| Objective: This experiment to explore the mechanism of male China oak silkworm liquid protect liver through observation of male China oak silkworm liquid on alcoholic liver fibrosis animal model role and expression of TGF-?1/ Smads, BAMBI, TIMP-1, CTGF and Toll-like receptors-4 and Connexin 43 in the liver; and analysis expression of the related indicators of EMT such as twist protein, E-cadherin and vimentin, to investigate the process of alcoholic liver fibrosis whether EMT occurs and Effect of male China oak silkworm liquid extracts alcoholic liver fibrosis. To provide a sufficient theoretical basis for the clinical antagonistic effect of alcoholic liver fibrosis.Methods: 1. C57 male mice(SPF) were randomly divided into groups. respectively the control group, model group, high dose group of male China oak silkworm liquid and low dose group of male China oak silkworm liquid. After the three groups were gavage with alcohol to establish the model of alcoholic liver fibrosis, weighing 1 times a week, 16 weeks doing tests,mice were sacrificed,take out the liver then weighing, Calculated the liver index. 2. Determination lever of ALT and AST in Serological method. 3. HE staining to observe the change of liver histopathology. 4. Masson staining was used to observe the area of collagen fibers. 5. Immunohistochemistry was used to detect liver tissue TGF-?1, CTGF, TIMP-1, BAMBI, TLR4, Connexin 43, Twist, E-Cadherin and Vimentin protein expression levels. 6. Detect liver tissue TGF-?1, SMAD2/3, BAMBI, TLR4, Twist, E-Cadherin and Vimentin protein expression by Western Blot method.Results: 1. The effect of male China oak silkworm liquid on general growth of mice Compared with the other groups state, the model group was in poor condition. high-dose group of male China oak silkworm liquid and low-dose group of male China oak silkworm liquid body weight of mice within a certain period of time has increased, the hair is neat and bright lights, state fair.2. Liver index and the test results of ALT and AST in serum. In model group was obviously higher than that in control group about the liver index(P<0.01); The liver index of in high-dose group of male China oak silkworm liquid and low-dose group of male China oak silkworm liquid compared with the model group were decreased obviously(P<0.01). The content of ALT and AST Compared with control group,in model group were increased, The content of AST in high-dose group of male China oak silkworm liquid was increased obviously(P<0.01,P<0.05); The content of ALT and AST compared with the model group,in high-dose group of male China oak silkworm liquid and low-dose group of male China oak silkworm liquid were obviously decreased(P < 0.01). 3. In liver tissues the results of HE staining and Masson staining HE staining showed that in the high-dose group of male China oak silkworm liquid and low-dose group of male China oak silkworm liquid, liver cell fatty degeneration and swelling phenomenon; at the same time, showing focal necrosis, it is thus clear to see the inflammatory cell infiltration and so on. compared with the model group, the necrosis degree and extent of necrosis were significantly reduced, low-dose group of male China oak silkworm necrosis degree and extent of necrosis is light than the high-dose group of male China oak silkworm liquid. The model group had the change of fibrosis. Masson staining of collagen fibers showed that: the collagen fiber area of in model group was increased more obvious than the control group(P< 0.01); the collagen fiber area of in high-dose group of male China oak silkworm liquid and low-dose group of male China oak silkworm liquid compared with the model group, were decreased obviously. 4. Results of immunohistochemical methods Compared with the control group, the expression of TGF beta 1, TIMP-1, CTGF, Bambi, TLR4, Twist and vimentin in model group, high-dose group of male China oak silkworm liquid and low-dose group of male China oak silkworm liquid were obviously increased, the expression of Connexin 43 was obviously decreased; the expression of E-cadherin protein in model group and high-dose group of male China oak silkworm liquid were obviously decreased(P<0.01). The expression of TGF beta 1, TIMP-1, CTGF, Bambi, TLR4, twist and vimentin in high-dose group of male China oak silkworm liquid and low-dose group of male China oak silkworm liquid compared with the model group were decreased and connexin 43 and E-cadherin expression were obviously increased(P<0.01, P<0.05). 5. The expression level of protein in liver tissues was detected by Western Blot method The relative expression level of TGF beta 1, SMAD2/3, TLR4, Twist and vimentin in model group and high-dose group of male China oak silkworm liquid compared with the control group were quantity increased, The relative expression level of E-cadherin protein was decreased; The relative expression level of TGF beta 1 in low-dose group of male China oak silkworm liquid was quantity increased; The relative expression of Bambi in model group was obviously increased(P<0.01). The relative expression level of TGF beta 1, Bambi and twist protein in high-dose group of male China oak silkworm liquid and low-dose group of male China oak silkworm liquid compared with the model group were decreased; The relative expression level of Smad2/3, TLR4 and vimentin in low-dose group of male China oak silkworm liquid was reduced(P<0.01, P<0.05).Conclusions: 1. Using alcohol to fill the stomach can induce the occurrence of alcoholic liver fibrosis in C57 mice. 2. The male China oak silkworm liquid decreased significantly the ALT and AST levels in serum induced by alcohol irrigation stomach, it showed protective effects against liver injury induced by alcohol in mice. The effect to protect the liver in the low-dose of male China oak silkworm liquid is better than in the high-dose of male China oak silkworm liquid. 3. EMT can occur in the process of alcoholic liver fibrosis, EMT play an major role in the process of alcohol induced liver fibrosis. 4. The male China oak silkworm liquid has obvious inhibitory effect on EMT,it might be related to down regulation the expression of twist and vimentin, up regulation of Connexin 43 and E-cadherin expression. 5. The male China oak silkworm liquid was on the alcohol-induced liver fibrosis in mice has a significant preventive effect, The specific mechanism might be related to the regulation of TGF-?1/Smad2/3, down regulation of TIMP, CTGF, Bambi and TLR4 to restrain the activation and proliferation of HSC and EMT. |
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