| Background:Liver fibrosis is a dynamic wound healing response of liver to various chronic liver injuries. Continuous chronic liver injuries would lead to a progressive accumulation of scarring proteins and finally alter normal tissue structure and function by inducing fibrosis, cirrhosis and ultimately liver failure. There has been considerable progresses on the research of the pathogenesis of liver fibrosis recent years, but few important breakthrough on the treatment. Therefore we believe that maybe other factors participates in the development of liver fibrosis. Professor Lixin Liu discovered that IGFBPrP1 is probably a new pathogenic factor in the formation of liver fibrosis. In human livers with fibrosis and cirrhosis, the expression of IGFBPrP1 increases along with the degree of liver fibrosis. Recombinant-IGFBPrP1 can activate HSC in vitro, which is dose dependant in a certain extent. Anti-IGFBPrP1 antibody can reverse HSC's over-production of collagen I induced by TGFβ1. In order to investigate the function and mechanism of IGFBPrP1 in the development of liver cirrhosis, we designed this research program.In order to make clear whether anti-IGFBPrP1 have protective function to liver tissues of fibrotic mouse and to explore its mechanism, we designed this study.Objective:To investigate the protective effect of anti-IGFBPrP1 antibody in the liver of mice with hepatic fibrosis induced by thioacetamide, and explore whether its acting mechanism is related to TGF-β1/Smad3 signal pathway.Methods:Thirty male Kunming mice were randomly divided into five groups, including the normally control group (A); TAA-four-week adding anti-IGFBPrP1 antibody treated one week group(B) and TAA-four-week group(C); TAA-five-week adding anti-IGFBPrP1 antibody treated one week group(D) and TAA-five-week group(E). (1)The value of both alanine aminotransferase (ALT) and lactic acid dehydrogenase (LDH) from abdominal aorta was analyzed by biochemical methods.(2) The morphological changes of liver fibrosis were observed with eyes.(3) The morphological changes of liver fibrosis were observed with both HE stain and Masson stainning.(4) Examine the expressions of alpha-smooth muscle actin (α-SMA), Collagen I, fibronectin (FN), transforming growth factor-beta 1(TGF-β1) and Smad3 of Hepatic tissues with immunohistochemistry. The relationships of Smad3 withα-SMA, Collagen I, FN, TGF-β1 were analyzed. (5)Examine the expressions of fibronectin (FN) and Smad3 in the fibrosis liver with Western blot. (6)The apoptosis rate of hepatic cells was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling.Results:(1) The content of both ALT and LDH in the serum was significantly increased in TAA-four-week group (C) and TAA-five-week group (E). It was significantly decreased in TAA-four-week adding anti-IGFBPrP1 antibody treated one week group (B) compared with that of TAA-four-week group (C) (F=389.107, P<0.01; F=389.104, P<0.01); so does that of TAA-five-week adding anti-IGFBPrP1 antibody treated one week group (D) compared with in TAA-five-week group (E) (F=570.905, P<0.01; F=570.908, P<0.01).(2) The Masson staining analysis: Compared with normal group (A), content of collagen fiber in TAA-four-week group (C) and TAA-five-week group (E)was significantly increased (3.80%±0.09% vs 0.13%±0.02%; 13.08%±0.13% vs 0.13%±0.02%). It was significantly decreased in TAA-four-week adding anti-IGFBPrP1 antibody treated one week group (B) compared with that of TAA-four-week group (C)(0.22%±0.02% vs 3.80%±0.09%, H=15.158, P<0.01), so does that of TAA-five-week adding anti-IGFBPrP1 antibody treated one week group (D) compared with in TAA-five-week group (E)(6.31%±0.06% vs 13.08%±0.13%,H=15.158, P<0.01).(3)The expressions ofα-SMA, Collagen I, FN, TGF-β1 and Smad3 in hepatic tissues were significantly increased in TAA-four-week group (C) and TAA-five-week group (E). These expressions were significantly decreased in TAA-four-week adding anti-IGFBPrP1 antibody treated one week group (B) compared with that of TAA-four-week group (C)(Collagen I: F=191.110, P<0.01;α-SMA, FN, TGF-β1, Smad3: H=15.158, P<0.01); so does that of TAA-five-week adding anti-IGFBPrP1 antibody treated one week group (D) compared with that of TAA-five-week group (E)(Collagen I: F=326.701, P<0.01;α-SMA, FN, TGF-β1: H=15.158, P<0.01; Smad3: H=14.749, P<0.01).(4) The expression of Smad3 have positive correlation with that ofα-SMA(r=0.876, P<0.01), Collagen I(r=0.750, P<0.01), FN(r=0.917, P<0.01), TGF-β1(r=0.872, P<0.01) in A, B and C group; The expression of Smad3 have positive correlation with that ofα-SMA(r=0.833, P<0.01), Collagen I(r=0.825, P<0.01), FN(r=0.878, P<0.01), TGF-β1(r=0.864, P<0.01) in A, D and E group.(5) The changes of levels of FN and Smad3 in hepatic tissues examined by Western blot were significantly increased in TAA-four-week group (C) and TAA-five-week group (E) (FN: 0.15±0.01; Smad3: 0.09±0.01). Those were significantly decreased in TAA-four-week adding anti-IGFBPrP1 antibody treated one week group (B) compared with that of in TAA-four-week group (C) (FN: 0.32±0.02 vs 0.65±0.01, H=15.158, P<0.01; Smad3: 0.16±0.01 vs 0.44±0.04, H=15.158, P<0.01), also in decreased in TAA-five-week adding anti-IGFBPrP1 antibody treated one week group (D) compared with that of in TAA-five-week group (E) (FN: 0.80±0.02 vs 1.59±0.02, H=15.158, P<0.01; Smad3: 0.54±0.04 vs 0.65±0.05, H=14.749, P<0.01).(6)The apoptotic rate of hepatic cells in TAA-four-week group (C) was significantly increased than that of the normal control group (A) (0.57%±0.06%). The apoptotic rate of hepatic cells in TAA-four-week adding anti-IGFBPrP1 antibody treated one week group (B) was signifycantly reduced than that of TAA-four-week group (C)(1.97%±0.15% vs 7.16%±0.10%, H=15.174, P<0.01).Conclusion:(1) Anti-IGFBPrP1 antibody can suppresses the progression of hepatic fibrosis through many ways, including inhibiting the activation of HSC and reducing the expressions of Collagen I and FN.(2) Anti-IGFBPrP1 antibody can suppresses the progression of hepatic fibrosis through reducing hepatocyte apoptosis and so on.(3) The possible mechanism maybe relate to the TGF-β1/Smad3 signal path.
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