【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(duì)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(jī)制,為其應(yīng)用于肝纖維化的治療提供實(shí)驗(yàn)依據(jù)。方法:本研究通過膽管結(jié)扎(BDL)誘導(dǎo)小鼠肝損傷及肝纖維化建立在體實(shí)驗(yàn)?zāi)Ｐ?并以甘氨鵝脫氧膽酸鈉(GCDC)處理HL-7702細(xì)胞建立體外肝損傷模型,通過在體和離體實(shí)驗(yàn)探討積雪草酸的抗肝纖維化作用及機(jī)制。在體實(shí)驗(yàn):結(jié)扎C57小鼠的總膽管,建立淤膽型肝損傷模型,分為假手術(shù)對(duì)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(biāo)水平來檢測肝功能。稱量小鼠肝臟及脾臟組織,并計(jì)算肝臟指數(shù)和脾臟指數(shù)。檢測肝組織內(nèi)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(yīng)激。將肝臟組織切片HE染色、Masson染色進(jìn)行病理學(xué)檢查,TUNEL染色檢測肝細(xì)胞凋亡;α-SMA(α-平滑肌肌動(dòng)蛋白)免疫組化及Western blot檢測肝纖維化程度。采用實(shí)時(shí)熒光定量PCR測定炎癥因子及肝纖維化因子相關(guān)基因的mRNA水平,分析積雪草酸對(duì)炎癥、肝纖維化的改善作用;測定膽汁酸代謝相關(guān)的基因表達(dá)水平,分析膽汁酸代謝調(diào)節(jié)途徑。Western blot法檢測Bax、Bcl-2的表達(dá)水平,分析積雪草酸的抗凋亡機(jī)制;檢測核因子E2相關(guān)因子2(Nrf2)、血紅素氧合酶-1(HO-1)的蛋白表達(dá)水平,研究積雪草酸的抗氧化機(jī)制。離體實(shí)驗(yàn):采用GCDC作用于HL-7702細(xì)胞建立體外肝損傷模型,分為對(duì)照組、模型組和不同濃度的積雪草酸處理組,MTT法檢測細(xì)胞活性,Annexin V/PI雙染法通過流式細(xì)胞儀檢測細(xì)胞凋亡,DCFH-DA熒光探針法檢測細(xì)胞中活性氧(ROS)的含量,細(xì)胞免疫熒光和蛋白免疫印跡法檢測Nrf2的蛋白表達(dá)水平。結(jié)果:在BDL小鼠模型中,我們發(fā)現(xiàn)BDL模型組小鼠肝臟腫大、欠光澤,肝組織出現(xiàn)膠原沉積、炎性細(xì)胞浸潤的現(xiàn)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(shù)和脾臟指數(shù)。BDL模型組小鼠血清中天冬氨酸轉(zhuǎn)氨酶(AST)、丙氨酸轉(zhuǎn)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(biāo)的水平,高劑量效果更明顯。同時(shí),積雪草酸能夠減少肝組織α-SMA的表達(dá);降低纖維化因子Ⅲ型膠原蛋白(Col3a1)、波形蛋白(Vim)、肌動(dòng)蛋白(Acta2)、轉(zhuǎn)化生長因子(TGF-β1)mRNA的表達(dá)水平;降低促炎因子包括前列腺素內(nèi)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(dá)水平。積雪草酸給藥作用后,上調(diào)Bcl2,下調(diào)Bax表達(dá),減少肝組織細(xì)胞的凋亡。積雪草酸給藥能夠抑制BDL小鼠肝組織的氧化應(yīng)激水平,表現(xiàn)為MDA含量的降低,SOD、GSH和Catalase水平的提高,促進(jìn)Nrf2和HO-1的蛋白表達(dá)。此外,積雪草酸給藥能夠調(diào)控BDL小鼠的膽汁酸代謝途徑相關(guān)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(xì)胞生長因子(FGF15)的mRNA水平,減少肝組織內(nèi)膽汁酸淤積。離體實(shí)驗(yàn)表明積雪草酸能夠有效的保護(hù)GCDC誘導(dǎo)的HL-7702細(xì)胞損傷。實(shí)驗(yàn)結(jié)果顯示,積雪草酸處理可以顯著增加細(xì)胞存活率(P0.001);減少細(xì)胞內(nèi)ROS生成,降低細(xì)胞凋亡率,抑制作用均呈濃度依賴性增強(qiáng);促進(jìn)HL-7702細(xì)胞表達(dá)Nrf2蛋白。結(jié)論:在體實(shí)驗(yàn)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(yīng)激,抑制炎癥反應(yīng),調(diào)控膽汁酸代謝,改善肝組織形態(tài)和功能;在細(xì)胞水平上,積雪草酸對(duì)抗GCDC誘導(dǎo)的HL-7702細(xì)胞損傷,抑制ROS的增加,減少細(xì)胞凋亡,促進(jìn)Nrf2的表達(dá)。本論文研究結(jié)果揭示積雪草酸有改善膽汁性肝纖維化的作用,其機(jī)制可能與抗凋亡以及Nrf2介導(dǎo)的抗氧化和調(diào)控膽汁酸代謝有關(guān)。
[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(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 李亞芳;霍麗娟;;肝纖維化藥物治療的研究進(jìn)展[J];國際消化病雜志;2016年04期

2 李萍;楊秋輝;王俊嶺;江智龍;;吡菲尼酮對(duì)牛血清蛋白誘導(dǎo)的肝纖維化大鼠肝損害的保護(hù)作用觀察[J];實(shí)用肝臟病雜志;2015年01期

3 Gülsüm ?zlem Elpek;;Cellular and molecular mechanisms in the pathogenesis of liver fibrosis:An update[J];World Journal of Gastroenterology;2014年23期

4 Albert J Czaja;;Hepatic inflammation and progressive liver fibrosis in chronic liver disease[J];World Journal of Gastroenterology;2014年10期

5 Maria J Perez;Oscar Briz;;Bile-acid-induced cell injury and protection[J];World Journal of Gastroenterology;2009年14期

6 Nuket Mas;Ilker Tasci;Bilgin Comert;Ramazan Ocal;Mehmet Refik Mas;;Ursodeoxycholic acid treatment improves hepatocyte ultrastructure in rat liver fibrosis[J];World Journal of Gastroenterology;2008年07期

，

本文編號(hào)：2226945