| Background and aims: Morbidity of alcoholic chronic panreatitis(ACP) is becoming more and more high. Mechanism of ACP has attracted more and more attention. Recent studies have shown that lipopolysaccharide(LPS) play an important role in the development of various diseases. LPS could induce multiple organ injury by binding with pattern recognition receptor TLR4 and producing serious inflammatory reaction. However the effect and mechanism of LPS-TLR4 pathway on ACP has not been fully clarified. The purpose of this study was to elucidate the role and mechanism of LPS-TLR4 on alcohol-related pancreatic fibrosis in alcoholic Sprague-Dawley(SD) rat model and pancreatic stellate cell(PSC) biology.Methods: Forty-eight male SD rats(120±40g) were randomly divided into 2 groups and fed with Lieber-De Carli alcohol diet and control diet individually for 10 weeks. At beginning of 8th week, the two group rats were further divided into four groups including control diet group, control diet plus LPS group, alcoholic diet group and alcoholic diet plus LPS group. On the 6th day of eighth week. both control diet plus LPS and alcoholic diet plus LPS groups were injected with LPS(3 mg/kg) once per week for three weeks. PSC cell-line(RP-2 cell) were used in this study. HE staining was used to determine the severity of pancreatic injury. Aniline blue staining method was to evaluating the degree of pancreatic fibrosis. FSP-1 and F4/80 were detected by immunohistochemical method to recognize PSC and macrophage in pancreatic tissues. The expressions of TGF-β1 and TLR4 in PSCs or macrophages were detected by double immunofluorescence staining method. ELISA was used to detect the content of Collagen 1A1 of pancreas. q RT-PCR was used to detect the levels of TNF-α, IL-1, IL-6, MCP-1 and RANTES m RNAs. In vitro stuty, Western blot was used to detect Collagen I, Bambi, p Smad, p65 and p50 proteins. RT-q PCR was used to detect the levels of Bambi and Collagen I m RNAs.Results: In vivo study showed that LPS could enhance the severity of pancreatic inflammation and fibrosis in the rats fed with either alcoholic diet or control diet and that LPS could induce the proliferation of PSCs and macrophages as well as over-expression of TGF-β1 in macrophages and that LPS could increase the levels TNF-ɑ, IL-6 MCP-1 and RANTES. In vitro studies indicated that LPS was able to enhance the production of collagen I m RNA and protein in TGF-β1-treated RP-2 cells, the mechanism of which was that LPS could reduce the production of Bambi m RNA and protein which was able to modify TGF-β signals by acting as a pseudo-receptor. The results also showed that LPS binding with TLR4 could in turn active My D88 dependent NF-κB signaling but couldn’t influence TGF-β/Smad signaling. The LPS-TLR4-My D88-NF-k B signaling pathway might be associated with RP-2 cell-mediated cytokine and collagen I production.Conclusion: LPS is a key player in the fibrogensis of ACP. LPS is able to activate PSC and macrophage to produce inflammatory factors and collagen I. LPS-TLR4 pathway enhance TGF-β1-induced collagen I production in PSCs by inhibiting Bambi expression. Therefore, it might be an important pathway for anti-pancreatic fibrosis by inhibiting LPS-TLR4 pathway. |
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