【作者】 王茜；

【导师】 朱依谆；

【作者基本信息】 复旦大学 ， 疾病蛋白组学， 2009， 博士

【摘要】 【目的】大蒜已被证实具有心脏保护作用,其活性成分主要为含硫化合物。本研究通过研究大蒜有机硫化合物及其类似物的心脏保护作用,比较不同的半胱氨酸衍生物对心肌细胞损伤的保护作用,筛选优势候选化合物,并探讨其对缺血性损伤心肌细胞的抗氧化和抗凋亡作用,进一步阐明其作用途径和机制,为临床有效治疗缺血性心脏病研发新药提供理论基础。【方法】1.以原代培养乳鼠心肌细胞为研究对象,细胞缺氧6 h复氧3 h制模,分为正常对照组、缺氧复氧模型组、七个半胱氨酸衍生物干预组。MTT法检测细胞存活率,检测细胞内及培养液中乳酸脱氢酶（lactate dehydrogenase,LDH）活性、细胞上清液H₂S浓度,并测定超氧化物歧化酶（superoxide dismutase,SOD）、过氧化氢酶（catalase,CAT）、还原型谷胱甘肽（GSH）、氧化型谷胱甘肽（GSSG）、脂质过氧化产物（MDA）和凋亡相关因子Caspase-3活性,采用RT-PCR方法观察CuZn-SOD和Mn-SOD、Caspase-3及胱硫醚-γ-裂解酶（cystathione-γ-lyase,CSE）mRNA的表达,Hoechst染色观察凋亡形态学变化。2.结扎大鼠左冠状动脉制备急性心肌梗死（myocardial infarction,MI）模型,分为假手术组、MI模型组以及SPC、SAC和SPRC预处理组。观察死亡率及梗死面积,检测大鼠血浆H₂S浓度和心室肌组织CSE活性,观察血清酶学变化及心肌组织中抗氧化酶SOD和谷胱甘肽过氧化物（glutathione peroxidase,Gpx）活性、GSH和GSSG氧化还原状态以及MDA含量,采用RT-PCR法观察SOD各分型及CSE mRNA的表达。3.采用急性心肌梗死大鼠模型,分为假手术组、MI模型组、炔丙基甘氨酸（Propargylglycine,PAG）干预组、SPRC预处理组及SPRC+PAG干预组。伊文氏兰与TTC双染检测心肌梗死面积,定位心脏梗死区和梗死周边区,并监测鼠尾动脉压和心率观察心功能改变,通过RT-PCR和western blot的方法观察凋亡相关因子mRNA和蛋白表达;4.采用急性心肌梗死大鼠模型,随机分为SPRC组和对照组。分别给予SPRC 50mg/kg/d或生理盐水灌胃,6周后处死动物。经右颈总动脉插管检测心室内压及动脉压,观察体重变化及全心湿重、肺重与体重比值,检测血清中SOD活性及MDA水平,并观察凋亡相关因子和心室重构相关的蛋白和mRNA的表达。【结果】第1部分:七种半胱氨酸衍生物均能一定程度的提高细胞存活率,减少细胞内LDH的漏出,增加GSH水平和CAT活性;SAC显著提高Mn-SOD和CuZn-SOD活性,并上调其mRNA表达;SPC、SBC、SPEC和SAMC显著提高CuZn-SOD的活性;SEC和SPRC则增加Mn-SOD的活性和mRNA表达。SAC、SEC、SPC、SAMC和SPRC降低Caspase-3活性。七种化合物中除了SEC外,细胞上清液中H₂S浓度显著上升,细胞内CSE mRNA表达上调。第2部分:SPC、SAC和SPRC预处理大鼠死亡率和梗死面积显著降低,与模型组相比有统计学意义（P＜0.05）,并伴随血清中H₂S浓度升高。另外,三个化合物均可增加SOD和GPx活性、维持心肌细胞内GSH水平、并降低MDA浓度。PAG为CSE抑制剂,可阻断三个化合物对心肌的保护作用。第3部分:SPRC预处理可改善心功能,维持心肌细胞内ATP水平,减小梗死面积。SPRC增加Bcl-2蛋白表达,与模型组相比,分别是模型组梗死周边区的1.8倍和梗死区的3倍;SPRC还可下调Bax蛋白表达,是模型组梗死周边区的0.88倍和梗死区的0.65倍。SPRC对Bcl-2和Bax mRNA的表达与其对蛋白表达的影响趋势相同。SPRC还可诱导CSE在心脏梗死及周边组织的表达,尤其梗死区更为显著,与模型组相比有统计学意义。SPRC增加血清中H₂S浓度,其抗凋亡活性可被PAG阻断。第4部分:SPRC组与模型组比较全心湿重与体重的比值有减小趋势,但没有统计学意义（P＞0.05）。模型组大鼠肺重与体重的比值较假手术组相比显著增加,SPRC组大鼠的肺重/体重较模型组明显降低（P＜0.05）,LVEDP升高程度减低,LVSP和±LVdp/dt_max较模型组均有升高,收缩功能改善;SPRC可增加血清中SOD活性,并降低MDA含量;形态学显示SPRC组心肌细胞变形与坏死改变程度较轻,范围较小,坏死周边区炎性细胞浸润明显减少;SPRC干预组大鼠血清H₂S浓度增加,在梗死周边区和非梗死区SPRC均呈现增加CSE活性的趋势。SPRC对大鼠梗死周边区和非梗死区凋亡相关因子Bax、Caspase-9和Caspase-3有显著的抑制作用;并下调心室重构相关的TGF-β1和胶原蛋白ⅠmRNA表达。【结论】1.半胱氨酸衍生物对乳鼠心肌细胞缺氧复氧诱导损伤具有保护作用,尤其是三碳化合物SPC、SAC和SPRC;2.SPC、SAC和SPRC对急性心肌梗死大鼠有预防性心脏保护作用,其作用机制与抗氧化和促进内源性H₂S生成相关,其中尤以SPRC心脏保护作用最为显著。3.SPRC的心脏保护作用与其调控线粒体凋亡途径关键酶和蛋白的表达、减少缺血心肌细胞凋亡有关;4.SPRC对心肌细胞凋亡的调控可能是通过激活CSE/H₂S通路,从而介导内源性H₂S生成所致。5.SPRC可介导内源性H2S生成,抑制心肌细胞凋亡和心室重构,对心肌梗死后心力衰竭的发生有治疗作用,值得深入研究。更多还原

【Abstract】 Objectives:To find novel compound which is useful to treat ischemic heart diseases,we compared the protective effects of 7 cysteine-containing compounds on neonatal rat cardiomyocytes（NRCs） injury induced by hypoxia/reoxygenation（H/R）, including S-allyl-L-cysteine（SAC）,S-ethyl-L-cysteine（SEC）,S-propyl-L-cysteine （SPC）,S-allylmercapto-L-cysteine（SAMC） and their analogues S-butyl-L-cysteine （SBC）,S-pentyl-L-cysteine（SPEC）,and S-propargyl-L-cysteine（SPRC）.Furthmore, to elucidate the role of SPRC,SAC and SPC on ischemic heart of rats and to explore the involved pathways,we evaluated the pharmacological effects of three cysteine containing compounds on H₂S production,apoptosis and antioxidant defences in an acute myocardial infarction（MI） rat model.Besides,we determined the therapeutical role of SPRC on chronic heart failure after myocardial infarction.This study will provide novel evidence for treatment of ischemic heart diseases.Methods:1.Methylthiazol tetrazoium（MTT） assay for cell viability,lactate dehydrogenase（LDH） release as an index of cytotoxicity and malondialdehyde（MDA） content indicating lipid peroxidation were measured.To study the mechanism, intracellular glutathione（GSH） level in NRCs,OH radical scavenging activity and activities of antioxidant enzymes including superoxide dismutase（SOD）,catalase （CAT）were determined.Hoechst 33342 staining was performed to observe apoptosis. Reverse transcriptional PCR（RT-PCR） were used to detect mRNA expression.2.SD rats were subjected to left coronary artery occlusion.Infarct size and mortality were determined.Plasma H₂S level and cystathionase-γ-lyase（CSE） activity in the ventricular tissues were determined spectrophotometrically.The enzymatic activities of SOD and glutathione peroxidase（Gpx）,glutathione redox status and MDA content were also determined.3.The infarct size was measured by Evan’s blue and TTC double staining and myocardial function reflected by blood pressure.The myocardial tissues in peri-infarct and infarct areas were used for gene and protein expression studies as well as myocardial metabolites analysis.4.Hemodynamic indexes including LVSP,LVEDP and±dp/dtmax were eassayed in each group after operation 6 weeks.The hearts and lungs were harvested to test heart weight index or lung weight index.HE staining was used to evaluate the pathological changes of heart following ischemia.Results:Part 1:Intervention with seven cysteine-containing compounds significantly protected the cardiomyocytes against H/R injury.All of the 7 compounds increased GSH level,and CAT activities.SAC restored both SODs activities and mRNA expression.SPC,SBC,SPEC and SAMC significantly preserved CuZn-SOD activity.And SEC and SPRC increased Mn-SOD activity and gene expression.SAC, SEC,SPC,SAMC and SPRC inhibit caspase-3 activity and apoptosis.These agents, except SEC,also significantly increased CSE mRNA expression and H₂S concentration in culture media.Part 2:SPC,SAC and SPRC treated animals demonstrated reduced mortality and infarct size as compared to the MI vehicle control group（P＜0.05）;this correlating with an increase in plasma H₂S concentrations.In addition,all three compounds were found to preserve SOD and GPx activities and tissue GSH content whilst reducing the formation of the lipid peroxidation product MDA in ventricular tissues.Propargylglycine,a selective inhibitor of CSE,abolished the protective effects of each compound in the current model.Part 3:The protective effects of SPRC were confirmed by significant reduction in infarct size,improvement in cardiac function and well preserved myocardial ATP level.Interestingly,SPRC was shown to increase Bcl-2 protein expression by 1.8- fold in peri-infarct area and 3.0-fold in infarct area and decrease Bax protein expression by 0.88-fold in peri-infarct and 0.65-fold in infarct region when compared to MI rats.The mRNA expressions of Bcl-2 and Bax showed similar patterns as the protein expressions. SPRC induced CSE in both areas,especially in infarct area compared with MI rats. Moreover,Plasma H₂S concentration was significantly higher in SPRC-pretreated group.These effects of SPRC were abolished by PACt Part 4:Heart weight index of SPRC-treated rats was decreased,but had no significant difference when compare with CHF vehicle rats（P＜0.05）.But the lung weight index was increased when compared to Sham group,and SPRC sharply decreased the lung weight index when compare with CHF vehicle group（P＜0.05）.Experimental results showed the increase of LVSP and±LVdp/dt_max in SPRC treated group.Cardiac function improved in SPRC-treated rats.SPRC preserved SOD activity and decreased MDA level with an increase in plasma H₂S concentrations in the plasma.There was no significant difference of Bcl-2 protein and mRNA expression in all treated groups.But SPRC decreased Bax,Caspase-9 and Caspase-3 mRNA expression in peri-infarct and remote area.And SPRC could inhibit the expression of TGF-β₁ and Collagen I,there are obvious statistical differences compared with CHF vehicle group（P＜0.05）Conclusion:1.The present study demonstrated that the cysteine-containing compounds have protective effects on NRCs H/R injury via the antioxidant and anti-apoptotic pathway,modulated by endogenous H₂S.The cysteine-containing compounds may be considered as valuable agents for ischemia heart disease, especially the three carbon chain compounds,such as SPC,SAC and SPRC.2.SPC, SAC and SPRC have cardioprotective effects in MI by reducing the deleterious effects of oxidative stress and by modulating the endogenous levels of H₂S and preserving the activities of antioxidant defencive enzymes like SOD.3.SPRC is efficacious in response to MI by inhibiting apoptosis.The properties are associated with the modulation of H₂S signaling pathways.4.The results indicated that SPRC have the cardioprotective effects on chronic heart failure after myocardial infarction, which was showed by improved heart function,decreased apoptosis in remote and peri-infarct area,and inhibit ventricular remodeling.Endogenous H₂S have partly involved in the cardioprotective effects of SPRC.更多还原