Study On The Immune Regulatory Function Of Kupffer Cell Of Patients With Autoimmune Hepatitis By The Intestinal Mucosal Barrier

Background and Objects:The pathogenesis of autoimmune hepatitis (AIH) was still unknown. Attack against liver cell self antigens following the breakdown of immune tolerance were believed to be the major mechanism. Kupffer cells (KC), the resident liver macrophages, with the function of TLR signal pathway, played an important role in immune tolerance induction and maintenance. Recent studies have demonstrated that intestinal-derived toxin could promote production of cytokines and disfunction of biological system of the liver. Based on these observations, we hypothesized that intestinal injury and increased intestinal permeability combined with bacteria translocation could promote. TLR4-dependent KC activation as an early event in the pathogenesis of AIH.Methods:Part1The mucous membrane of duodenum were observed by electron microscope and immunohistochemistry technique was used to examine the tight junction proteins Zonula occludens-1(ZO-1)、Occludin.16SrDNA fluorescent quantitative PCR was applied in determining the content of bifidobacterium, lactobacillus, escherichia coli and enterococcus in feces. And the level of endotoxin (LPS) and alanine aminotransferase (ALT) were detected in the peripheral blood.Part2Western blot and double immunofluorescence staining techniques were used to examine the expression and the resource of TLR4in liver tissues. Part3Western blot and double immunofluorescence staining techniques were used to examine the expression and the resource of Racl signaling makers (Vavl and PAK1) in liver tissues. The function of antigen presentation of peripheral blood monocyte was analyzed by CD80and HLA-DR using flow cytometry. Phagocytosis of peripheral blood monocytes was analyzed by challenging cells with fluorescein isothiocyanate (FITC) labeled Escherichia coli (E.coli) using flow cytometry.Results:Part1(1) The level of serum ALT in AIH group was obviously higher than that in control group (P<0.05).(2) The structure of the duodenum mucosa is obviously broken, the mucosal microvilli structure and tight junction is significantly broken in the AIH group than those in control group.(3) ZO-1and occludin were localized along the apical region of the lateral plasma membrane representing the region of tight junctions in surface and crypt epithelial cells. The positive stainings of ZO-1and occludin of AIH group were obviously decreased (P<0.05).(4) The amount of anaerobes represented by the bifidobacterium and lactobacillus declined (P <0.05). However, the amount of aerobes represented by escherichia coli and enterococcus did not change obviously, Bifidobacteria/Escherichia coli (B/E) which indicated a balance of intestinal flora declined (P<0.05). The results showed the intestinal dysbacteriosis occured in patients with AIH.(5) The level of plasma endotoxin of AIH group were obviously increased than that in control group (P<0.05). Part2The expression of TLR4in liver tissue of AIH group was obviously higher than that of control group(P<0.05), and the expression of TLR4on Kupffer cells of AIH group was obviously higher than that of control group. Part3(1) The expressions of VAV1and PAK1in liver tissue of AIH group were obviously higher(P <0.05), and the expression of VAV1and PAK1on Kupffer cells of AIH group was obviously higher than that of control group.(2) The peripheral blood monocytes antigen presentation function of AIH group was lower than control group (P<0.05).(3) The peripheral blood monocytes phagocytic function of AIH group was lower than control group (P<0.05).Conclusions:AIH was associated with increased intestinal permeability. Intestinal-derived LPS might activate KC through TLR4signaling in early stages of AIH. The intestinal mucosal barrier fuction might be an important part in the etiology of liver injury. Thus, disfunction of intestinal mucosal barrier might bring a new vision to treatment or prevention of AIH via KC.