MicroRNA-363-3p/sphingosine-1-phosphate receptor 1 axis inhibits sepsis-induced acute lung injury via the inactivation of nuclear factor kappa-B ligand signaling

Infection-associated inflammation and coagulation are critical pathologies in sepsis-induced acute lung injury (ALI). This study aimed to investigate the effects of microRNA-363-3p (miR-363-3p) on sepsis-induced ALI and explore the underlying mechanisms. A cecal ligation and puncture-induced septic mouse model was established. The results of this study suggested that miR-363-3p was highly expressed in lung tissues of septic mice. Knockdown of miR-363-3p attenuated sepsis-induced histopathological damage, the inflammation response and oxidative stress in lung tissues. Furthermore, knockdown of miR-363-3p reduced the formation of platelet-derived microparticles and thrombin generation in blood samples of septic mice. Downregulation of miR-363-3p suppressed sphingosine-1-phosphate receptor 1 (S1PR1) expression in lung tissues and subsequently inactivated the nuclear factor kappa-B ligand (NF-κB) signaling. A luciferase reporter assay confirmed that miR-363-3p directly targeted the 3’-untranslated region of the mouse S1pr1 mRNA. Collectively, our study suggests that inactivation of NF-κB signaling is involved in the miR-363-3p/S1PR1 axis-mediated protective effect on septic ALI.     

Study on the Mechanism of BMSCs in Regulating NF-κB Signal Pathway by Targeting miR-449a to Improve the Inflammatory Response to Peripheral Nerve Injury

Objective:To evaluate the mechanism of Bone Marrow Mesenchymal Stem Cells (BMSCs) in regulating NF-κB signal pathway by targeting miR-449a.Methods:Stem cells were transfected by over-expressing and inhibiting miR-449a to detect the levels and viability of miR-449a in stem cells after transfection. Stem cells and neurons were co-cultured in vitro to evaluate the in vitro mechanism of stem cells over-expressing miR-449a on neurons.Results:After the addition of neurons, the neuronal activity of miR-449a over-expression group increased significantly, the expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, and the inflammatory cytokines (TNF-α and IL-1β) decreased significantly (P<0.05). In vivo experiments in rats also showed that rats were unresponsive, did not chirp or elude after being stimulated. After stem cell therapy, the weight and response of rats gradually returned to normal levels. miR-449a expression significantly increased in the stem cell + miR-449a over-expression group, expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, inflammatory cytokines (TNF-α and IL-1β) significantly decreased, and cell activity significantly increased (P<0.05).Conclusions:BMSCs can modulate NF-κB signaling pathway by targeting miR-449a, so as to reduce the inflammatory response to peripheral nerve injury and repair nerve injury.     

Protective effects of the notoginsenoside R1 on acute lung injury by regulating the miR-128-2-5p/Tollip signaling pathway in rats with severe acute pancreatitis

Notoginsenoside R1 (NG-R1), the extract and the main ingredient of Panax notoginseng, has anti-inflammatory effects and can be used in treating acute lung injury (ALI). In this study, we explored the pulmonary protective effect and the underlying mechanism of the NG-R1 on rats with ALI induced by severe acute pancreatitis (SAP). MiR-128-2-5p, ERK1, Tollip, HMGB1, TLR4, IκB, and NF-κB mRNA expression levels were measured using real-time qPCR, and TLR4, Tollip, HMGB1, IRAK1, MyD88, ERK1, NF-κB65, and P-IκB-α protein expression levels using Western blot. The NF-κB and the TLR4 activities were determined using immunohistochemistry, and TNF-α, IL-6, IL-1β, and ICAM-1 levels in the bronchoalveolar lavage fluid (BALF) using ELISA. Lung histopathological changes were observed in each group. NG-R1 treatment reduced miR-128-2-5p expression in the lung tissue, increased Tollip expression, inhibited HMGB1, TLR4, TRAF6, IRAK1, MyD88, NF-κB65, and p-IκB-α expression levels, suppressed NF-κB65 and the TLR4 expression levels, reduced MPO activity, reduced TNF-α, IL-1β, IL-6, and ICAM-1 levels in BALF, and alleviated SAP-induced ALI. NG-R1 can attenuate SAP-induced ALI. The mechanism of action may be due to a decreased expression of miR-128-2-5p, increased activity of the Tollip signaling pathway, decreased activity of HMGB1/TLR4 and ERK1 signaling pathways, and decreased inflammatory response to SAP-induced ALI. Tollip was the regulatory target of miR-128-2-5p.     

Inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway

HMGB1 is a highly conserved nuclear protein that is rapidly released into the extracellular environment during infection or tissue damage. In osteoarthritis, HMGB1 acts as a pro-inflammatory cytokine inducing a positive feedback loop for synovial inflammation and cartilage degradation. The aim of this study was to explore the role of HMGB1 in inflammation and catabolism of temporomandibular joint osteoarthritis (TMJOA) and whether inhibition of HMGB1 affects TMJOA. Human synovial fibroblasts were incubated with HMGB1, the expression of pro-inflammatory cytokines and catabolic mediators were measured by Western blot and ELISA. NF-κB signaling pathway involvement was studied by the NF-κB inhibitor and detected by Western blotting and immunofluorescence staining. TMJOA was induced by an injection of complete Freund’s adjuvant (CFA) into anterosuperior compartment of rat’s joint. An anti-HMGB1 antibody was used to assess the effect to HMGB1 in the synovium and cartilage of the CFA-induced TMJOA rats by hematoxylin and eosin, Safranin O, Masson trichrome staining, immunohistochemistry and immunofluorescence. HMGB1 markedly increased the production of MMP13, ADAMTS5, IL-1β and IL-6 through activating NF-κB signaling pathway in human synovial fibroblasts. In vivo, application of the HMGB1 neutralizing antibody effectively ameliorated the detrimental extent of TMJOA. Furthermore, the HMGB1 neutralizing antibody reduced the expression of NF-κB, pro-inflammatory cytokines and catabolic mediators in the synovium and cartilage of CFA-induced TMJOA rats. HMGB1 inhibition alleviates TMJOA by reducing synovial inflammation and cartilage catabolism possibly through suppressing the NF-κB signaling pathway and may become a therapeutic method against TMJOA.Key words: HMGB1 neutralizing antibody, temporomandibular joint osteoarthritis, inflammation, catabolism, NF-κB     

Effect of Carbon Monoxide-Releasing Molecules II-liberated CO on Suppressing Inflammatory Response in Sepsis by Interfering with Nuclear Factor Kappa B Activation

Sepsis continues to be a challenge in clinic. The rates of mortality in sepsis patients remain high. The present study aimed to investigate the effects and the underlying mechanisms of carbon monoxide-releasing molecules II (CORM-2)-liberated CO on suppressing inflammatory response in sepsis. It was shown that treatment of septic mice with CORM-2 attenuated PMN accumulation, downregulated cytokines production, inhibited expressions of iNOS and NF-κB activity in the lung and liver. In parallel, CORM-2 prevented activation of NF-κB in LPS-stimulated HUVEC. This was accompanied by a decrease in ROS and NO production, expression of ICAM-1 and subsequent PMN adhesion to HUVEC. These findings demonstrated that CORM-released CO attenuates inflammatory responses by interfering with NF-κB activation and therefore decreasing the expression of ICAM-1 and NO production, attenuating the oxidative stress and inflammation in sepsis.     

Activation of Nuclear Factor Kappa B in the Hepatic Stellate Cells of Mice with Schistosomiasis Japonica

Schistosomiasis japonica is a serious tropical parasitic disease in humans, which causes inflammation and fibrosis of the liver. Hepatic stellate cells (HSCs) are known to play an important role in schistosome-induced fibrosis, but their role in schistosome-induced inflammation is still largely unknown. Here, we use a murine model of schistosomiasis japonica to investigate the role that nuclear factor kappa B (NF-κB), a critical mediator of inflammatory responses, plays in schistosome-induced inflammation. We revealed that NF-κB was significantly activated in HSCs at the early stage of infection, but not at later stages. We also show that the expression levels of several chemokines regulated by NF-κB signaling (Ccl2, Ccl3 and Ccl5) were similarly elevated at early infection. TLR4 signaling, one of the strongest known inducers of NF-κB activation, seemed not activated in HSCs post-infection. Importantly, we found that levels of miR-146 (a known negative regulator of NF-κB signaling) in HSCs opposed those of NF-κB signaling, elevating at later stage of infection. These results indicate that HSCs might play an important role in the progression of hepatic schistosomiasis japonica by linking liver inflammation to fibrosis via NF-κB signaling. Moreover, our work suggests that miR-146 appeared to regulate this process. These findings are significant and imply that manipulating the function of HSCs by targeting either NF-κB signaling or miR-146 expression may provide a novel method of treating hepatic schistosomiasis japonica.     

Abietic acid attenuates sepsis-induced lung injury by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway to inhibit M1 macrophage polarization

Lung injury is one of the leading causes of death in sepsis. Abietic acid (AA) has demonstrated anti-inflammatory and bacteriostatic properties. Herein, we established a mouse model of sepsis by cecal ligation and puncture, and intraperitoneally injected AA to treat. Lung injury was assessed by H&E staining and the inflammation in bronchoalveolar lavage fluid (BALF) were assessed by counting the number of inflammatory cells and detecting the content of inflammatory factors. Meanwhile, we also designed to study the effect of AA on lipopolysaccharide (LPS)-induced inflammatory response and macrophage marker gene expression in RAW264.7 cells in vitro. In this study, we found that AA inhibited LPS-induced secretion of inflammatory mediators (IL-1β, TNF-α, IL-6 and MIP-2), and decreased the expression of M1 macrophage e markers (CD16 and iNOS) and p-p65 protein, while increased the expression of M2 macrophage markers (CD206 and Arg-1) in RAW264.7 cells in vitro. In vivo, the therapy of AA not only rescued septic animals, but also attenuated lung injury in sepsis mice. Moreover, AA decreased the number of total cells, neutrophils and macrophages, the conceration of total protein, and the levels of inflammatory mediators in BALF of sepsis mice. Further, we found that AA inhibited M1 macrophage polarization and blocked nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in BALF of sepsis mice. In conclusion, Abietic acid attenuates sepsis-induced lung injury, and its mechanism may be related to reducing inflammation by inhibiting NF-κB signaling to inhibit M1 macrophage polarization.     

MiR-146b protect against sepsis induced mice myocardial injury through inhibition of Notch1

Myocardial dysfunction is a major cause of death in sepsis. MicroRNA-146b (miR-146b) has been reported to be related to myocardial disease. However, the role of miR-146b in sepsis as well as myocardial injury is still unclear. Septic cardiac dysfunction in mice was induced by cecal ligation and puncture (CLP) and miR-146b was found increased significantly in the myocardium tissue of CLP mice. It was found that up-regulation of miR-146b by agomiR injection suppressed expression of IL-1β in mice as well as myocardium apoptosis in CLP mice. However, suppression of miR-146b by antagomiR injection had inverse effects. Notch1 was identified as a target gene of miR-146b by bioinformatics analysis. And it was verified that in cardiomyocytes, the decrease of miR146b led to increase of both the mRNA and protein level of Notch1 and vice versa. In septic mice serum stimulated cardiomyocytes, up-regulation of miR-146b decreased the level of Notch1 and Hes1. The knockout of Notch1 in transgenic mice showed that the deficiency of Notch1 improved myocardial injury induced by CLP operation. The apoptosis of cardiomyocytes was relieved and the expression of IL-1β was decreased. In conclusion, miR-146b targets to Notch1 and protected cardiomyocytes against inflammation and apoptosis.     

Anisomycin protects against sepsis by attenuating IκB kinase-dependent NF-κB activation and inflammatory gene expression

Anisomycin is known to inhibit eukaryotic protein synthesis and has been established as an antibiotic and anticancer drug. However, the molecular targets of anisomycin and its mechanism of action have not been explained in macrophages. Here, we demonstrated the anti-inflammatory effects of anisomycin both in vivo and in vitro. We found that anisomycin decreased the mortality rate of macrophages in cecal ligation and puncture (CLP)- and lipopolysaccharide (LPS)-induced acute sepsis. It also declined the gene expression of proinflammatory mediators such as inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β as well as the nitric oxide and proinflammatory cytokines production in macrophages subjected to LPS-induced acute sepsis. Furthermore, anisomycin attenuated nuclear factor (NF)-κB activation in LPS-induced macrophages, which correlated with the inhibition of phosphorylation of NF-κB-inducing kinase and IκB kinase, phosphorylation and IκBα proteolytic degradation, and NF-κB p65 subunit nuclear translocation. These results suggest that anisomycin prevented acute inflammation by inhibiting NF-κB-related inflammatory gene expression and could be a potential therapeutic candidate for sepsis.     

Chronic Alcohol-Induced microRNA-155 Contributes to Neuroinflammation in a TLR4-Dependent Manner in Mice

Introduction

Alcohol-induced neuroinflammation is mediated by pro-inflammatory cytokines and chemokines including tumor necrosis factor-α (TNFα), monocyte chemotactic protein-1 (MCP1) and interleukin-1-beta (IL-1β). Toll-like receptor-4 (TLR4) pathway induced nuclear factor-κB (NF-κB) activation is involved in the pathogenesis of alcohol-induced neuroinflammation. Inflammation is a highly regulated process. Recent studies suggest that microRNAs (miRNAs) play crucial role in fine tuning gene expression and miR-155 is a major regulator of inflammation in immune cells after TLR stimulation.

Aim

To evaluate the role of miR-155 in the pathogenesis of alcohol-induced neuroinflammation.

Methods

Wild type (WT), miR-155- and TLR4-knockout (KO) mice received 5% ethanol-containing or isocaloric control diet for 5 weeks. Microglia markers were measured by q-RTPCR; inflammasome activation was measured by enzyme activity; TNFα, MCP1, IL-1β mRNA and protein were measured by q-RTPCR and ELISA; phospho-p65 protein and NF-κB were measured by Western-blotting and EMSA; miRNAs were measured by q-PCR in the cerebellum. MiR-155 was measured in immortalized and primary mouse microglia after lipopolysaccharide and ethanol stimulation.

Results

Chronic ethanol feeding up-regulated miR-155 and miR-132 expression in mouse cerebellum. Deficiency in miR-155 protected mice from alcohol-induced increase in inflammatory cytokines; TNFα, MCP1 protein and TNFα, MCP1, pro-IL-1β and pro-caspase-1 mRNA levels were reduced in miR-155 KO alcohol-fed mice. NF-κB was activated in WT but not in miR-155 KO alcohol-fed mice. However increases in cerebellar caspase-1 activity and IL-1β levels were similar in alcohol-fed miR-155-KO and WT mice. Alcohol-fed TLR4-KO mice were protected from the induction of miR-155. NF-κB activation measured by phosphorylation of p65 and neuroinflammation were reduced in alcohol-fed TLR4-KO compared to control mice. TLR4 stimulation with lipopolysaccharide in primary or immortalized mouse microglia resulted in increased miR-155.

Conclusion

Chronic alcohol induces miR-155 in the cerebellum in a TLR4-dependent manner. Alcohol-induced miR-155 regulates TNFα and MCP1 expression but not caspase-dependent IL-1β increase in neuroinflammation.     

MicroRNA-449b-5p targets HMGB1 to attenuate hepatocyte injury in liver ischemia and reperfusion

MicroRNAs (miRNAs) participate in the pathological process of liver ischemia/reperfusion (I/R) injury. MiR-449b-5p is the target miRNA of high mobility group box 1 (HMGB1). Its role and molecular mechanism in liver I/R injury remain unidentified. In this study, we found a protective effect of miR-449b-5p against hepatic I/R injury. HMGB1 expression significantly increased, whereas miR-449b-5p dramatically decreased in patients after liver transplant and in L02 cells exposed to hypoxia/reoxygenation (H/R). A dual-luciferase reporter assay confirmed the direct interaction between miR-449b-5p and the 3′ untranslated region of HMGB1 messenger RNA. We also found that overexpression of miR-449b-5p significantly promoted cell viability and inhibited cell apoptosis of L02 cells exposed to H/R. Moreover, miR-449b-5p repressed HMGB1 protein expression and nuclear factor-κB (NF-κB) pathway activation in these L02 cells. In an in vivo rat model of hepatic I/R injury, overexpression of miR-449b-5p significantly decreased alanine aminotransferase and aspartate aminotransferase and inhibited the HMGB1/NF-κB pathway. Our study thus suggests that miR-449b-5p alleviated hepatic I/R injury by targeting HMGB1 and deactivating the NF-κB pathway, which may provide a novel and promising therapeutic target for hepatic I/R injury.     

MiRNA-891a-5p mediates HIV-1 Tat and KSHV Orf-K1 synergistic induction of angiogenesis by activating NF-κB signaling

Co-infection with HIV-1 and Kaposi''s sarcoma-associated herpesvirus (KSHV) is the cause of aggressive AIDS-related Kaposi''s sarcoma (AIDS-KS) characterized by abnormal angiogenesis. The impact of HIV-1 and KSHV interaction on the pathogenesis and extensive angiogenesis of AIDS-KS remains unclear. Here, we explored the synergistic effect of HIV-1 Tat and KSHV oncogene Orf-K1 on angiogenesis. Our results showed that soluble Tat or ectopic expression of Tat enhanced K1-induced cell proliferation, microtubule formation and angiogenesis in chorioallantoic membrane and nude mice models. Mechanistic studies revealed that Tat promoted K1-induced angiogenesis by enhancing NF-κB signaling. Mechanistically, we showed that Tat synergized with K1 to induce the expression of miR-891a-5p, which directly targeted IκBα 3′ untranslated region, leading to NF-κB activation. Consequently, inhibition of miR-891a-5p increased IκBα level, prevented nuclear translocation of NF-κB p65 and ultimately suppressed the synergistic effect of Tat- and K1-induced angiogenesis. Our results illustrate that, by targeting IκBα to activate the NF-κB pathway, miR-891a-5p mediates Tat and K1 synergistic induction of angiogenesis. Therefore, the miR-891a-5p/NF-κB pathway is important in the pathogenesis of AIDS-KS, which could be an attractive therapeutic target for AIDS-KS.     

Gprc5a-deficiency confers susceptibility to endotoxin-induced acute lung injury via NF-κB pathway

Susceptibility to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) varies greatly among patients in sepsis/septic shock. The genetic and biochemical reasons for the difference are not fully understood. G protein coupled receptor family C group 5 member A (GPRC5A), a retinoic acid target gene, is predominately expressed in the bronchioalveolar epithelium of lung. We hypothesized that Gprc5a is important in controlling the susceptibility to ALI or ARDS. In this study, we examined the susceptibility of wild-type and Gprc5a-knockout (ko) mice to induced ALI. Administration of endotoxin LPS induced an increased pulmonary edema and injury in Gprc5a-ko mice, compared to wild-type counterparts. Consistently, LPS administration induced higher levels of inflammatory cytokines (IL-1β and TNFα) and chemokine (KC) in Gprc5a-ko mouse lungs than in wild-type. The enhanced pulmonary inflammatory responses were associated with dysregulated NF-κB signaling in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs. Importantly, selective inhibition of NF-κB through expression of the super-repressor IκBα in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs alleviated the LPS-induced pulmonary injury, and inflammatory response. Thus, Gprc5a is critical for lung homeostasis, and Gprc5a deficiency confers the susceptibility to endotoxin-induced pulmonary edema and injury, mainly through NF-κB signaling in bronchioalveolar epithelium of lung.