【作者】 骆助林；

【导师】 田伏洲；

【作者基本信息】 第三军医大学 ， 外科学， 2013， 博士

【摘要】 研究背景和目的：非酒精性脂肪性肝病（nonalcoholic fatty liverdisease, NAFLD）是指除酒精和其他明确的损肝因素所致外，以弥漫性肝细胞大泡性脂肪变为主要特征的临床病理综合征,包括非酒精性单纯性脂肪肝以及由其演变的非酒精性脂肪性肝炎（nonalcoholicsteatohepatitis, NASH）和肝硬化。其中NASH是以肝细胞丧失、脂肪变性、气球样变、炎性坏死及纤维化形成为特征的病理状态。 NASH常见于II型糖尿病、高血压、高脂血症及肥胖患者。NASH的发病机制非常复杂，且未完全阐明。目前被多数学者所接受的是1998年由Day CP所提出的“二次打击学说”。在“初次打击”诱发肝细胞脂质沉积的前提下，以氧化应激和炎症反应为轴心的“二次打击”在NASH的发生发展中起重要作用。NASH的持续存在已成为肝硬化甚至肝癌的重要因素之一。其中15%-20%的NASH患者在10-20年内可进展为肝硬化。随着中国物质生活水平的日益提高，NASH的患病人数持续增加，其年龄也呈现低龄化趋势。针对NASH的药物研究也成为当今医学研究的热点之一。气体信号分子硫化氢（H₂S），作为一种气体递质，不需借助任何特殊的运输工具就可以自由快速地通过细胞膜， H₂S在心血管、呼吸、消化、神经等系统中具有重要生理作用。研究发现H₂S具有抗氧化应激、抗炎症反应，抗凋亡以及抗纤维化作用。内源性H₂S是否参与NASH发病过程，现在尚无研究。本课题的研究目的就是阐明内源性H₂S在大鼠NASH发病过程中的变化；并进一步通过给以外源性的H₂S进行治疗，阐明H₂S在大鼠NASH发病过程中的作用，同时为NASH的药物研究提供基础。方法：第一部分：观察大鼠NASH发病过程中内源性H₂S的变化营养诱发动物模型（食物喂养法）是NASH动物模型建立最常用的方法。其中蛋氨酸/胆碱缺乏饲料（，methionine-choline deficient diet,MCD）和高脂饲料（high-fatdiet,HFD）为诱导NASH的两种常见饲料。1. MCD诱导形成NASH大鼠模型，分1天，7天，3周，6周，8周时间梯度观察血清中H₂S水平，肝脏中H₂S的生成能力；应用RT-PCR法和Western blotting法考察肝脏中H₂S生成酶如胱硫醚-β-合酶（CBS）和胱硫醚-γ-裂解酶（CSE）的基因转录和蛋白表达的变化。2. HFD诱导形成NASH大鼠模型，分1天，7天，3周，6周，8周时间梯度观察血清中H₂S水平，肝脏中H₂S的生成能力；应用RT-PCR法和Western blotting法分别考察肝脏中H₂S生成酶如CBS和CSE的基因转录和蛋白表达的变化。第二部分：外源性H₂S对大鼠NASH肝损伤的保护作用1．MCD诱导形成NASH大鼠模型，同时腹腔注射硫氢化钠（NaHS，H₂S供体）治疗8周，观察肝脏病理损伤、肝功能损伤指标；观察肝细胞凋亡情况。2．HFD诱导形成NASH大鼠模型，同时腹腔注射硫氢化钠（NaHS，H₂S供体）治疗8周，观察肝脏病理损伤、肝功能损伤指标；观察肝细胞凋亡情况。第三部分：H₂S大鼠缓解NASH肝损伤的作用机制1．MCD诱导形成NASH大鼠模型，同时腹腔注射硫氢化钠（NaHS，H₂S供体）治疗8周，观察氧化应激相关指标与炎症反应相关指标；观察肝脏脂质沉积以及脂肪酸代谢基因表达；2．HFD诱导形成NASH大鼠模型，同时腹腔注射硫氢化钠（NaHS，H₂S供体）治疗8周，观察氧化应激相关指标与炎症反应相关指标；观察肝脏脂质沉积以及脂肪酸代谢基因表达；结果：第一部分：大鼠NASH发病过程中内源性H₂S的变化1. MCD饲料诱导形成NASH大鼠模型，3周后，血清和肝脏中H₂S水平降低，肝脏中CBS和CSE的mRNA水平和蛋白表达水平下降。2. HFD诱导形成NASH大鼠模型，6周时，发现血清和肝脏中H₂S水平降低，肝脏中CBS和CSE的mRNA水平和蛋白表达水平下降。第二部分：外源性H₂S对大鼠NASH肝损伤的保护作用1. MCD饲料诱导形成NASH大鼠模型，同时腹腔注射硫氢化钠（NaHS，H₂S供体）治疗8周。（1）病理检测：HE染色见正常对照组大鼠肝小叶结构完整，肝索排列整齐，肝细胞大小均匀，无变性坏死；MCD模型组大鼠肝脏肝小叶失去正常结构，肝板排列紊乱，肝细胞普遍脂肪变性，炎症坏死及出血明显，炎症细胞侵润，肝细胞气球样变。H₂S治疗组炎症细胞侵润和肝细胞气球样变得到明显减轻。（2）凋亡检测：TUNEL检测发现MCD模型组大鼠肝脏中凋亡细胞数目增加。H₂S治疗组与模型组相比，凋亡细胞数目减少。（3）肝脏损伤指标：MCD模型组大鼠血清中丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）水平增加；H₂S治疗组与模型组相比，ALT和AST水平降低。2. HFD诱导形成NASH大鼠模型，同时腹腔注射硫氢化钠（NaHS，H₂S供体）治疗8周。（1）病理检测：HE染色见正常对照组大鼠肝小叶结构完整，肝索排列整齐，肝细胞大小均匀，无变性坏死；HFD模型组大鼠肝脏肝小叶失去正常结构，肝板排列紊乱，肝细胞普遍脂肪变性，炎症坏死及出血明显，炎症细胞侵润，肝细胞气球样变。H₂S治疗组炎症细胞侵润和肝细胞气球样变得到明显减轻。天狼星红（Sirius Red）染色发现HFD模型组大鼠脏肝纤维化明显，H₂S治疗大鼠脏肝纤维化得以缓解。（2）凋亡检测：TUNEL检测发现HFD模型组大鼠肝脏中凋亡细胞数目增加；H₂S治疗组与HFD模型组相比，凋亡细胞数目减少。（3）肝脏损伤指标：HFD模型组大鼠血清中丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）水平增加；H₂S治疗组与模型组相比，ALT和AST水平降低。（4）胰岛素耐受：HFD模型组大鼠空腹血糖水平增加，空腹胰岛素水平变化不明显，胰岛素抵抗指数增加；H₂S治疗降低了HFD模型组大鼠的空腹血糖水平，降低了胰岛素抵抗指数。第三部分：外源性H₂S大鼠缓解NASH肝损伤的作用机制1．MCD饲料诱导形成NASH大鼠模型，同时腹腔注射硫氢化钠（NaHS，H₂S供体）治疗8周。（1）氧化应激相关指标：MCD模型组大鼠肝脏中丙二醛（MDA，脂质过氧化的标志分子）水平增加， CYP2E1和HO-1的mRNA和蛋白表达水平增加；H₂S治疗组与模型组相比，MDA水平降低，CYP2E1的mRNA和蛋白表达水平降低，而HO-1的mRNA和蛋白表达水平进一步增加。（2）炎症反应相关指标：MCD模型组大鼠肝脏中TNFα和IL-6的mRNA和蛋白表达水平增加，细胞浆中IκBα蛋白水平降低，细胞核中NFκB p65蛋白水平增加。H₂S治疗组与模型组相比，大鼠肝脏中TNFα和IL-6的mRNA和蛋白表达水平降低，细胞浆中IκBα蛋白水平增加，细胞核中NFκB p65蛋白水平减少。（3）肝脏脂质沉积以及相关脂肪酸代谢基因的表达：MCD模型组大鼠肝脏中胆固醇和甘油三脂的水平明显增加，PPARα, SREBP-1c, FAS,和L-FABP的mRNA水平降低，CD36, TLR-2和TLR-4的mRNA水平增加；H₂S治疗降低了MCD模型组大鼠肝脏中胆固醇和甘油三脂的水平，增加了PPARα和L-FABP的mRNA水平，降低了CD36,TLR-2和TLR-4的mRNA水平，进一步降低了SREBP-1c和FAS的mRNA水平。2．HFD诱导形成NASH大鼠模型，同时腹腔注射硫氢化钠（NaHS，H₂S供体）治疗8周。（1）肝脏损伤指标：HFD模型组大鼠血清中丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）水平增加；H₂S治疗组与模型组相比，ALT和AST水平降低。（2）氧化应激相关指标：HFD模型组大鼠肝脏中丙二醛（MDA，脂质过氧化的标志分子）水平增加， CYP2E1和HO-1的mRNA和蛋白表达水平增加；H₂S治疗组与HFD模型组相比，MDA水平降低， CYP2E1的mRNA和蛋白表达水平降低，而HO-1的mRNA和蛋白表达水平进一步增加。（3）炎症反应相关指标：HFD模型组大鼠肝脏中TNFα和IL-6的mRNA和蛋白表达水平增加，细胞浆中IκBα蛋白水平降低，细胞核中NFκB p65蛋白水平增加。H₂S治疗组与模型组相比，大鼠肝脏中TNFα和IL-6的mRNA和蛋白表达水平降低，细胞浆中IκBα蛋白水平增加，细胞核中NFκB p65蛋白水平减少。（6）肝脏脂质沉积以及相关脂肪酸代谢基因的表达：HFD模型组大鼠肝脏中胆固醇和甘油三脂的水平明显增加，PPARα, SREBP-1c, FAS,和L-FABP的mRNA水平降低，CD36, TLR-2和TLR-4的mRNA水平增加；H₂S治疗降低了HFD模型组大鼠肝脏中胆固醇和甘油三脂的水平，增加了PPARα和L-FABP的mRNA水平，降低了CD36,TLR-2和TLR-4的mRNA水平，进一步降低了SREBP-1c和FAS的mRNA水平。结论：本课题探索了H₂S在NASH（NASH）发病过程中的变化以及作用机制：一、在MCD或HFD诱导的NASH大鼠模型中，大鼠血液和肝脏中的H₂S水平都减少。H₂S水平的明显变化提示该分子可能参与NASH的发病过程。二、在MCD或HFD诱导的NASH大鼠模型中给以外源性H₂S治疗发现，NASH的病变状态明显减弱，凋亡减少，胰岛素耐受情况得到好转（仅限于HFD诱导的NASH模型），肝脏中脂质沉积水平降低，氧化应激和炎症反应减轻。结果提示NASH过程中H₂S水平的降低可能是NASH恶化的因素之一，内源性的H₂S本身可能是体内的防御分子之一，提示了应用外源性H₂S治疗NASH患者的潜在价值，为临床的H₂S治疗应用提供理论依据。另外也广泛探讨了H₂S作为一种肝脏保护分子的作用机制，其机制涉及对氧化应激，炎症反应，胰岛素耐受和脂肪酸代谢蛋白的调控。更多还原

【Abstract】 Objective：Non-alcoholic fatty liver disease （NAFLD） is the most major chronic liver dysfunctionin the world and its prevalence in the China is increasing with epidemics of obesity,diabetes, and metabolic syndrome. NAFLD is defined as the spectrum of benign fatty liver（steatosis） to necro-inflammation and fibrosis or non-alcoholic steatohepatitis （NASH） thatcan lead to cirrhosis and hepatocellular cancer, in the absence of excessive alcoholingestion.In the transition from benign steatosis to NASH,“two hit theory” is assumed to berequired, with the first hit being lipid accumulation in hepatocytes increasing the sensitivityof the liver to the second hit such as oxidative stress and pro-inflammatory cytokines,which are expected to be the promising targets in the treatment of this disease.Hydrogen sulfide （H₂S） was best known as a foul smelling and toxic gas before beingrecognized as an important mediator in several biological systems including neurological,cardiovascular, and gastrointestinal systems. H₂S is endogenously produced in mammaliantissues by cystathionine γ-lyase （CSE） and cystathionine β-synthase （CBS）.Given that H₂S displays anti-oxidative, anti-inflammatory and cytoprotective activities,an anti-fibrotic effect against pulmonary fibrosis, and a protective effect against CCl4-induced injury in liver, we hypothesized that H₂S might attenuate the methionine-choline-deficient diet （MCD） or high-fat diet （HFD） induced NASH in rats.Methods：Part I:1. A MCD rat model was prepared. Rats were divided into three experimental groupsand fed for8weeks as follows:（1） control rats;（2） MCD-fed rats. Measurement of H₂Slevels in plasma and livers of rats was performed by using ELIT Ion Analyzer. The mRNAexpression and protein expression of CBS and CSE were determined by RT-PCR method and Western blotting analysis, respectively.2. A HFD rat model was prepared. Rats were divided into three experimental groupsand fed for8weeks as follows:（1） control rats;（2） HFD-fed rats. Measurement of H₂Slevels in plasma and livers of rats was performed by using ELIT Ion Analyzer. The mRNAexpression and protein expression of CBS and CSE were determined by RT-PCR methodand Western blotting analysis, respectively.Part II:1. A MCD rat model was prepared. Rats were divided into three experimental groupsand fed for8weeks as follows:（1） control rats;（2） MCD-diet-fed rats;（3） MCD-diet-fedrats treated with NaHS （intraperitoneal injection of0.1ml/kg/d of0.28mol/l NaHS, adonor of H₂S）. Assessments of steatohepatitis by HE staining, apoptosis by TUNEL, ALTand AST activities were performed to investigate the effect of H₂S on MCD-inducedNASH.2. A HFD rat model was prepared. Rats were divided into three experimental groupsand fed for8weeks as follows:（1） control rats;（2） HFD-diet-fed rats;（3） HFD-diet-fed ratstreated with NaHS （intraperitoneal injection of0.1ml/kg/d of0.28mol/l NaHS, a donor ofH₂S）. Assessments of steatohepatitis by HE staining, apoptosis by TUNEL, ALT and ASTactivities and insulin resistant were performed to investigate the effect of H₂S onHFD-induced NASH.Part III:1. A MCD rat model was prepared. Rats were divided into three experimental groupsand fed for8weeks as follows:（1） control rats;（2） MCD-diet-fed rats;（3） MCD-diet-fedrats treated with NaHS （intraperitoneal injection of0.1ml/kg/d of0.28mol/l NaHS, adonor of H₂S）. Oxidative stress, inflammation, and expression profiles of fatty acidmetabolism genes in livers were measured to investigate the mechanism of the H₂Sprotective effect on HFD-induced NASH.2.A HFD rat model was prepared. Rats were divided into three experimental groupsand fed for8weeks as follows:（1） control rats;（2） HFD-diet-fed rats;（3） HFD-diet-fed ratstreated with NaHS （intraperitoneal injection of0.1ml/kg/d of0.28mol/l NaHS, a donor ofH₂S）. Oxidative stress, inflammation, and expression profiles of fatty acid metabolism genes in livers were measured to investigate the mechanism of the H₂S protective effect onHFD-induced NASH.Results：Part I:1. After3weeks, MCD led to reduced plasma H₂S level and hepatic H₂S content inrats. MCD suppressed CBS and CSE mRNA and protein expression in livers of rats.2. After6weeks, HFD led to reduced plasma H₂S level and hepatic H₂S content inrats. HFD suppressed CBS and CSE mRNA and protein expression in livers of rats.Part II:1. Effect of treatment with exogenous H₂S in MCD-fed rats[1] Treatment with exogenous H₂S in MCD-fed rats increased plasma H₂S levels andhepatic H₂S content, further decreased hepatic CSE expression, but had no significant effecton hepatic CBS expression.[2] Serum levels of glucose, total cholesterol, and triglycerides were lower inMCD-fed rats than that in control rats. Treatment of MCD-fed rats with H₂S had nosignificant effect on serum glucose, total cholesterol, and triglycerides.[3] Hepatic content of cholesterol and triglycerides were higher in MCD-fed rats thanthat in control rats. Treatment with H₂S in MCD-fed rats resulted in a significant reductionof hepatic cholesterol and triglycerides.[4] HE staining revealed macrovesicular steatosis and inflammation in the livers ofMCD-fed rats, which was attenuated by treatment with H₂S. The increased TUNEL-positive cells revealed increased apoptosis in the livers of MCD-fed rats, which wasalleviated by treatment with H₂S. MCD led to increased activities of ALT and AST in serum,which was restored partly by treatment with H₂S.2. Effect of treatment with exogenous H₂S in HFD-fed rats[1] Treatment with exogenous H₂S in HFD-fed rats increased plasma H₂S levels andhepatic H₂S content, further decreased hepatic CSE expression, but had no significant effecton hepatic CBS expression.[2] Serum levels of glucose, total cholesterol, and triglycerides were higher inHFD-fed rats than that in control rats. Treatment of HFD-fed rats with H₂S reduced serum glucose, but had no significant effect on total cholesterol, and triglycerides in serum.[3] Hepatic content of cholesterol and triglycerides were higher in HFD-fed rats thanthat in control rats. Treatment with H₂S in HFD-fed rats resulted in a significant reductionof hepatic cholesterol and triglycerides.[4] HE staining revealed macrovesicular steatosis and inflammation in the livers ofHFD-fed rats, which was attenuated by treatment with H₂S. The increased TUNEL-positivecells revealed increased apoptosis in the livers of HFD-fed rats, which was alleviated bytreatment with H₂S. HFD led to increased activities of ALT and AST in serum, which wasrestored partly by treatment with H₂S.Part III:1. Effect of treatment with exogenous H₂S in MCD-fed rats[1] MCD led to increased MDA formation in livers, which were restored by treatmentwith H₂S. Hepatic mRNA and protein expression of CYP2E1and HO-1were higher inMCD-fed rats than that in control rats. Treatment with H₂S reduced CYP2E1expression,but further increased HO-1expression in livers of MCD-fed rats.[2] MCD induced hepatic NFκB activation, which was suppressed by treatment withH₂S. MCD led to upregulation of hepatic mRNA and protein expression of TNF-α and IL-6,which was restored by treatment with H₂S.[3] Hepatic mRNA levels of PPARα, SREBP-1c, FAS, and L-FABP weredownregulated; hepatic mRNA levels of CD36, TLR-2, and TLR-4were upregulated inMCD rats. Treatment of MCD-fed rats with H₂S increased hepatic mRNA levels of PPARαand L-FABP, and reduced hepatic mRNA levels of CD36, SREBP-1c, FAS, TLR-2, andTLR-4.2. Effect of treatment with exogenous H₂S in HFD-fed rats[1] HFD led to increased MDA formation in livers, which were restored by treatmentwith H₂S. Hepatic mRNA and protein expression of CYP2E1and HO-1were higher inHFD-fed rats than that in control rats. Treatment with H₂S reduced CYP2E1expression, butfurther increased HO-1expression in livers of HFD-fed rats.[2] HFD induced hepatic NFκB activation, which was suppressed by treatment withH₂S. HFD led to upregulation of hepatic mRNA and protein expression of TNF-α and IL-6,which was restored by treatment with H₂S. [3] Hepatic mRNA levels of PPARα, SREBP-1c, FAS, and L-FABP weredownregulated; hepatic mRNA levels of CD36, TLR-2, and TLR-4were upregulated inHFD rats. Treatment of HFD-fed rats with H₂S increased hepatic mRNA levels of PPARαand L-FABP, and reduced hepatic mRNA levels of CD36, SREBP-1c, FAS, TLR-2, andTLR-4.Conclusion：Endogenous H₂S formation was found suppressed in development of NASH inducedby MCD and HFD in rats.Treatment with H₂S could attenuate MCD and HFD-induced NASH in rats.Treatment with H₂S could abate MCD and HFD-induced oxidative stress in livers.Treatment with H₂S could inhibit MCD and HFD-induced inflammation in livers.Treatment with H₂S could attenuate HFD-induced insulin resistant in rats.Treatment with H₂S could alleviate MCD and HFD-induced lipid accumulation andhave a beneficial modulation on expression profiles of fatty acid metabolism genes inlivers.更多还原