柞蠶雄蛾液防治小鼠酒精性肝纖維化機制的實驗研究
發(fā)布時間：2018-10-15 09:03
【摘要】：目的:本實驗通過觀察柞蠶雄蛾液對酒精性肝纖維化動物模型的作用,以及轉化生長因子β1/Smads、結締組織生長因子、金屬蛋白酶組織抑制因子-1、骨形成蛋白和激活素的跨膜抑制劑、Toll樣受體與細胞間隙連接蛋白43在肝臟中表達變化的情況,探討柞蠶雄蛾液保護肝臟的作用機制;并且通過對EMT相關指標Twist因子、E-鈣黏蛋白與波形蛋白表達情況進行分析,探討酒精性肝纖維化過程中EMT的發(fā)生及柞蠶雄蛾液對酒精性肝纖維化的影響。旨在為臨床拮抗酒精性肝纖維化提供較為充分的理論依據。方法:1.將雄性SPF級小鼠C57隨機進行分組,組別為:空白對照組、模型組、柞蠶雄蛾液高劑量組和柞蠶雄蛾液低劑量組。后三組制備酒精性肝纖維化模型,每周稱重1次,連續(xù)干預16周后處死小鼠取肝臟,稱重計算肝指數(shù)。2.血清學ALT與AST測定。3.蘇木素-伊紅染色,觀察肝組織病理學改變。4.Masson法染色,觀察膠原纖維的面積。5.免疫組織化學法檢測肝組織中TGF-β1、CTGF、TIMP-1、BAMBI、TLR4、Connexin43、Twist、E-Cadherin與Vimentin的蛋白表達水平。6.Western blot方法檢測肝組織中TGF-β1、SMAD2/3、BAMBI、TLR4、Twist、E-Cadherin與Vimentin的蛋白表達水平。結果:1.柞蠶雄蛾液對小鼠一般生長狀況的影響模型組小鼠與同期其它各組比較狀態(tài)差。柞蠶雄蛾液高劑量組小鼠與柞蠶雄蛾液低劑量組小鼠體重在一定時間內有所增加,毛發(fā)較整齊光亮,狀態(tài)尚可。2.肝指數(shù)及血清中ALT及AST檢測結果模型組較之對照正常組,肝指數(shù)增大(P0.01);柞蠶雄蛾液高劑量組與柞蠶雄蛾液低劑量組較之模型組,肝指數(shù)降低(P0.01)。與對照正常組比較,模型組的ALT與AST升高,柞蠶雄蛾液高劑量組的AST升高(P0.01,P0.05)。與模型組比較,柞蠶雄蛾液高劑量組與柞蠶雄蛾液低劑量組的ALT與AST降低(P0.01)。3.肝組織HE染色及Masson染色結果HE染色結果顯示,柞蠶雄蛾液高劑量組與柞蠶雄蛾液低劑量組肝細胞出現(xiàn)脂肪變性,并有輕度腫脹現(xiàn)象;同時,呈現(xiàn)點灶狀的壞死,可見炎細胞浸潤等表現(xiàn)。柞蠶雄蛾液低劑量組壞死程度及范圍輕于柞蠶雄蛾液高劑量組,與模型組比較,其壞死程度及壞死范圍明顯減輕。模型組有纖維化改變。Masson染色結果顯示:與對照正常組比較,模型組的膠原纖維面積明顯增大(P0.01);膠原纖維面積在柞蠶雄蛾液高劑量組與柞蠶雄蛾液低劑量組中與模型組比較明顯減小(P0.01,P0.05)。4.免疫組織化學方法檢測結果與對照正常組比較,模型組、柞蠶雄蛾液高劑量組與柞蠶雄蛾液低劑量組TGF-β1、TIMP-1、CTGF、BAMBI、TLR4、Twist與Vimentin蛋白表達量明顯增高,Connexin 43蛋白表達量明顯降低;模型組與柞蠶雄蛾液高劑量組E-cadherin蛋白表達量明顯降低(P0.01)。與模型組比較,柞蠶雄蛾液高劑量組與柞蠶雄蛾液低劑量組TGF-β1、TIMP-1、CTGF、BAMBI、TLR4、Twist與Vimentin蛋白表達量明顯降低,Connexin 43與E-cadherin蛋白表達量明顯升高(P0.01,P0.05)。5.Western blot法檢測蛋白在肝組織中的表達水平與對照正常組比較:TGF-β1、SMAD2/3、TLR4、Twist與Vimentin蛋白相對表達量在模型組與柞蠶雄蛾液高劑量組中增高,E-cadherin降低;柞蠶雄蛾液低劑量組TGF-β1蛋白相對表達量增高;模型組的BAMBI蛋白表達量相對水平增高(P0.01)。與模型組比較:柞蠶雄蛾液高劑量組與柞蠶雄蛾液低劑量組的TGF-β1、BAMBI與Twist蛋白相對表達量降低;柞蠶雄蛾液低劑量組的Smad2/3、TLR4與Vimentin蛋白相對表達量降低(P0.01,P0.05)。結論:1.采用酒精灌胃可以誘導C57小鼠酒精性肝纖維化的發(fā)生。2.柞蠶雄蛾液能明顯降低酒精灌胃的小鼠血清中ALT與AST水平,減輕肝細胞壞死程度及范圍,對酒精性肝損傷具有保護作用,低劑量柞蠶雄蛾液的效果優(yōu)于高劑量柞蠶雄蛾液。3.酒精性肝纖維化發(fā)展過程中呈現(xiàn)上皮-間質轉化,上皮-間質轉化在酒精誘導的肝纖維化過程中發(fā)揮著重要作用。4.柞蠶雄蛾液對上皮-間質轉化的現(xiàn)象具有明顯的抑制作用,可能與下調Twist與Vimentin表達,上調Connexin 43與E-cadherin表達有關。5.柞蠶雄蛾液對酒精誘導的小鼠肝纖維化具有顯著的防治作用,其機制可能與調控TGF-β1/Smad2/3信號轉導通路,下調TIMP-1、CTGF、BAMBI及TLR4的表達抑制HSC的活化與增殖,抑制上皮-間質轉化有關。
[Abstract]:Objective: To observe the effect of male moth liquid on the animal model of alcoholic liver fibrosis, as well as the transmembrane inhibitor of transforming growth factor-1/ NSCs, connective tissue growth factor, metalloprotease tissue inhibitor-1, bone morphogenetic protein and activin. In order to study the mechanism of the liver function, the expression of E-cadherin and p27 protein in the liver was analyzed by the expression of Twist factor, E-cadherin and p27 protein in the liver by the expression of the Toll-like receptor and the intercellular junction protein 43 in the liver. To investigate the occurrence of alcohol and the effect of male moth liquid on alcoholic liver fibrosis during alcoholic liver fibrosis. It aims to provide a theoretical basis for clinical antagonizing alcoholic liver fibrosis. Method: 1. The male SPF mice C57 were randomly divided into two groups: blank control group, model group, high dose group and low dose group of male moth. The model of alcoholic liver fibrosis was prepared in three groups. The liver was weighed once a week, and the liver was weighed and calculated after 16 weeks of continuous intervention. Serological ALT and AST assay. 3. Histopathological changes of the liver were observed by hematoxylin-eosin staining. The area of collagen fibers was observed in 4. Masson method. Immunohistochemical method was used to detect the level of protein expression in liver tissues, such as the expression levels of IL-1, CTGF, TIMP-1, BAMBI, TLR4, Connexin43, Twist, E-Cadherin and Vimentin in liver tissue. Result: 1. The effect of male moth liquid on the general growth condition of mice was worse than that in other groups in the same period. The weight of mice with high dose group and low dose group of male moths increased in a certain period of time, and the hair was more tidy and bright, and the state was acceptable. Compared with the control group, the liver index increased (P <0.01) compared with the control group (P <0.01). Compared with the control group, the ALT and AST of the model group increased, and the AST of the high dose group was increased (P <0.01, P 0.05). Compared with the model group, the ALT and AST of the high-dose group and the low-dose group were decreased (P0.01). HE staining of liver tissue and HE staining of Masson staining showed that there was fatty degeneration in the high dose group and low dose group of male moths, and slight swelling was observed; meanwhile, the appearance of focal necrosis, visible inflammatory cell infiltration and so on were presented. The degree and extent of necrosis and necrosis of the low-dose group were significantly reduced compared with the model group. There were fibrosis changes in the model group. The results showed that the collagen fiber area of the model group increased significantly compared with the control group (P0.01), and the collagen fiber area was significantly decreased compared with the model group (P0.01, P0.05). The results of immunohistochemistry were compared with those of control group. Compared with control group, the expression of TIMP-1, TIMP-1, CTGF, BAMBI, TLR4, Twist and Vimentin in low dose group were significantly increased. The expression level of E-cadherin protein in the high dose group was significantly lower in the model group and the high dose group (P0.01). Compared with the model group, the expression of IL-1, TIMP-1, CTGF, BAMBI, TLR4, Twist and Vimentin in the high dose group and the low dose group of the male moth was significantly decreased, and the expression level of Connexin 43 and E-cadherin was significantly higher than that of the group E-cadherin (P0.01). The expression level of protein in liver tissue was compared with that of normal control group (P0.05). The expression level of BMBI protein in the low dose group was higher than that in the low dose group (P0.01). Compared with model group, the relative expression of BMBI and Twist protein in high dose group and low dose group of male moth decreased (P <0.01, P <0.05). Conclusion: 1. Alcohol gavage could induce the occurrence of alcoholic liver fibrosis in C57 mice. The male moth liquid can obviously reduce ALT and AST levels in serum of mice fed by alcohol, reduce necrosis degree and range of liver cells, have protective effect on alcoholic liver injury, and the effect of low dose of male moth liquid is better than that of high-dose male moth liquid. During the development of alcoholic liver fibrosis, epithelial-mesenchymal transition and epithelial-mesenchymal transition play an important role in the process of alcohol-induced liver fibrosis. It is possible to regulate the expression of Twist and Vimentin, and up-regulate the expression of connexin 43 and E-cadherin. The mechanism may regulate the expression of TIMP-1, CTGF, BAMBI and TLR4, inhibit the activation and proliferation of HSC, and inhibit the epithelial-mesenchymal transition.
【學位授予單位】：濟南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R259
本文編號：2272035
[Abstract]:Objective: To observe the effect of male moth liquid on the animal model of alcoholic liver fibrosis, as well as the transmembrane inhibitor of transforming growth factor-1/ NSCs, connective tissue growth factor, metalloprotease tissue inhibitor-1, bone morphogenetic protein and activin. In order to study the mechanism of the liver function, the expression of E-cadherin and p27 protein in the liver was analyzed by the expression of Twist factor, E-cadherin and p27 protein in the liver by the expression of the Toll-like receptor and the intercellular junction protein 43 in the liver. To investigate the occurrence of alcohol and the effect of male moth liquid on alcoholic liver fibrosis during alcoholic liver fibrosis. It aims to provide a theoretical basis for clinical antagonizing alcoholic liver fibrosis. Method: 1. The male SPF mice C57 were randomly divided into two groups: blank control group, model group, high dose group and low dose group of male moth. The model of alcoholic liver fibrosis was prepared in three groups. The liver was weighed once a week, and the liver was weighed and calculated after 16 weeks of continuous intervention. Serological ALT and AST assay. 3. Histopathological changes of the liver were observed by hematoxylin-eosin staining. The area of collagen fibers was observed in 4. Masson method. Immunohistochemical method was used to detect the level of protein expression in liver tissues, such as the expression levels of IL-1, CTGF, TIMP-1, BAMBI, TLR4, Connexin43, Twist, E-Cadherin and Vimentin in liver tissue. Result: 1. The effect of male moth liquid on the general growth condition of mice was worse than that in other groups in the same period. The weight of mice with high dose group and low dose group of male moths increased in a certain period of time, and the hair was more tidy and bright, and the state was acceptable. Compared with the control group, the liver index increased (P <0.01) compared with the control group (P <0.01). Compared with the control group, the ALT and AST of the model group increased, and the AST of the high dose group was increased (P <0.01, P 0.05). Compared with the model group, the ALT and AST of the high-dose group and the low-dose group were decreased (P0.01). HE staining of liver tissue and HE staining of Masson staining showed that there was fatty degeneration in the high dose group and low dose group of male moths, and slight swelling was observed; meanwhile, the appearance of focal necrosis, visible inflammatory cell infiltration and so on were presented. The degree and extent of necrosis and necrosis of the low-dose group were significantly reduced compared with the model group. There were fibrosis changes in the model group. The results showed that the collagen fiber area of the model group increased significantly compared with the control group (P0.01), and the collagen fiber area was significantly decreased compared with the model group (P0.01, P0.05). The results of immunohistochemistry were compared with those of control group. Compared with control group, the expression of TIMP-1, TIMP-1, CTGF, BAMBI, TLR4, Twist and Vimentin in low dose group were significantly increased. The expression level of E-cadherin protein in the high dose group was significantly lower in the model group and the high dose group (P0.01). Compared with the model group, the expression of IL-1, TIMP-1, CTGF, BAMBI, TLR4, Twist and Vimentin in the high dose group and the low dose group of the male moth was significantly decreased, and the expression level of Connexin 43 and E-cadherin was significantly higher than that of the group E-cadherin (P0.01). The expression level of protein in liver tissue was compared with that of normal control group (P0.05). The expression level of BMBI protein in the low dose group was higher than that in the low dose group (P0.01). Compared with model group, the relative expression of BMBI and Twist protein in high dose group and low dose group of male moth decreased (P <0.01, P <0.05). Conclusion: 1. Alcohol gavage could induce the occurrence of alcoholic liver fibrosis in C57 mice. The male moth liquid can obviously reduce ALT and AST levels in serum of mice fed by alcohol, reduce necrosis degree and range of liver cells, have protective effect on alcoholic liver injury, and the effect of low dose of male moth liquid is better than that of high-dose male moth liquid. During the development of alcoholic liver fibrosis, epithelial-mesenchymal transition and epithelial-mesenchymal transition play an important role in the process of alcohol-induced liver fibrosis. It is possible to regulate the expression of Twist and Vimentin, and up-regulate the expression of connexin 43 and E-cadherin. The mechanism may regulate the expression of TIMP-1, CTGF, BAMBI and TLR4, inhibit the activation and proliferation of HSC, and inhibit the epithelial-mesenchymal transition.
【學位授予單位】：濟南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R259
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