEGF and some inflammation biomarkers in evaluating clinical response and prognosis in patients with non-operative esophageal squamous cell carcinoma (ESCC).Methods: Peripheral blood of 143 patients with non-operative ESCC at our institute was dynamically collected at 5 time points including 1 day before radiotherapy, during radiotherapy (15f), at the end of radiotherapy, 1 month after radiotherapy, and 3 months after radiotherapy. VEGF expression in the peripheral blood was detected and related inflammation biomarkers such as GPS, CAR and CLR were counted. Logistic regression and Cox regression were implemented respectively to analyze the correlation of each predictor with clinical response and prognosis. The performance of combined testing was estimated using AUCs. Based on independent predictors, a nomogram prediction model was established to predict the probabilities of 1- and 2-year PFS of patients. The effectiveness of the nomogram model was characterized by C-index, AUC, calibration curves and DCA.Results: VEGF and CLR levels at the end of radiotherapy were independent predictors of clinical response, while VEGF and GPS levels at 3 months after radiotherapy were independent prognostic predictors. The efficacy of combined detection of VEGF and CLR is superior to the single detection in evaluating clinical response and prognosis. The nomogram showed excellent accuracy in predicting PFS.Conclusions: The combined detection of VEGF and CLR at the end of radiotherapy can be used to evaluate the clinical response of patients with non-operative ESCC, and the combined detection of VEGF and GPS 3 months after radiotherapy can be used to predict the prognosis. Implemented by nomogram model, it is expected to provide practical and reliable method to evaluate the clinical response and prognosis of patients with non-operative ESCC tool.

Vinpocetine is a derivative of vincamine. It has been used to prevent and treat cerebrovascular disorders such as stoke and dementia, and remains widely available in dietary supplements that often marketed as nootropics. Due to its excellent safety profile at therapeutic dose regimen, vinpocetine has raised research interest in its new applications in various experimental disease models. Here we review recent studies that uncovered novel functions of vinpocetine in cardiovascular diseases, including atherosclerosis, obesity, neointimal hyperplasia, vasoconstriction, pathological cardiac remodeling and ischemia stroke. Molecular mechanisms underlined the protective effects of vinpocetine are also discussed. These novel findings may suggest a broadened usage of vinpocetine against relevant cardiovascular diseases in human.

Abdominal aortic aneurysm (AAA), commonly occurring in the aged population, is a degenerative disease that dilate and weaken infrarenal aorta due to progressive degeneration of aortic wall integrity. Vinpocetine, a derivative of alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment in the aged population. Recent studies have indicated that vinpocetine antagonizes occlusive vascular disorders such as intimal hyperplasia and atherosclerosis. However, its role in vascular degenerative disease AAA remains unexplored. Herein, we determined the effect of vinpocetine on the formation of AAA as well as the intervention of pre-existing moderate AAA. AAA was induced by periaortic elastase application in C57BL/6J mice. Systemic vinpocetine treatment was applied daily via intraperitoneal injection. We showed that vinpocetine pre-treatment remarkably attenuated aneurysmal dilation assessed by diameter and volume. More importantly, vinpocetine also significantly suppressed the progression of pre-existing moderate AAA in a post-intervention model. Vinpocetine improved multiple cellular and molecular changes associated with AAA, such as elastin degradation, media smooth muscle cell depletion, collagen fibers remodeling and macrophage infiltration in aneurysmal tissues. Vinpocetine potently suppressed tumor necrosis factor-α-induced nuclear factor kappa-light-chain-enhancer of activated B cells activation and proinflammatory mediator expression in primary cultured macrophages in vitro, as well as in the aorta wall in vivo, suggesting vinpocetine conferred anti-AAA effect at least partially via the inhibition of inflammation. Taken together, our findings reveal a novel role of vinpocetine in AAA formation, development and progression. Given the excellent safety profile of vinpocetine, the present study suggests vinpocetine may be a novel therapeutic agent for AAA prevention and treatment.

Atopic dermatitis (AD) is a common inflammatory skin disorder that affects children and adults. Despite the pathology of AD involves in immune dysfunction and epidermal barrier function destruction has been found, the mechanism of immune activation and barrier damage remain largely unknown. In the present study, The TNF-α/IFN-γ-stimulated HaCaTs, organotypic AD-like 3D skin equivalents and AD-like mouse model were constructed. The mRNA, histological morphology, protein levels, cytokines were detected by real-time quantitative polymerasechain reaction (RT-qPCR), hematoxylin and eosin (HE) staining, Immunohistochemistry (IHC), immunoblotting, immunofluorescence (IF) staining, and enzyme linked immunosorbent assay (ELISA), respectively. Cell viability, cell cycle, and apoptosis were respectively calculated using a Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and flow cytometry. A dual-luciferase reporter gene system was used to investigate the relationship between miR-1294 and STAT3. Compared with the control group, the expression of miR-1294 decreased in TNF-α/IFN-γ-stimulated HaCaTs (P 0.001), AD-like skin model, and AD-like mouse model (P 0.001). Moreover, STAT3 was documented as a direct target of miR-1294. Inflammation (P 0.05) and epidermal barrier function destruction (P 0.05) in AD was suppressed by overexpression of miR-1294 but enhanced by STAT3 upregulation and its downstream NF-κB pathway. We also found miR-1294 upregulation inhibited inflammation and epidermal barrier function destruction via targeting STAT3 to suppress NF-κB pathway activation in AD.

Immunity and inflammation play critical roles in the pathogenesis of acute ischemic stroke. Therefore, immune intervention, as a new therapeutic strategy, is worthy of exploration. Here, we tested the inflammation modulator, vinpocetine, for its effect on the outcomes of stroke. For this multi-center study, we recruited 60 patients with anterior cerebral circulation occlusion and onset of stroke that had exceeded 4.5 h but lasted less than 48 h. These patients, after random division into two groups, received either standard management alone (controls) or standard management plus vinpocetine (30 mg per day intravenously for 14 consecutive days, Gedeon Richter Plc., Hungary). Vinpocetine treatment did not change the lymphocyte count; however, nuclear factor kappa-light-chain-enhancer of activated B cell activation was inhibited as seen not only by the increased transcription of IκBα mRNA but also by the impeded phosphorylation and degradation of IκBα and subsequent induction of pro-inflammatory mediators. These effects led to significantly reduced secondary lesion enlargement and an attenuated inflammation reaction. Compared to controls, patients treated with vinpocetine had a better recovery of neurological function and improved clinical outcomes during the acute phase and at 3-month follow-up. These findings identify vinpocetine as an inflammation modulator that could improve clinical outcomes after acute ischemic stroke. This study also indicated the important role of immunity and inflammation in the pathogenesis of acute ischemic stroke and the significance of immunomodulatory treatment. Clinical Trial Registration Information: www.clinicaltrials.gov . Identifier: NCT02878772

Asthma is a complex inflammatory disease of the airways and acupuncture is one of the effective therapies widely used to treat asthma in China. The aim of the study was to evaluate the regulatory role of acupuncture in airway inflammation and the hypothalamic-pituitary-adrenal (HPA) axis activity in OVA-induced murine asthma model. Our results demonstrated that acupuncture was effective in suppression of AHR, inhibition of total leukocyte, neutrophil, lymphocyte and eosinophil counts in BALF, attenuation of airway inflammation and TNF-α, IL-1β, IL-5 and eotaxin secretion. Furthermore, the HPA axis activity was also regulated by acupuncture, which included promotion of adrenocorticotropic hormone and cortisol secretion in the plasma. Our findings revealed that acupuncture could attenuate airway inflammation and regulate HPA axis and immunologic function in the OVA-induced murine asthma model, which may provide support to better understand the contribution of acupuncture to the regulation of airway inflammation and HPA axis activity in asthma.

Sepsis is a severe disease, which results from the excessive inflammatory response to the infection. Dysfunction of intestinal barrier is a crucial problem in various pathological conditions. Meanwhile, microRNAs exhibit significant roles in the modulation of many diseases, including sepsis. Multiple investigations indicate that miR-199a-5p participates in different human diseases. Nevertheless, little is known on the roles of miR-199a-5p in sepsis. Herein, we evaluated the mechanism of miR-199a-5p on the intestinal barrier dysfunction in sepsis. Intestinal mucosa permeability indicators including D-lactic acid, DAO, and FD-40 levels were determined, and they were greatly increased in sepsis. Then, we proved that miR-199a-5p was induced in sepsis mice tissues and isolated intestinal epithelial cells. Moreover, miR-199a-5p increased D-lactic acid, DAO, and FD-40 while inhibition of miR-199a-5p exhibited a reversed process. Additionally, we observed that miR-199a-5p affected the oxidative damage and inflammation in the intestine tissues from sepsis mice. The content of MDA was elevated whereas SOD was remarkably repressed in the miR-199a-5p mimic group. IL-6, IL-1β, and TNF-α were induced by miR-199a-5p overexpression while IL-10 was reduced by miR-199a-5p. Subsequently, surfactant protein D (SP-D) was predicted as the target of miR-199a-5p. The activation of NF-κB has been identified in sepsis. Herein, we demonstrated that inhibitor of miR-199a-5p contributed to IEC injury via targeting SP-D and inactivating the NF-κB pathway. These revealed miR-199a-5p exacerbated the intestinal barrier dysfunction via inhibiting SP-D and activating the NF-κB pathway in sepsis.

Sepsis and intestinal injury triggered by sepsis are common in intensive care units, which can contribute to a high mortality. lncRNAs can modulate gene expression, and they are closely involved in multiple diseases, including sepsis. In our present study, we investigated the biological function of MEG3 in sepsis, especially during the intestinal injury. Currently, we observed that in LPS-induced sepsis mouse models, the intestinal injury was triggered. Meanwhile, we reported that MEG3 was greatly decreased in vivo, with an increase of miR-129-5p and inhibition of SP-D. Then, MEG3 was overexpressed, and we found that its overexpression repressed the intestinal injury via downregulating miR-129-5p in sepsis mice. Moreover, TNF-α and IL-6 expression was elevated in intestinal tissues compared to the control groups. MEG3 restrained the activation of TNF-α and IL-6, in sepsis models. Subsequently, to induce the inflammatory injury of sepsis, human colorectal Caco2 cells were treated with 10 ng/ml LPS. 10 ng/ml LPS significantly inhibited Caco2 cell proliferation and increased the apoptosis. Additionally, MEG3 was decreased whereas miR-129-5p was obviously increased in Caco2 cells incubated with LPS. Interestingly, we showed that MEG3 repressed cell apoptosis partly and enhanced Caco2 cell proliferation. miR-129-5p overexpression could reverse the effect of MEG3 in vitro. Previously, we proved SP-D was reduced in sepsis and it depressed the intestinal injury in vivo. Finally, the correlation among MEG3, miR-129-5p, and SP-D was predicted and confirmed in our investigation. These findings indicated that MEG3 might be a potential target for intestinal damage caused by sepsis via regulating miR-129-5p and SP-D.

Sepsis is a severe clinical disease, which is resulted from the excessive host inflammation response to the infection. Growing evidence indicates that Staphylococcus aureus pneumonia is a significant cause of sepsis, which can lead to intestinal injury, inflammation, and apoptosis. Studies have shown that miR-182-5p can serve as a tumor oncogene or a tumor suppressive microRNA in various cancers, however, its biological role in sepsis is still uninvestigated. Here, we reported that miR-182-5p was obviously increased in S. aureus pneumonia mice models. Loss of miR-182-5p inhibited intestinal damage and intestinal apoptosis as indicated by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. In addition, we observed the lack of miR-182-5p altered the local inflammatory response to pneumonia in the intestine. Elevated tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were observed in intestinal tissue of pneumonia groups compared with the shams. Furthermore, miR-182-5p knockout (KO) pneumonia group demonstrated decreased levels of intestinal TNF-α and IL-6. Primary murine intestinal epithelial cells were isolated and cultured in our investigation. We exhibited downregulation of miR-182-5p repressed intestinal epithelial cells apoptosis and rescued the cell viability. Meanwhile, miR-182-5p caused elevated cell apoptosis and reduced cell proliferation. Moreover, the surfactant protein D (SP-D) binds with the bacterial pathogens and remove the pathogens and apoptotic bodies, which exhibits important roles in modulating immune responses. It was displayed in our study that SP-D was greatly decreased in pneumonia mice models. SP-D was predicted as a downstream target of miR-182-5p. These data concluded that miR-182-5p promoted intestinal injury in S. aureus pneumonia-induced sepsis via targeting SP-D.

Acetylcholine (Ach) is the key anti-inflammatory transmitter in the cholinergic anti-inflammatory pathway. In this study, critically ill patients treated with early enteral nutrition (EEN) were observed to explore whether EEN affected Ach levels and inflammation.One hundred thirteen patients were included in this prospective observational study. All patients were provided the early enteral nutrition protocol 24-48 h after admission to the intensive care unit (ICU). Blood samples were collected, and the plasma levels of Ach, cholecystokinin (CCK), and inflammatory markers (tumour necrosis factor alpha (TNF-α), interleukin 1beta (IL1-β), and IL6) were measured on Days 0, 1, 3, 5, and 7. Nutritional intervention data were recorded within one week, including the number of patients receiving nutrition, the number of days nutrition was provided, the caloric intake and protein intake, feeding intolerance and prokinetic drug administration. Other collected data included the sequential organ failure assessment score (SOFA score), the Acute Physiology and Chronic Health Evaluation (APACHE) II score, the use of mechanical ventilation (the number of patients and the duration), use of vasoactive drugs and the number of renal replacement treatments (RRT) received by each patient during their ICU stay. The primary outcome was 28-day mortality. Additionally, we analysed the correlation between plasma Ach levels and inflammation, as well as the correlation between plasma Ach and CCK levels. Moreover, a multivariate regression analysis was performed to examine the independent effects of different variables on 28-day mortality and Ach levels.The overall 28-day mortality was 28.3% (32/113). Eighty-two patients tolerated enteral nutrition. Compared with Day 0 15.6 (2.8) nmol/l, the plasma Ach level was significantly increased on Day 3 18.6 (6.7) nmol/l, Day 5 19.3 (6.2) nmol/l and Day 7 19.7 (4.3) nmol/l (p .001). Compared with Day 0176.2 (50.4) pg/ml, the plasma TNF-α level was significantly decreased on Day 3144.0 (77.4) pg/ml, Day 5127.3 (51.8) pg/ml and Day 7111.4 (42.5) pg/ml (p .05). Compared with Day 0, the plasma IL1-β level was significantly decreased on Day 7 (p .05). The plasma IL6 level was significantly decreased on Day 5 and Day 7 (p .05) compared with Day 0. Compared with Day 0, the plasma CCK level was significantly increased on Day 3, 5 and 7 (p .001). The correlation analysis revealed negative correlations between Ach levels and inflammation (p .001), and a positive correlation between CCK and Ach levels (r = 0.775, p .001). A comparison of patients who did or did not tolerate EEN revealed significant differences in the plasma levels of Ach, TNF-α, IL6 and CCK (p .05). Significant differences in plasma levels of Ach, TNF-α, IL1-β, IL6 and CCK were observed between 28-day survivors and non-survivors (p .05). The multivariate logistic regression analysis identified vasopressor support, RRT, the administration of EEN, SOFA score, APACHE II score at ICU admission and plasma Ach levels as independent determinants of 28-day mortality. Additionally, the multivariate linear regression analysis identified EEN, plasma lactate, mechanical ventilation, the SOFA score and plasma CCK levels as independent determinants of plasma Ach levels.The administration of EEN to critically ill patients contributed to the increased plasma Ach levels and decreased inflammatory markers. The effect of EEN on Ach levels is partially attributed to the increase in CCK levels. Elevated plasma Ach levels indicate a better prognosis. Clinical trials identifier: NCT03612206.

Nonalcoholic fatty liver disease (NAFLD), as a common chronic liver disorder, is prevalent in the world. Recent evidence demonstrates that the "gut-liver axis" is related well to the progression of NAFLD, which regards gut microbiota and the intestinal barrier as two critical factors correlated with NAFLD. Diammonium glycyrrhizinate (DG), a compound of the natural bioactive pentacyclic triterpenoid glycoside, is the main component of licorice root extracts. The anti-inflammatory and liver protection effects of DG have already been reported, but to date, the mechanism has not been fully elucidated. In this research, we observed that DG reduced body weight, liver steatosis, as well as hepatic inflammation in NAFLD model mice induced by a high-fat diet. Illumina sequencing of the 16S rRNA revealed that DG intervention notably altered the composition of the gut microbiota in NAFLD mice. The richness of gut microbiota was significantly increased by DG. Specifically, DG reduced the Firmicutes-to- Bacteroidetes ratio and the endotoxin-producing bacteria such as Desulfovibrio and elevated the abundance of probiotics such as Proteobacteria and Lactobacillus. DG could augment the levels of short-chain fatty acid (SCFA)-producing bacteria such as Ruminococcaceae and Lachnospiraceae and promote SCFA production. In addition, DG supplementation dramatically alleviated the intestinal low-grade inflammation. Meanwhile, DG improved the expression of tight junction proteins, the goblet cell number, and mucin secretion and sequentially enhanced the function of intestinal barrier. Collectively, the prevention of NAFLD by DG might be mediated by modulating gut microbiota and restoring the intestinal barrier.

Phosphodiesterase 4B (PDE4B) plays an important role in inflammation. Recently we have reported that roflumilast as a PDE4-selective inhibitor, synergizes with nontypeable Haemophilus influenzae (NTHi) to up-regulate PDE4B expression in vitro and in vivo. Clinical evidence and our previous results suggest that synergistic induction of PDE4B could be counterproductive for suppressing inflammation or may contribute to tolerance to roflumilast. We thus investigated if dexamethasone inhibits the synergistic induction of PDE4B by roflumilast and NTHi as well as inflammation. Here, dexamethasone markedly suppressed the synergistic induction of PDE4B in human lung epithelial cells and in vivo. We also found that dexamethasone further suppressed NTHi-induced inflammatory response in vitro and in vivo. Moreover, Compound A, as a dissociating non-steroidal glucocorticoid receptor (GR) ligand, inhibited the synergistic induction of PDE4B, thereby suggesting the requirement of dexamethasone-mediated GR activation in the suppression of PDE4B expression. Taken together, our data suggest that dexamethasone may help attenuate inflammation and tolerance through suppressing the PDE4B expression in chronic obstructive pulmonary disease (COPD) patients using roflumilast.

Inflammation is a hallmark of many important human diseases. Appropriate inflammation is critical for host defense; however, an overactive response is detrimental to the host. Thus, inflammation must be tightly regulated. The molecular mechanisms underlying the tight regulation of inflammation remain largely unknown. Ecotropic viral integration site 1 (EVI1), a proto-oncogene and zinc finger transcription factor, plays important roles in normal development and leukemogenesis. However, its role in regulating NF-κB–dependent inflammation remains unknown. In this article, we show that EVI1 negatively regulates nontypeable Haemophilus influenzae- and TNF-α–induced NF-κB–dependent inflammation in vitro and in vivo. EVI1 directly binds to the NF-κB p65 subunit and inhibits its acetylation at lysine 310, thereby inhibiting its DNA-binding activity. Moreover, expression of EVI1 itself is induced by nontypeable Haemophilus influenzae and TNF-α in an NF-κB–dependent manner, thereby unveiling a novel inducible negative feedback loop to tightly control NF-κB–dependent inflammation. Thus, our study provides important insights into the novel role for EVI1 in negatively regulating NF-κB–dependent inflammation, and it may also shed light on the future development of novel anti-inflammatory strategies.

Objective— Cyclophilin A (CyPA, encoded by Ppia ) is a proinflammatory protein secreted in response to oxidative stress in mice and humans. We recently demonstrated that CyPA increased angiotensin II (Ang II)–induced reactive oxygen species (ROS) production in the aortas of apolipoprotein E ( Apoe ) −/− mice. In this study, we sought to evaluate the role of CyPA in Ang II–induced cardiac hypertrophy. Methods and Results— Cardiac hypertrophy was not significantly different between Ppia +/+ and Ppia −/− mice infused with Ang II (1000 ng/min per kg for 4 weeks). Therefore, we investigated the effect of CyPA under conditions of high ROS and inflammation using the Apoe −/− mice. In contrast to Apoe −/− mice, Apoe −/− Ppia −/− mice exhibited significantly less Ang II–induced cardiac hypertrophy. Bone marrow cell transplantation showed that CyPA in cells intrinsic to the heart plays an important role in the cardiac hypertrophic response. Ang II–induced ROS production, cardiac fibroblast proliferation, and cardiac fibroblast migration were markedly decreased in Apoe −/− Ppia −/− cardiac fibroblasts. Furthermore, CyPA directly induced the hypertrophy of cultured neonatal cardiac myocytes. Conclusion— CyPA is required for Ang II–mediated cardiac hypertrophy by directly potentiating ROS production, stimulating the proliferation and migration of cardiac fibroblasts, and promoting cardiac myocyte hypertrophy.

Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF), have been shown to stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent data suggest that steady laminar flow decreases EC apoptosis and blocks TNF-mediated EC activation. EC apoptosis is likely important in the process termed "plaque erosion" that leads to platelet aggregation. Steady laminar flow inhibits EC apoptosis by preventing cell cycle entry, by increasing antioxidant mechanisms (e.g., superoxide dismutase), and by stimulating nitric oxide-dependent protective pathways that involve enzymes PI3-kinase and Akt. Conversely, our laboratory has identified nitric oxide-independent mechanisms that limit TNF signal transduction. TNF regulates gene expression in EC, in part, by stimulating mitogen-activated protein kinases (MAPK) which phosphorylate transcription factors. We hypothesized that fluid shear stress modulates TNF effects on EC by inhibiting TNF-mediated activation of MAP kinases. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm2) on TNF-stimulated activity of two MAP kinases: extracellular signal regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK). Flow alone stimulated ERK1/2 activity, but decreased JNK activity compared to static controls. TNF (10 ng/ml) alone activated both ERK1/2 and JNK maximally at 15 minutes in human umbilical vein EC (HUVEC). Pre-exposing HUVEC for 10 minutes to flow inhibited TNF activation of JNK by 46%, but it had no significant effect on ERK1/2 activation. Incubation of EC with PD98059, a specific mitogen-activated protein kinase kinase inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Flow-mediated inhibition of JNK was unaffected by 0.1 mM L-nitroarginine, 100 pM 8-bromo-cyclic GMP, or 100 microM 8-bromo-cyclic AMP. Transfection studies with dominant negative constructs of the protein kinase MEK1 and MEK5 suggested an important role for BMK1 in flow-mediated regulation of TNF signals. In summary, the atheroprotective effects of steady laminar flow on the endothelium involve multiple synergistic mechanisms.

Background— Oxidative stress, generated by excessive reactive oxygen species, promotes cardiovascular disease. Cyclophilin A (CyPA) is a 20-kDa chaperone protein secreted from vascular smooth muscle cells (VSMCs) in response to reactive oxygen species that stimulates VSMC proliferation and inflammatory cell migration in vitro; however, the role CyPA plays in vascular function in vivo remains unknown. Methods and Results— We tested the hypothesis that CyPA contributes to vascular remodeling by analyzing the response to complete carotid ligation in CyPA knockout mice, wild-type mice, and mice that overexpress CyPA in VSMC (VSMC-Tg). After carotid ligation, CyPA expression in vessels of wild-type mice increased dramatically and was significantly greater in VSMC-Tg mice. Reactive oxygen species–induced secretion of CyPA from mouse VSMCs correlated significantly with intracellular CyPA expression. Intimal and medial hyperplasia correlated significantly with CyPA expression after 2 weeks of carotid ligation, with marked decreases in CyPA knockout mice and increases in VSMC-Tg mice. Inflammatory cell migration into the intima was significantly reduced in CyPA knockout mice and increased in VSMC-Tg mice. Additionally, VSMC proliferation assessed by Ki67 + cells was significantly less in CyPA knockout mice and was increased in VSMC-Tg mice. The importance of CyPA for intimal and medial thickening was shown by strong correlations between CyPA expression and the number of both inflammatory cells and proliferating VSMCs in vivo and in vitro. Conclusions— In response to low flow, CyPA plays a crucial role in VSMC migration and proliferation, as well as inflammatory cell accumulation, thereby regulating flow-mediated vascular remodeling and intima formation.

Steady laminar blood flow protects vessels from atherosclerosis. We showed that flow decreased tumor necrosis factor-α (TNF)-mediated VCAM1 expression in endothelial cells (EC) by inhibiting JNK. Here, we determined the relative roles of MEK1, MEK5 and their downstream kinases ERK1/2 and BMK1 (ERK5) in flow-mediated inhibition of JNK activation. Steady laminar flow (shear stress = 12 dyn/cm2) increased BMK1 and ERK1/2 activity in EC. Pre-exposing EC for 10 min to flow inhibited TNF activation of JNK by 58%. A key role for BMK1, but not ERK1/2 was shown. (1) Incubation of EC with PD184352, at concentrations that blocked ERK1/2, but not BMK1, had no effect on flow inhibition of TNF-mediated JNK activation. (2) BIX02188, a MEK5-selective inhibitor, completely reversed the inhibitory effects of flow. These findings indicate that flow inhibits TNF-mediated signaling events in EC by a mechanism dependent on activation of MEK5–BMK1, but not MEK1–ERK1/2. These results support a key role for the MEK5–BMK1 signaling pathway in the atheroprotective effects of blood flow.

Unidirectional laminar flow is atheroprotective, in part by inhibiting cytokine-mediated endothelial cell (EC) inflammation and apoptosis. Previously, we showed that flow inhibited TNF-alpha signaling by preventing activation of JNK. Recently, PKCzeta was identified as the PKC isoform most strongly regulated by flow pattern, with increased PKCzeta activity in regions of disturbed flow versus unidirectional flow. Interestingly, PKCzeta is cleaved by caspases after TNF-alpha stimulation to generate a 50-kDa truncated form (CATzeta, catalytic domain of PKCzeta) with a higher kinase activity than the full-length protein. We hypothesized that flow would inhibit TNF-alpha-mediated PKCzeta cleavage and thereby CATzeta formation. We found that PKCzeta activity was required for TNF-alpha-mediated JNK and caspase-3 activation in ECs. PKCzeta was rapidly cleaved to generate CATzeta in cultured bovine and human aortic ECs and in intact rabbit vessels stimulated with TNF-alpha. This truncated form of PKCzeta enhanced JNK and caspase-3 activation. Interestingly, PKCzeta cleavage was prevented by inhibitors of PKCzeta, JNK, and caspase activities, suggesting that these enzymes, via regulating CATzeta formation, modulate caspase-3 activity in ECs. Finally, we found that flow reduced caspase-dependent processing of PKCzeta and caspase-3 activation. These results define a novel role for PKCzeta as a shared signaling mediator for flow and TNF-alpha, and important for flow-mediated inhibition of proinflammatory and apoptotic events in ECs.

Activating transcription factor 3 (ATF3) is an adaptive-response protein induced by various environmental stresses and is implicated in the pathogenesis of many disease states. However, the role of ATF3 SUMOylation in hypertension-induced vascular injury remains poorly understood. Here we investigated the function of ATF3 SUMOylation in vascular endothelial cells (ECs). The expression of ATF3 and small ubiquitin-like modifier 1 (SUMO1) was increased in angiotensin II (Ang II)-induced human umbilical vein endothelial cells (HUVECs). Microscopic analyses further revealed that the expression of ATF3 and SUMO1 is upregulated and colocalized in the endothelium of thoracic aortas from Ang II-induced hypertensive mice. However, Ang II-induced upregulation of ATF3 and SUMO1 in vitro and in vivo was blocked by Ang II type I receptor antagonist olmesartan. Moreover, Ang II induced ATF3 SUMOylation at lysine 42, which is SUMO1 dependent. ATF3 SUMOylation attenuated ATF3 ubiquitination and in turn promoted ATF3 protein stability. ATF3 or SUMO1 knockdown inhibited Ang II-induced expression of inflammatory molecules such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8. Wild type ATF3 but not ATF3-K42R (SUMOylation defective mutant) reduced the production of nitric oxide (NO), a key indicator of EC function. Consistently, ginkgolic acid, an inhibitor of SUMOylation, increased NO production in HUVECs and significantly improved vasodilatation of aorta from Ang II-induced hypertensive mice. Our findings demonstrated that ATF3 SUMOylation is involved in Ang II-induced EC inflammation and dysfunction in vitro and in vivo through inhibiting ATF3 ubiquitination and increasing ATF3 protein stability.

Objectives— Atherosclerosis preferentially occurs in areas of turbulent flow, whereas laminar flow is atheroprotective. Inflammatory cytokines have been shown to stimulate adhesion molecule expression in endothelial cells that may promote atherosclerosis, in part, by stimulating c-Jun N -terminal kinase (JNK) and nuclear factor (NF)-κB transcriptional activity. Methods and Results— Because Src kinase family and Src homology region 2-domain phosphatase-2 (SHP-2) may regulate JNK activation, we studied the effect of shear stress on endothelial inflammation and JNK. Human umbilical vein endothelial cells preexposed to flow showed decreased tumor necrosis factor (TNF)-α–induced c-Jun and NF-κB transcriptional activation. TNF-α–mediated JNK, c-Jun, and NF-κB activation required Src and SHP-2 activity. Shear stress significantly inhibited SHP-2 phosphatase activity without affecting TNF-α–induced Src family kinase activation. Because MEKK3 and Gab1 are critical for TNF-α–induced c-Jun and NF-κB activation, we determined the role of SHP-2 phosphatase activity in MEKK3 signaling. A catalytically inactive form of SHP-2 increased MEKK3/Gab1 interaction and inhibited MEKK3 (but not MEKK1)-mediated c-Jun and NF-κB activation. Conclusions— These results suggest that SHP-2 is a key mediator for the inhibitory effects of shear stress on TNF-α signaling in part via regulating MEKK3/Gab1 interaction, MEKK3 signaling, and subsequent adhesion molecule expression.

Background: Atherosclerosis is the major cause of cardiovascular disease and stroke, which are among the top 10 leading causes of death worldwide. Pathogen-associated molecular patterns (PAMPs) can activate toll-like receptors (TLRs) and activate nuclear factor kappa B (NFκB) signaling, a central pathway in inflamma...

Inflammation is a hallmark of many diseases, such as atherosclerosis, chronic obstructive pulmonary disease, arthritis, infectious diseases, and cancer. Although steroids and cyclooxygenase inhibitors are effective antiinflammatory therapeutical agents, they may cause serious side effects. Therefore, developing unique antiinflammatory agents without significant adverse effects is urgently needed. Vinpocetine, a derivative of the alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment. Its role in inhibiting inflammation, however, remains unexplored. Here, we show that vinpocetine acts as an antiinflammatory agent in vitro and in vivo. In particular, vinpocetine inhibits TNF-α–induced NF-κB activation and the subsequent induction of proinflammatory mediators in multiple cell types, including vascular smooth muscle cells, endothelial cells, macrophages, and epithelial cells. We also show that vinpocetine inhibits monocyte adhesion and chemotaxis, which are critical processes during inflammation. Moreover, vinpocetine potently inhibits TNF-α- or LPS-induced up-regulation of proinflammatory mediators, including TNF-α, IL-1β, and macrophage inflammatory protein-2, and decreases interstitial infiltration of polymorphonuclear leukocytes in a mouse model of TNF-α- or LPS-induced lung inflammation. Interestingly, vinpocetine inhibits NF-κB–dependent inflammatory responses by directly targeting IKK, independent of its well-known inhibitory effects on phosphodiesterase and Ca 2+ regulation. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned for the treatment of many inflammatory diseases.

Inflammation and oxidative stress are pathogenic mediators of many diseases, but molecules that could be therapeutic targets remain elusive. Inflammation and matrix degradation in the vasculature are crucial for abdominal aortic aneurysm (AAA) formation. Cyclophilin A (CypA, encoded by Ppia) is highly expressed in vascular smooth muscle cells (VSMCs), is secreted in response to reactive oxygen species (ROS) and promotes inflammation. Using the angiotensin II (AngII)-induced AAA model in Apoe-/- mice, we show that Apoe-/-Ppia-/- mice are completely protected from AngII-induced AAA formation, in contrast to Apoe-/-Ppia+/+ mice. Apoe-/-Ppia-/- mice show decreased inflammatory cytokine expression, elastic lamina degradation and aortic expansion. These features were not altered by reconstitution of bone marrow cells from Ppia+/+ mice. Mechanistic studies showed that VSMC-derived intracellular and extracellular CypA are required for ROS generation and matrix metalloproteinase-2 activation. These data define a previously undescribed role for CypA in AAA formation and suggest CypA as a new target for treating cardiovascular disease.

Shear stress–induced extracellular signal-regulated kinase (ERK)5 activation and the consequent regulation of Kruppel-like factor 2 and endothelial nitric oxide synthase expression represents one of the antiinflammatory and vascular tone regulatory mechanisms maintaining normal endothelial function. Endothelial dysfunction is a major initiator of atherosclerosis, a vascular pathology often associated with diabetes. Small ubiquitin-like modifier (SUMO) covalently attaches to certain residues of specific target transcription factors and could inhibit its activity. We investigated whether H 2 O 2 and AGE (advanced glycation end products), 2 well-known mediators of diabetes, negatively regulated ERK5 transcriptional activity and laminar flow–induced endothelial nitric oxide synthase expression through ERK5 SUMOylation. H 2 O 2 and AGE induced endogenous ERK5 SUMOylation. In addition, ERK5 SUMOylation was increased in the aortas from diabetic mice. ERK5 transcriptional activity, but not kinase activity, was inhibited by expression of Ubc9 (SUMO E2 conjugase) or PIAS1 (E3 ligase), suggesting the involvement of ERK5 SUMOylation on its transcriptional activity. Point-mutation analyses showed that ERK5 is covalently modified by SUMO at 2 conserved sites, Lys6 and Lys22, and that the SUMOylation defective mutant of ERK5, dominant negative form of Ubc9 (DN-Ubc9), and small interfering RNA PIAS1 reversed H 2 O 2 and AGE–mediated reduction of shear stress–mediated ERK5/myocyte enhancer factor 2 transcriptional activity, as well as promoter activity of Kruppel-like factor 2. Finally, PIAS1 knockdown reversed the inhibitory effect of H 2 O 2 in shear stress–induced Kruppel-like factor 2 and endothelial nitric oxide synthase expression. These data clearly defined SUMOylation-dependent ERK5 transcriptional repression independent of kinase activity and suggested this process as among the molecular mechanisms of diabetes-mediated endothelial dysfunction.

Peroxisome proliferator-activated receptors (PPAR) decrease the production of cytokine and inducible nitric-oxide synthase (iNOS) expression, which are associated with aging-related inflammation and insulin resistance. Recently, the involvement of the induction of heme oxygenase-1 (HO-1) in regulating inflammation has been suggested, but the exact mechanisms for reducing inflammation by HO-1 remains unclear. We found that overexpression of HO-1 and [Ru(CO)(3)Cl(2)](2), a carbon monoxide (CO)-releasing compound, increased not only ERK5 kinase activity, but also its transcriptional activity measured by luciferase assay with the transfection of the Gal4-ERK5 reporter gene. This transcriptional activity is required for coactivation of PPARdelta by ERK5 in C2C12 cells. [Ru(CO)(3)Cl(2)](2) activated PPARdelta transcriptional activity via the MEK5/ERK5 signaling pathway. The inhibition of NF-kappaB activity by ERK5 activation was reversed by a dominant negative form of PPARdelta suggesting that ERK5/PPARdelta activation is required for the anti-inflammatory effects of CO and HO-1. Based on these data, we propose a new mechanism by which CO and HO-1 mediate anti-inflammatory effects via activating ERK5/PPARdelta, and ERK5 mediates CO and HO-1-induced PPARdelta activation via its interaction with PPARdelta.

Bcr is a serine/threonine kinase and is highly expressed in the neointima after vascular injury. Because angiotensin II (Ang II) and inflammation play important roles in the development of intimal proliferation, we hypothesized that Bcr is an important regulator of Ang II-mediated nuclear factor (NF)-κB activation. By transfecting vascular smooth muscle cells (VSMCs) with dominant negative Bcr (DN-Bcr), we demonstrated that DN-Bcr inhibited Ang II-mediated NF-κB activation in VSMCs (82 ± 10% inhibition, n = 3, p n = 3, p p p p