积雪草酸对小鼠胆管结扎诱导的肝纤维化的治疗作用及机制的研究

发布时间：2018-09-06 16:45

【摘要】：目的:肝纤维化是许多肝脏疾病发展成为肝硬化的重要病理过程,目前还没有疗效良好的治疗药物。本课题研究积雪草酸对胆汁性肝纤维化小鼠的治疗作用,并探讨积雪草酸的作用机制,为其应用于肝纤维化的治疗提供实验依据。方法:本研究通过胆管结扎(BDL)诱导小鼠肝损伤及肝纤维化建立在体实验模型,并以甘氨鹅脱氧胆酸钠(GCDC)处理HL-7702细胞建立体外肝损伤模型,通过在体和离体实验探讨积雪草酸的抗肝纤维化作用及机制。在体实验:结扎C57小鼠的总胆管,建立淤胆型肝损伤模型,分为假手术对照组、模型组、积雪草酸15 mg/kg组和30 mg/kg组,每组6只。各组小鼠连续灌胃给药5天。测定血清生化指标水平来检测肝功能。称量小鼠肝脏及脾脏组织,并计算肝脏指数和脾脏指数。检测肝组织内MDA(丙二醛),SOD(超氧化物气化酶)和Catalase(过氧化氢酶)和GSH(还原型谷胱甘肽)分析小鼠肝组织氧化应激。将肝脏组织切片HE染色、Masson染色进行病理学检查,TUNEL染色检测肝细胞凋亡;α-SMA(α-平滑肌肌动蛋白)免疫组化及Western blot检测肝纤维化程度。采用实时荧光定量PCR测定炎症因子及肝纤维化因子相关基因的mRNA水平,分析积雪草酸对炎症、肝纤维化的改善作用;测定胆汁酸代谢相关的基因表达水平,分析胆汁酸代谢调节途径。Western blot法检测Bax、Bcl-2的表达水平,分析积雪草酸的抗凋亡机制;检测核因子E2相关因子2(Nrf2)、血红素氧合酶-1(HO-1)的蛋白表达水平,研究积雪草酸的抗氧化机制。离体实验:采用GCDC作用于HL-7702细胞建立体外肝损伤模型,分为对照组、模型组和不同浓度的积雪草酸处理组,MTT法检测细胞活性,Annexin V/PI双染法通过流式细胞仪检测细胞凋亡,DCFH-DA荧光探针法检测细胞中活性氧(ROS)的含量,细胞免疫荧光和蛋白免疫印迹法检测Nrf2的蛋白表达水平。结果:在BDL小鼠模型中,我们发现BDL模型组小鼠肝脏肿大、欠光泽,肝组织出现胶原沉积、炎性细胞浸润的现象,积雪草酸给药后能够改善BDL小鼠的肝组织形态和病理特征,降低肝脏指数和脾脏指数。BDL模型组小鼠血清中天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)、碱性磷酸酶(AKP)、羟脯氨酸(Hyp)、总胆固醇(T-CHO)、总胆红素(TBIL)和总胆汁酸(TBA)的水平显著上升,积雪草酸给药能够显著降低这些生化指标的水平,高剂量效果更明显。同时,积雪草酸能够减少肝组织α-SMA的表达;降低纤维化因子Ⅲ型胶原蛋白(Col3a1)、波形蛋白(Vim)、肌动蛋白(Acta2)、转化生长因子(TGF-β1)mRNA的表达水平;降低促炎因子包括前列腺素内过氧化物合成酶2(Pgst2)、IL-6、TNF-α、趋化因子配体3(CCL3)的mRNA表达水平。积雪草酸给药作用后,上调Bcl2,下调Bax表达,减少肝组织细胞的凋亡。积雪草酸给药能够抑制BDL小鼠肝组织的氧化应激水平,表现为MDA含量的降低,SOD、GSH和Catalase水平的提高,促进Nrf2和HO-1的蛋白表达。此外,积雪草酸给药能够调控BDL小鼠的胆汁酸代谢途径相关基因,法尼酯X受体(FXR)、胆固醇7α-羟化酶(CYP7α1)、小分子异源二聚体伴侣(SHP)、成纤维细胞生长因子(FGF15)的mRNA水平,减少肝组织内胆汁酸淤积。离体实验表明积雪草酸能够有效的保护GCDC诱导的HL-7702细胞损伤。实验结果显示,积雪草酸处理可以显著增加细胞存活率(P0.001);减少细胞内ROS生成,降低细胞凋亡率,抑制作用均呈浓度依赖性增强;促进HL-7702细胞表达Nrf2蛋白。结论:在体实验水平,积雪草酸具有显著改善BDL鼠肝纤维化的作用,抑制氧化应激,抑制炎症反应,调控胆汁酸代谢,改善肝组织形态和功能;在细胞水平上,积雪草酸对抗GCDC诱导的HL-7702细胞损伤,抑制ROS的增加,减少细胞凋亡,促进Nrf2的表达。本论文研究结果揭示积雪草酸有改善胆汁性肝纤维化的作用,其机制可能与抗凋亡以及Nrf2介导的抗氧化和调控胆汁酸代谢有关。
[Abstract]:Objective: Hepatic fibrosis is an important pathological process in which many liver diseases develop into liver cirrhosis, and there is no effective drug to treat it. In this study, hepatic injury and fibrosis induced by bile duct ligation (BDL) in mice were established in vivo. HL-7702 cells were treated with sodium glycogen deoxycholate (GCDC) to establish an in vitro hepatic injury model. The anti-hepatic fibrosis effect and mechanism of asiatica acid were investigated in vitro and in vivo. The model of cholestatic liver injury was established and divided into sham operation control group, model group, 15 mg/kg Asiatic acid group and 30 mg/kg Asiatic acid group, 6 mice in each group. SOD (superoxide vaporase) and Catalase (catalase) and GSH (reduced glutathione) were used to analyze oxidative stress in liver tissue of mice. Real-time fluorescence quantitative PCR was used to detect the mRNA levels of inflammatory factors and hepatic fibrosis factor-related genes, to analyze the effect of asiatica acid on inflammation and hepatic fibrosis, to determine the expression level of genes related to bile acid metabolism, and to analyze the regulation pathway of bile acid metabolism. Anti-apoptosis mechanism; detection of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) protein expression level, to study the antioxidant mechanism of asiatica acid. In vitro experiment: HL-7702 cells were treated with GCDC to establish in vitro liver injury model, divided into control group, model group and different concentrations of asiatica acid treatment group, MTT method to detect cell viability. Annexin V/PI double staining was used to detect apoptosis by flow cytometry, the content of reactive oxygen species (ROS) was detected by DCFH-DA fluorescence probe, and the expression of Nrf2 protein was detected by immunofluorescence and Western blotting. Collagen deposition, inflammatory cell infiltration and asiatica acid administration can improve the liver morphology and pathological characteristics of BDL mice, reduce liver index and spleen index. In BDL model group, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AKP), hydroxyproline (Hyp), total cholesterol (T-CHO), total bilirubin Asiatic acid can significantly reduce the levels of these biochemical indicators, especially at high doses. Asiatic acid can also reduce the expression of alpha-SMA in liver tissue, reduce the expression of fibrosis factor type III collagen (Col3a1), vimentin (Vim), actin (Acta2), and transform growth factor. TGF-beta 1 mRNA expression level; pro-inflammatory factors including prostaglandin endoperoxide synthase 2 (Pgst 2), IL-6, TNF-a, chemokine ligand 3 (CCL3) mRNA expression level. Asiatic acid after administration, up-regulate Bcl 2, down-regulate Bax expression, reduce apoptosis of liver tissue cells. Asiatic acid administration can inhibit the oxygen content in liver tissue of BDL mice. In addition, asiatic acid could regulate the expression of genes related to bile acid metabolism pathway in BDL mice, such as FXR, CYP7alpha-hydroxylase (CYP7alpha1), small molecule heterodimer chaperone (SHP), fibroblasts. Asiatic acid can effectively protect HL-7702 cells from GCDC-induced injury. The results showed that Asiatic acid treatment can significantly increase the cell survival rate (P 0.001), reduce ROS production and apoptosis rate, and inhibit the proliferation of HL-7702 cells. CONCLUSION: Asiatic acid can significantly improve hepatic fibrosis, inhibit oxidative stress, inhibit inflammation, regulate bile acid metabolism, improve liver tissue morphology and function in BDL mice in vivo, and antagonize GCDC-induced injury in HL-7702 cells. The results of this study revealed that asiaticosic acid could improve the expression of Nrf2 and inhibit the increase of ROS. The mechanism may be related to anti-apoptosis, Nrf2-mediated antioxidation and regulation of bile acid metabolism.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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