柞蚕雄蛾液防治小鼠酒精性肝纤维化机制的实验研究
发布时间：2018-10-15 09:03
【摘要】：目的:本实验通过观察柞蚕雄蛾液对酒精性肝纤维化动物模型的作用,以及转化生长因子β1/Smads、结缔组织生长因子、金属蛋白酶组织抑制因子-1、骨形成蛋白和激活素的跨膜抑制剂、Toll样受体与细胞间隙连接蛋白43在肝脏中表达变化的情况,探讨柞蚕雄蛾液保护肝脏的作用机制;并且通过对EMT相关指标Twist因子、E-钙黏蛋白与波形蛋白表达情况进行分析,探讨酒精性肝纤维化过程中EMT的发生及柞蚕雄蛾液对酒精性肝纤维化的影响。旨在为临床拮抗酒精性肝纤维化提供较为充分的理论依据。方法:1.将雄性SPF级小鼠C57随机进行分组,组别为:空白对照组、模型组、柞蚕雄蛾液高剂量组和柞蚕雄蛾液低剂量组。后三组制备酒精性肝纤维化模型,每周称重1次,连续干预16周后处死小鼠取肝脏,称重计算肝指数。2.血清学ALT与AST测定。3.苏木素-伊红染色,观察肝组织病理学改变。4.Masson法染色,观察胶原纤维的面积。5.免疫组织化学法检测肝组织中TGF-β1、CTGF、TIMP-1、BAMBI、TLR4、Connexin43、Twist、E-Cadherin与Vimentin的蛋白表达水平。6.Western blot方法检测肝组织中TGF-β1、SMAD2/3、BAMBI、TLR4、Twist、E-Cadherin与Vimentin的蛋白表达水平。结果:1.柞蚕雄蛾液对小鼠一般生长状况的影响模型组小鼠与同期其它各组比较状态差。柞蚕雄蛾液高剂量组小鼠与柞蚕雄蛾液低剂量组小鼠体重在一定时间内有所增加,毛发较整齐光亮,状态尚可。2.肝指数及血清中ALT及AST检测结果模型组较之对照正常组,肝指数增大(P0.01);柞蚕雄蛾液高剂量组与柞蚕雄蛾液低剂量组较之模型组,肝指数降低(P0.01)。与对照正常组比较,模型组的ALT与AST升高,柞蚕雄蛾液高剂量组的AST升高(P0.01,P0.05)。与模型组比较,柞蚕雄蛾液高剂量组与柞蚕雄蛾液低剂量组的ALT与AST降低(P0.01)。3.肝组织HE染色及Masson染色结果HE染色结果显示,柞蚕雄蛾液高剂量组与柞蚕雄蛾液低剂量组肝细胞出现脂肪变性,并有轻度肿胀现象;同时,呈现点灶状的坏死,可见炎细胞浸润等表现。柞蚕雄蛾液低剂量组坏死程度及范围轻于柞蚕雄蛾液高剂量组,与模型组比较,其坏死程度及坏死范围明显减轻。模型组有纤维化改变。Masson染色结果显示:与对照正常组比较,模型组的胶原纤维面积明显增大(P0.01);胶原纤维面积在柞蚕雄蛾液高剂量组与柞蚕雄蛾液低剂量组中与模型组比较明显减小(P0.01,P0.05)。4.免疫组织化学方法检测结果与对照正常组比较,模型组、柞蚕雄蛾液高剂量组与柞蚕雄蛾液低剂量组TGF-β1、TIMP-1、CTGF、BAMBI、TLR4、Twist与Vimentin蛋白表达量明显增高,Connexin 43蛋白表达量明显降低;模型组与柞蚕雄蛾液高剂量组E-cadherin蛋白表达量明显降低(P0.01)。与模型组比较,柞蚕雄蛾液高剂量组与柞蚕雄蛾液低剂量组TGF-β1、TIMP-1、CTGF、BAMBI、TLR4、Twist与Vimentin蛋白表达量明显降低,Connexin 43与E-cadherin蛋白表达量明显升高(P0.01,P0.05)。5.Western blot法检测蛋白在肝组织中的表达水平与对照正常组比较:TGF-β1、SMAD2/3、TLR4、Twist与Vimentin蛋白相对表达量在模型组与柞蚕雄蛾液高剂量组中增高,E-cadherin降低;柞蚕雄蛾液低剂量组TGF-β1蛋白相对表达量增高;模型组的BAMBI蛋白表达量相对水平增高(P0.01)。与模型组比较:柞蚕雄蛾液高剂量组与柞蚕雄蛾液低剂量组的TGF-β1、BAMBI与Twist蛋白相对表达量降低;柞蚕雄蛾液低剂量组的Smad2/3、TLR4与Vimentin蛋白相对表达量降低(P0.01,P0.05)。结论:1.采用酒精灌胃可以诱导C57小鼠酒精性肝纤维化的发生。2.柞蚕雄蛾液能明显降低酒精灌胃的小鼠血清中ALT与AST水平,减轻肝细胞坏死程度及范围,对酒精性肝损伤具有保护作用,低剂量柞蚕雄蛾液的效果优于高剂量柞蚕雄蛾液。3.酒精性肝纤维化发展过程中呈现上皮-间质转化,上皮-间质转化在酒精诱导的肝纤维化过程中发挥着重要作用。4.柞蚕雄蛾液对上皮-间质转化的现象具有明显的抑制作用,可能与下调Twist与Vimentin表达,上调Connexin 43与E-cadherin表达有关。5.柞蚕雄蛾液对酒精诱导的小鼠肝纤维化具有显著的防治作用,其机制可能与调控TGF-β1/Smad2/3信号转导通路,下调TIMP-1、CTGF、BAMBI及TLR4的表达抑制HSC的活化与增殖,抑制上皮-间质转化有关。
[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.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R259
本文编号：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.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R259
【参考文献】
相关期刊论文 前10条
1 杨坤;田珍珍;王淑华;修明;郭乡羚;齐丽娜;李敏;孙丽;高润平;;LPS-TLR4信号通路在慢性酒精摄入大鼠模型肝纤维化发生中的作用及其机制[J];吉林大学学报(医学版);2015年04期
2 葛文松;范建高;李光明;;小分子干扰RNA沉默高迁移率族蛋白B1抑制肝星状细胞Ⅰ型和Ⅲ型胶原合成[J];实用肝脏病杂志;2015年04期
3 姜艳红;张维东;;酒精性肝纤维化中主要信号转导通路的研究[J];国际消化病杂志;2015年03期
4 张羽飞;李厚忠;高晓虹;袁晓环;初彦辉;;CTGF-siRNA对TGF-β1诱导的肝星状细胞胶原分泌的影响[J];中国食物与营养;2014年08期
5 孙玉生;林波;王思谦;刘悦;张优敬;郑乃芮;皇甫超申;;慢性酒精摄入所致的肝细胞上皮-间充质转化参与小鼠肝纤维化形成[J];中国病理生理杂志;2014年01期
6 吕继业;李金志;孙继红;张桂春;黄玉波;薛强;朱兴友;王宝贵;张桂英;刘姗姗;;柞蚕雄蛾营养成分的测定与评价[J];北方蚕业;2013年03期
7 徐文清;胡卫列;肖戈;黄福;吴浩明;;E-cadherin及Vimentin在肾上腺皮质癌中的表达及其意义[J];实用癌症杂志;2013年03期
8 谢潮鑫;孟猛;殷先锋;何凤玲;叶汉深;谢栋;;天然虾青素对抗肾纤维化及细胞凋亡的作用[J];南方医科大学学报;2013年02期
9 彭莉;肖建华;;TLR介导的信号通路在肝纤维化中的作用[J];微生物学免疫学进展;2011年04期
10 刘宇红;才洪良;穆丽雅;;多烯磷脂酰胆碱联合阿托伐他汀治疗酒精性肝病的临床观察[J];中国药房;2010年16期
，本文编号：2272035
本文链接：https://www.wllwen.com/zhongyixuelunwen/2272035.html
最近更新
教材专著
