Regulatory Role And Mechanisms Of NLRC5 In Controlling Inflammatory Cytokines Release And Fibrotic Response In Hepatic Stellate Cells Through NF-κB And TGF-β1/Smad Signal Pathway

The liver has important physiological functions. Recent years studies have shown that a variety of cytokines are involved in the onset process of chronic liver disease, such as: viral hepatitis, alcoholic liver damage, fatty liver, hepatocellular carcinoma, etc. The important process is that the influence of damage factor can activate liver Kupffer cells(KC) to release a large number of cytokines, through regulating hepatic stellate cells(HSC) in the relevant signal pathways, then promote the activation of HSC cells proliferation and excessive deposition in the liver, which is mainly composed of collagen synthesis of ECM, cause liver pathological change, finally develop into liver cirrhosis and even liver cancer. A study found that HSC may play an important role in the process of liver inflammation. It was well known that virus infection, drug and so on can lead to liver damage. After this process, HSC secrete a variety of cytokines, such as IL-1, IL-6 and IL-10 etc. These cytokines can be recognised by HSC membrane receptor specificity, then activate some within the nuclear transcription factors by intracellular signaling transduction pathways, promoting transcription and expression of the related target gene. Meanwhile, activation of HSC can produce a large number ofproinflammatory factor and pro-fibrosis factor and through paracrine and autocrine pathway, further stimulate inactive into HSC into muscle fibroblasts, which make liver fibrosis form positive feedback. Inflammation was regard as a factor, which can start and form liver fibrosis. Therefore, Inhibition of inflammation helps to prevent the occurrence of liver fibrosis. Moreover, HSC is mainly considered as the effector cell of the liver fibrosis, but its role in the inflammatory response is not clear. In view of the above research foundation, further explore the molecular mechanism of inflammatory response in HSC, help to find new targets and strategies, which can be used to stem liver inflammation, and further block or reverse liver fibrosis.In recent years, studies have shown that the activation of HSC is the key in the formation of liver fibrosis, and the key regulating factor of the process is the nuclear factor kappa B(NF-κB). HSCs from static to activate often were accompanied by the NF-κB. In recent years, a large number of researches have shown that the inhibiting NF-κB drug can be used to reverse the experimental liver fibrosis, which can induce HSC apoptosis. Therefore, it became a new hot spot of anti-fibrosis treatment. When NF-κB activity was activated in HSCs of liver fibrosis, inflammatory factors: TNF-α and IL-6 expression were increased, enlarging liver inflammation injury response. The activity of NF-κB can reduce HSC apoptosis, promote the activation, proliferation and move. The NF-κB can inhibit a variety of types of cell apoptosis, and active in the state to activate HSCs persist. Therefore, inhibiting the NF-κB activity can promote the apoptosis of HSCs, which will likely become a new strategy to reverse liver fibrosis and have a significant impact. Meanwhile, NF-κB promote the inflammatory transcription factor expression, has become the key control factors of a link process of inflammation-fibrosis-tumor. In inflammation and fibrosis process, NF-κB plays an important role in the formation process. It could be a important target to find its endogenous regulation factor for the prevention and control liver inflammation and fibrosis. Recent literatures reported that the protein molecules NLRC5, one member ofthe natural immune system receptor(NOD-like receptor, NLR) family, is involved in suppressing a kind of very important compounds of the NF-κB signaling pathway in innate immune cells, which can effectively regulate the activity of immune cells, preventing sustained inflammation caused by damage to the body itself. In addition, the previous research found that NLRC5 can negative regulate the secretion of proinflammatory factor IL-6, TNF-α in RAW264.7 cells. But the function of NLRC5 regulation in liver inflammation and fibrosis has not been reported, particularly for NF-κB signaling pathway and the related factors of inflammation related function in HSC. So this study focuses on the role of NLRC5 in liver inflammation and fibrosis, especially the role of HSC proliferation, providing a new target for preventing liver fibrosis, to clarify the related molecular mechanism of liver fibrosis formation, research liver fibrosis therapy drug, reduce the happening of liver cirrhosis and primary hepatocellular carcinoma(HCC), which is of great significance. The main content is contained as follows:1. NLRC5 expression in fibrotic liver tissueHuman liver hemangioma tissue was chose as normal liver group. The tissue(2 cm beyond cancer tissue) of liver cirrhosis adjacent hepatitis patients and HCC patients was chose as liver fibrosis group. Another mouse liver fibrosis model was induced by CCl4. Immunohistochemistry, RT-PCR and Western blot were used to detect the expression of NLRC5 gene and protein. In addition, immunosuppressive double staining method was used to observe localization of NLRC5. The results showed that compared with normal liver group, NLRC5 gene and protein significantly were increased in human fibrotic liver tissue, and mainly was expressed in HSC.2. Effect of NLRC5 on HSC activation and secretion inflammatory cytokines in LX-2 cells.In vitro experiments with human-derived portion of LX- 2 cells were used for the study. The result showed that compared with the non-induced group, TGF-β1 increased NLRC5 m RNA and protein expression levels in LX-2 cells, indicating that NLRC5 was involved in HSC activation, proliferation and fibrosis process. NLRC5-si RNA and Scrambled-si RNA were transfected into the LX-2 cells by LipofectamineTM2000 respectively. Real-time q PCR and Western blot analysis showed that, after NLRC5 was blocked specifically, HSC activation marker α-SMA was significantly decreased. Further studies showed that the Colla-1 expression level can be inhibited by NLRC5-si RNA. p EGFP-C2-NLRC5 and p EGFP-C2 were transfected by LipofectamineTM2000 into LX-2 cells, the NLRC5 over-expression group have significantly performance contrast to the silence group ’s results. The above result indicated that NLRC5 may promote the fibrosis progress. Furthermore, we observed whether NLRC5 can regulate secretion inflammatory cytokines in LX-2 cells. After treating LX-2 cells with TNF-α, the expression level of NLRC5 was significantly increased. When LX-2 cells were stimulated by TNF-α, IL-1β, and IL-6 expression levels were significantly increased. NLRC5 silencing inhibited the proliferation of LX-2 cells. In addition, IL-1β and IL-6 expression were significantly decreased. On the other hand, we transfected p EGFP-C2-NLRC5 and p EGFP-C2 by LipofectamineTM2000 respectively, the over-expression NLRC5 group result was contrast the performance of the silence group’s.3. Effect of NLRC5 on cell proliferation, cycle and apoptosis in LX-2 cellsMTT method results showed that compared to normal LX-2 cells, the proliferation of LX-2 cells pretreated with NLRC5-si RNA was significantly inhibited. In addition, flow cytometry results showed that the apoptosis of LX-2 cells pretreated NLRC5-si RNA was significantly improved, apoptosis related protein(DR4, DR5, and Caspase-3 protein) expression were increased significantly.4. Effect of NLRC5 on NF-κB and TGF-β1/Smad signaling pathway.Firstly, we studied the regulating role of NLRC5 in NF-κB signaling pathway. After LX-2 cells were stimulated by TGF-β1, Luciferase assay result showed that NF-κB signaling pathway is negatively regulated by NLRC5. By extracting the cytoplasmic protein and nucleo protein, Western blot result showed that transfect LX-2 cells with NLRC5-si RNA by LipofectamineTM2000, the expression level of nucleus P65 protein in the NLRC5-si RNA group was significantly higher than control group. The similar result was also observed by immunostaining method. LX-2 cells proliferation can be regulated by NLRC5 and its function was related with NF-κB signaling pathway. After use of NF-κB inhibitor PDTC, NLRC5 was found obviously decrease in the the TGF-β1 group, suggesting that NF-κB signaling pathway can produce NLRC5. Therefore, NLRC5 with the NF-κB signaling pathway can form a negative feedback loop. Furthermore, we study the role of NLRC5 for NF-κB signaling pathway, when secretion inflammatory cytokines of LX-2 cells was induced by TNF-α. The results showed that NF-κB signaling pathway significantly was active in LX-2 cells induced by TNF-α. The expression of NF-κB can be significantly activated by NLRC5-si RNA. NF-κB signaling pathway is negatively regulated by NLRC5. This finding has been further confirmed by Western blot. The study further examined the role of TGF-β1/Smad signaling pathway in NLRC5 regulation process and found that p-Smad2 and P-Smad3 can be inhibited by NLRC5 silencing. In conclusion, NLRC5 can regulate secretion inflammatory cytokines of LX-2 cells and its mechanism was related to NF-κB signaling pathway. Moreover, when NLRC5 participate in liver fibrosis, NF-κB signaling pathway can be negatively regulated by NLRC5 and TGF-β1/Smad signaling pathway can be positively regulated by NLRC5.