The Role Of Neutrophil Elastase In The Activiation Of Kupffer Cells In Mice With Nonalcoholic Steatohepatitis And The Effect Of Berberine

Many studies including our previous studies had confirmed the central role of toll-like receptor 4(TLR4) signal pathway activation in kupffer cells. A growing body of evidence have demonstrated neutrophils infiltrate in liver as one of the key histological characteristics of nonalcoholic steatohepatitis(NASH) in both animal models and human subjects. In our studies, firstly we confirmed that neutrophils play an active role in the process of NASH via the activation of kupffer cells.Secondly, we found increased NE and its inhibitor al-antitrypsin (A1AT)is closely associated with liver Inflammation in patients with NASH and could serve as a novel marker to predict NASH in humans. Then, NE inhibitor Sivelestat was employed to delineate the function of NE in NASH and the activation of kupffer cells using reliable rodent NASH, and recombinant mouce NE was also employed to activate kupffer cells in vitro which was depengding on TLR4. At last, we observed that mice with NASH treated with berberine showed alleviated inflammation,lower NE levels and less kupffer cell activitation.Part 1 The Role of Neutrophil Elastase in the Activiation of Kupffer Cells in Mice with NASHStudy 1 Neutrophils Play a Crucial Role in the Early Stage of NASH and the Activation of Kupffer CellsObjective To investigate the role of neutrophils in the process of NASH and its underling mechanisms.Methods C57BL/6J mice were fed with either standard chow (SC) or methionine/choline-defecient (MCD) diet for 1,2,4,8weeks respectively. Anti-Ly6G antibody was employed to deplete neutrophils. Immunohistochemistry (IHC) staining and RT-PCR with CD68 and F4/80 was employed to test the activation of kupffer cells.Results MCD diet receiving mice with neutrohpil depletion had much lower serum ALT activity, liver inflammation and pro-inflammation mRNA levels in the early stage of NASH (1 and 2 weeks) when compared to non-neutrophil depleted mice. As disease proceeding (4 and 8 weeks), neurphil-depletion did not make lower serum ALT levels and liver lesions due to activation of kupffer cells.Conclusion Neutrophils play a crucial role in the early stage of NASH and the activation of kupffer cells.Study 2 The role of the NE-A1AT system in the histological progression of NAFLD Objective To investigate the role of the NE-A1AT system in the histological progression of NAFLD, and to evaluate the ability of it to predictNASH.Methods A total of 252 adults (NAFLD group, n=202; Healthy group, n=50) were recruited. Clinical biochemical characteristics, NE and A1AT concentrations were measured in all subjects. Among the NAFLD group,86 patients had previously undergone liver biopsy and information on histological characteristics was consequently available. The area under receiver operating characteristic curve (AUC) was used to determine the predictive accuracy of the NE-A1AT system for NASH.Results NAFLD patients had an elevated serum NE concentration and a reduced A1AT level with consequent NE/A1AT imbalance. NE increased in the early stage of steatosis, preceded the decline in A1AT, dating from the onset of NASH (NAS 3-4), and subsequent NE/A1AT increased in the presence of NASH. Nonetheless, this increase began to resolve as the disease state progressed to advanced fibrosis. AiAT had a sensitivity (SEN) of 83.8% and a specificity (SP) of 83.3% with the optimal cut-off of-1459.43, NE/A1AT had a SEN of 88.8% and a SP of 83.3% with cut-off of 0.363 to predict NASH.Conclusion An increased NE:A1AT ratio is closely associated with liver Inflammation in patients with NASH and could serve as a novel marker to predict NASH in humans.Study 3 Sivelestat Alleviated Inflammatory of Nonalcoholic Steatohepatitis in Mice by Reducing the Activtion of Kupffer CellsObjective To investigate the role of neutrophil elastase inhibitor sivelestat in treating non-alcoholic steatohepatitis and its underling mechanisms.Methods 4-week male C57BL/6J ApoE-/- mice were fed with Standard chow(SC) or High-fat high-cholesterol (HFIIC) diet, then each group of mice were subsequently divided into two subgroups, one was injected i.p. with sivelestat,another was injected with saline as control from 6 weeks. Serum biochemical parameters were measured, histological data were evaluated by HE and oil red O staining, and gene expression levelswere examined by Quantitative RT-PCR.Results Mice fed with HFHC diet developed typical NASH features compared to those fed with SC. Compared to mice fed with HFHC diet without sivelestat, those mice treated with neutrophil elastase inhibitor sivelestat had much lower glucose, lipid profile and ALT activity. In addition, the histological lesions including steatosis, hepatocellular ballooning and lobular inflammatory was alleviated markedly, accompanied by obvious reduction of NAS scores(5.71±1.11 vs 3.16±1.16, P< 0.05). The production of pro inflammatory cytokine monocyte chemoattractant protein (MCP-1) and tumor necrosis factor-a (TNF-a) was aslo decreased correspondingly in mice treated with sivelestat. By IHC staining and mRNA levels analysis of CD68 and F4/80, we found that kupffer cells activation was much lower in mice treated with sivelastat.Conclusion Sivelestat alleviated inflammatory of nonalcoholic steatohepatitis in mice by inhibiting the activtion of kupffer cells.Study 4 The Role of Recombinant NE in the Activation of Kuppfer CellsObjective To confirm the role of NE in the activation of kupffer cells in vitro.Methods Kupffer cells were isolated from TLR4-WT and TLR4mut mice respectively then cultured for 3 days. Cells were treated with100 nM recombinant mouse NE for 6 hours prior to RNA extraction, heated inactivated NE was used as control. Then we tested mRNA levels of MCP-1,TNF-a and IL-1β.Results ThemRNA levels of MCP-1,TNF-a and IL-1β was much higher in kupffer cells which isolated from TLR4-WT mice than they were isolated from TLR4mut mice.Conclusion Recombinant mouse NE activated kuppfer cells in vitro and it depends on TLR4.Part 2 The Role of berberine in NASH and the activation of kupffer cellsObjective To investigate the efficacy of berberine (BBR) in NASH and its role in the activation of kupffer cells which depending on NE-TLR4.Methods 8-week male C57BL/6J ApoE-/- mice were divided into 3 groups:Standard chow(SC) diet+drinking water gavege; High-fat high-cholesterol (HFHC) diet+dringking water gavage, HFHC diet+BBR gavege (200mg/kg). After 12 weeks, all mice were executed.Serum biochemical parameters were measured, histological data were evaluated by HE and oil red O staining, and gene expression levelswereexamined by Quantitative RT-PCR.Results Mice fed with HFHC diet developed typical NASH features compared to those fed with SC. Mice treated with BBR had lower ALT, AST, and TC levels when compared to HFHC diet singlely. BBR lowered the steatosis, lobular inflammation, ballooning and the pro-inflammation levels as well. NE concentration and activities was also lowered in liver treated with BBR, which was consistently with kupffer cell activation.Conclusion BBR alleviates NASH depending on NE-TLR4 pathway which mediates the activation of kupffer cells.