Experimental Study Of Enterogenous And Exogenous Endotoxins On Alcohol-Induced Rat Pancreatic Fibrosis

Pancreatic fibrosis is a key pathological feature of alcoholic chronic pancreatitis(ACP).Pancreatic stellate cell(PSC)play a critical role in the fibrogenesis of ACP.Quiescent PSCs are activated by ethanol,its metabolites and reactive oxidation species resulting in their conversion into myofibroblastlike cells that express the cytoskeletal protein alpha-smooth muscle actin(α-SMA),have the capacity to proliferate,synthesize extracellular matrix(ECM)proteins including type I collagen(Col1).The fibrogenic process of ACP is believed to be initiated by a cytokine-based interplay between activated macrophage(Mφ)and PSC that followed alcohol-induced tissue injury.The fact that only 5-10% of alcoholics eventually develop overt pancreatitis indicates that ethanol alone is insufficient to lead to clinical pancreatitis.Both rodent animal models and human research have shown that alcohol consumption induces intestinal dysbiosis and hyperpermeability resulting in endotoxemia.Over the last decade,bacterial endotoxin lipopolysaccharide(LPS)has been recognized as a trigger of fibrogenesis and PSC activation during ACP.Recently we showed enhanced levels of chemotactic cytokines including monocyte chemotactic protein 1(MCP-1),macrophage inflammatory protein-1 alpha(MIP-1α),regulated upon activation normal T cell expressed and secreted factor(RANTES)and profibrogenic factor transforming growth factor β1(TGF-β1)in the sera of ACP patients with endotoxemia.In vitro studies our results showed that LPS activates Toll-like receptor(TLR4)on macrophages and PSCs that produce chemotactic cytokines(MCP-1,MIP-1α and RANTES)and profibrogenic TGF-β1.Despite the known detrimental effects of LPS in the pathogenesis of ACP,there still exist limitations for exogenous LPS use to evaluate the role of endotoxin in the pathogenesis of ACP.Currently it is still not clear whether endotoxin is an initiating event or an aggravating factor or both in the pathogenesis of pancreatic fibrosis in ACP.Additionally the interplay between Mφs and PSCs as well as the molecular link between pancreatic inflammation and fibrogenesis in ACP need to be further clarified.In this study we firstly investigated the relationship between enterogenous endotoxin in portal vein and pancreatic Col1 content in chronic alcohol(ALC)-fed rats.Secondly,we designed to determine mechanisms of ACP by which LPS enhances TGF-β1 signaling and pancreatic fibrosis using an in vivo model of chronic ALC feeding challenged with repeated LPS injection and in vitro Mφ and PSC models.Our study could be divided into two parts:Part 1: Role of Enterogenous Endotoxin on Type I Collagen Production in Chronic ALC-Fed Rat PancreasPURPOSE: The aim of this study was to assess the relationship between the concentration of LPS in the portal vein and pancreatic Col1 content in chronic ALC-fed rats.METHODS: Sixty-six male Sprague-Dawley rats were randomly divided into two groups and fed with Lieber-De Carli isocaloric control(CON)liquid diet or ALC(15 g/kg/d)liquid diet.11 CON or ALC rats were euthanized at the end of week 8,9 or 10.The plasma LPS from portal vein was determined by dynamic turbidimetric assay.Pancreatic inflammatory injury was and fibrosis were assessed by H&E staining and aniline blue collagen staining respectively.Immunostains for α-SMA and F4/80 were examined to identify PSC and macrophage(Mφ);Pancreatic type I collagen alpha 1(Col1A1)and TLR4 m RNA were detected by RT-q PCR while TLR4 protein was examined by Western blot;Pancreatic chemokines and TGF-β1 were determined by ELISA.RESULTS: Pancreatic inflammatory scores were increased in 10-week ALC-fed rats compared with CON-diet-fed rats,but there was no significant difference in collagen deposition between two groups.The levels of portal vein LPS and pancreatic TLR4 and Col1A1 m RNA and protein were increased in a time-dependent fashion in ALC rats,with the highest levels occurring at 10 weeks.Additionally,by 8 weeks,pancreatic TLR4 and Col1A1 m RNA in ALCrats were significantly increased as compared to CON rats,whereas portal vein LPS remained unchanged.The number of PSCs and Mφs and expression of chemokines(MCP-1,MIP-1α and RANTES),TGF-β1,or Col1A1 were also significantly increased,each of which was positively correlated with the level of portal vein LPS in 10-week ALC rats.CONCLUSION: These results suggest that endogenous LPS is associated with ALC-induced fibrosis in pancreatitis and targeting of bacterial endotoxin may be a promising therapeutic strategy for ACP.Part 2: Exogenous Lipopolysaccharide Enhanced Rat Pancreatic Fibrosis Through Up-regulation of Transforming Growth Factor β1SignalingPURPOSE: The aim of this study was to investigate the mechanisms by which LPS modulates TGF-β1 signaling and rat pancreatic fibrosis.METHODS: Twenty-two male Sprague-Dawley rats fed with a LieberDe Carli alcohol(ALC)liquid diet for 10 weeks with or without LPS challenge during the last 3 weeks.All rats were killed at 24 h after last LPS challenge or normal saline.Pancreatic tissues were fixed in 10% neutral buffer formalin forpreparation of paraffin sections or snap-frozen in liquid nitrogen.A stable activated rat PSC line(RP-2 cell)was used in this study.To prepare monocyte-derived Mφs,Rat peripheral blood mononuclear cells(PBMCs)were isolated using lymphocyte separation medium and then cultured in RPMI 1640 culture medium containing 10 ng/ml GM-CSF for 6 day.Pancreatic fibrosis was assessed by aniline blue collagen staining.Immunohistochemistry was used to detect the expression of TLR4,BAMBI,α-SMA and F4/80,the later two of which were used to identify PSC and Mφ in the pancreases respectively;Double immunofluorescence labeling antibodies for TLR4 or TGF-β1 and F4/80 or α-SMA were used to identify TLR4+Mφs/TLR4+PSC or TGF-β1+Mφ/TGF-β1+PSC.RT-q PCR was used to determine the levels of Col1A1,α-SMA,TLR4 and BAMBI m RNAs in the RP-2 cells;Western blot was used to examine the levels of Col1 and α-SMA proteins and to assess TGF-β1/Smad2/3and TLR4/My D88/NF-k B signaling pathways in RP-2 cells;ELISA was used to determine the concentrations of Col1A1 and TGF-β1 proteins in the supernatants from pancreatic samples or cultured Mφs and RP-2 cells.RESULTS: The results demonstrated that repeated LPS injection resulted in significantly more collagen production and PSC activation compared to rats fed with ALC alone.LPS administration caused over expression of pancreatic TLR4 or TGF-β1 which was paralleled by an increased number of TLR4-positive or TGF-β1-positive Mφs or PSCs in ALC-fed rats.In vitro,production of TLR4 or TTGF-β1 in Mφs or RP-2 cells was up-regulated by LPS.LPS alone or in combination with TGF-β1 significantly increased the production of Col1 and α-SMA and phosphorylation of Smad2 and Smad3 in serum-starved RP-2 cells.The expression of TGF-β1 pseudoreceptor BAMBI was inhibited by LPS,which was antagonized by Si-TLR4 RNA or by inhibitors of My D88/NF-k B.Furthermore,knockdown of BAMIB with Si-BAMIB RNA significantly increased TGF-β1 signaling in RP-2 cells.CONCLUSION: These findings indicate that LPS increases TGF-β1production through paracrine and autocrine mechanisms.LPS enhances TGF-β1 signaling in PSCs by repressing BAMBI via TLR4/My D88/NF-k B activation.