積雪草酸對小鼠膽管結扎誘導的肝纖維化的治療作用及機制的研究
發(fā)布時間：2018-09-06 16:45
【摘要】：目的:肝纖維化是許多肝臟疾病發(fā)展成為肝硬化的重要病理過程,目前還沒有療效良好的治療藥物。本課題研究積雪草酸對膽汁性肝纖維化小鼠的治療作用,并探討積雪草酸的作用機制,為其應用于肝纖維化的治療提供實驗依據。方法:本研究通過膽管結扎(BDL)誘導小鼠肝損傷及肝纖維化建立在體實驗模型,并以甘氨鵝脫氧膽酸鈉(GCDC)處理HL-7702細胞建立體外肝損傷模型,通過在體和離體實驗探討積雪草酸的抗肝纖維化作用及機制。在體實驗:結扎C57小鼠的總膽管,建立淤膽型肝損傷模型,分為假手術對照組、模型組、積雪草酸15 mg/kg組和30 mg/kg組,每組6只。各組小鼠連續(xù)灌胃給藥5天。測定血清生化指標水平來檢測肝功能。稱量小鼠肝臟及脾臟組織,并計算肝臟指數和脾臟指數。檢測肝組織內MDA(丙二醛),SOD(超氧化物氣化酶)和Catalase(過氧化氫酶)和GSH(還原型谷胱甘肽)分析小鼠肝組織氧化應激。將肝臟組織切片HE染色、Masson染色進行病理學檢查,TUNEL染色檢測肝細胞凋亡;α-SMA(α-平滑肌肌動蛋白)免疫組化及Western blot檢測肝纖維化程度。采用實時熒光定量PCR測定炎癥因子及肝纖維化因子相關基因的mRNA水平,分析積雪草酸對炎癥、肝纖維化的改善作用;測定膽汁酸代謝相關的基因表達水平,分析膽汁酸代謝調節(jié)途徑。Western blot法檢測Bax、Bcl-2的表達水平,分析積雪草酸的抗凋亡機制;檢測核因子E2相關因子2(Nrf2)、血紅素氧合酶-1(HO-1)的蛋白表達水平,研究積雪草酸的抗氧化機制。離體實驗:采用GCDC作用于HL-7702細胞建立體外肝損傷模型,分為對照組、模型組和不同濃度的積雪草酸處理組,MTT法檢測細胞活性,Annexin V/PI雙染法通過流式細胞儀檢測細胞凋亡,DCFH-DA熒光探針法檢測細胞中活性氧(ROS)的含量,細胞免疫熒光和蛋白免疫印跡法檢測Nrf2的蛋白表達水平。結果:在BDL小鼠模型中,我們發(fā)現BDL模型組小鼠肝臟腫大、欠光澤,肝組織出現膠原沉積、炎性細胞浸潤的現象,積雪草酸給藥后能夠改善BDL小鼠的肝組織形態(tài)和病理特征,降低肝臟指數和脾臟指數。BDL模型組小鼠血清中天冬氨酸轉氨酶(AST)、丙氨酸轉氨酶(ALT)、堿性磷酸酶(AKP)、羥脯氨酸(Hyp)、總膽固醇(T-CHO)、總膽紅素(TBIL)和總膽汁酸(TBA)的水平顯著上升,積雪草酸給藥能夠顯著降低這些生化指標的水平,高劑量效果更明顯。同時,積雪草酸能夠減少肝組織α-SMA的表達;降低纖維化因子Ⅲ型膠原蛋白(Col3a1)、波形蛋白(Vim)、肌動蛋白(Acta2)、轉化生長因子(TGF-β1)mRNA的表達水平;降低促炎因子包括前列腺素內過氧化物合成酶2(Pgst2)、IL-6、TNF-α、趨化因子配體3(CCL3)的mRNA表達水平。積雪草酸給藥作用后,上調Bcl2,下調Bax表達,減少肝組織細胞的凋亡。積雪草酸給藥能夠抑制BDL小鼠肝組織的氧化應激水平,表現為MDA含量的降低,SOD、GSH和Catalase水平的提高,促進Nrf2和HO-1的蛋白表達。此外,積雪草酸給藥能夠調控BDL小鼠的膽汁酸代謝途徑相關基因,法尼酯X受體(FXR)、膽固醇7α-羥化酶(CYP7α1)、小分子異源二聚體伴侶(SHP)、成纖維細胞生長因子(FGF15)的mRNA水平,減少肝組織內膽汁酸淤積。離體實驗表明積雪草酸能夠有效的保護GCDC誘導的HL-7702細胞損傷。實驗結果顯示,積雪草酸處理可以顯著增加細胞存活率(P0.001);減少細胞內ROS生成,降低細胞凋亡率,抑制作用均呈濃度依賴性增強;促進HL-7702細胞表達Nrf2蛋白。結論:在體實驗水平,積雪草酸具有顯著改善BDL鼠肝纖維化的作用,抑制氧化應激,抑制炎癥反應,調控膽汁酸代謝,改善肝組織形態(tài)和功能;在細胞水平上,積雪草酸對抗GCDC誘導的HL-7702細胞損傷,抑制ROS的增加,減少細胞凋亡,促進Nrf2的表達。本論文研究結果揭示積雪草酸有改善膽汁性肝纖維化的作用,其機制可能與抗凋亡以及Nrf2介導的抗氧化和調控膽汁酸代謝有關。
[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.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285.5
本文編號：2226945
[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.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285.5
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