硫化氢对大鼠急性心肌缺血的影响
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摘要
心肌缺血是指心脏的血液灌注减少,导致心脏的供氧减少,心肌能量代谢不正常,不能支持心脏正常工作的一种病理状态,严重威胁人类的身心健康,因而对于缺血心肌保护的研究是倍受关注的课题。大量动物实验及临床试验均表明,细胞凋亡是急性心肌缺血损伤发病机制的重要环节之一,线粒体在细胞整合各种凋亡刺激信号的过程中处于枢纽地位。
作为继一氧化氮(nitric oxide,NO)和一氧化碳(carbon monoxide,CO)之后的第三种重要化学递质和细胞内信号分子—硫化氢(hydrogen sulfide,H2S)一直倍受医学界的关注。内源性H2S主要由半胱氨酸等含硫氨基酸在胱硫醚-β-合成酶(cystathionine-β-synthase,CBS)和胱硫醚-γ-裂解酶(cystathionine-γ-lyase,CSE)作用下生成的,人们对其在体内各种生理和病理过程的调节作用进行了大量研究,发现其在舒张血管、抑制血管重构和保护心肌等方面具有重要的生理作用,并且与多种心血管疾病相关。近年来研究发现,H2S在心肌缺血再灌注损伤中起着十分重要的作用,对缺血再灌注损伤心肌具有保护作用,但关于H2S在心肌缺血中的作用,尚未见报道。因此,本实验通过建立心肌缺血动物模型,首先观察在心肌缺血过程中H2S和CSE的时程性变化,在此基础上给予H2S供体硫氢化钠(sodium hydrosulfide,NaHS),观察其对心肌损伤的改善作用,并探讨其心肌保护作用机制,为其临床应用提供实验依据。
第一部分急性心肌缺血大鼠内源性硫化氢/胱硫醚-γ-裂解酶体系的变化
目的:观察大鼠急性心肌缺血过程中不同时间内源性H2S/CSE体系的变化。
方法:健康雄性SD大鼠(270±20g)共80只,随机分为6组①假手术组;②缺血1 h组;③缺血3 h组;④缺血6h组;⑤缺血9h组;⑥缺血12h组,假手术组共40只(不同时间点各8只)。大鼠麻醉后连接心电图电极,记录术前Ⅱ导联心电图,消毒手术区域,结扎左冠状动脉前降支2分钟后再次记录心电图的变化,以S-T段较术前抬高0.15mv以上作为模型成功,假手术组只穿线不结扎。分别于1 h、3 h、6 h、9 h和12 h后Powerlab/8s多导生理仪记录各组大鼠缺血末左室收缩峰压(LVSP)、左室舒张末压(LVEDP)、左室压力变化速率(±dp/dtmax)、计算左室发展压(LVDP),经颈总动脉取血并迅速开胸取心脏,采用去蛋白法检测血浆中H2S含量,亚甲基蓝法检测心肌组织中CSE的活性,电镜观察心肌组织结构的变化。
结果:
1假手术组大鼠LVEDP、±dp/dtma、LVDP在缺血后1h～12h之间无明显变化。与相应的假手术组比较,缺血1h时大鼠上述各项指标均无明显变化,缺血3h时±dp/dtmax、LVDP明显降低(P<0.05或P<0.01),LVEDP明显升高(P<0.01),随着时间的延长,缺血6h、9h和12h时LVDP、±dp/dtmax明显降低,LVEDP明显升高(P<0.05或P<0.01)。
2假手术组大鼠血浆中H2S含量和心肌组织CSE活性在缺血后1h～12h之间无明显变化。与相应的假手术组比较,缺血1h时大鼠血浆中H2S含量和心肌组织CSE活性无明显变化,随着时间的延长,缺血3h、6h、9h和12h大鼠血浆中H2S含量和心肌组织CSE活性均明显降低(P<0.05或P<0.01)。
3电镜下观察心肌组织的超微结构,假手术组大鼠心肌细胞结构清晰,肌原纤维排列整齐,线粒体膜完整,嵴清晰。缺血1h组大鼠心肌组织的超微结构基本正常。缺血3h、6h、9h和12h组大鼠心肌组织超微结构明显受损,核周胞质有水肿,核膜消失,肌原纤维断裂,线粒体嵴和膜溶解、消失,胞浆内糖原包涵体增多,提示代谢障碍。甚至有的线粒体髄样化,呈同心圆排列,电子密度增高。
结论:大鼠缺血1h时心肌组织未出现明显损伤,缺血3h至12h时均存在组织损伤,内源性H2S含量和CSE活性均明显降低。提示H2S/CSE体系可能参与急性心肌缺血的病理生理过程。
第二部分硫化氢对心肌缺血大鼠线粒体功能及细胞凋亡的影响
目的:观察H2S对大鼠急性心肌缺血的影响并从线粒体功能及细胞凋亡方面探讨其作用机制。
方法:健康成年雄性SD大鼠(270±20g)共40只,随机分为5组,每组8只,分别为Ⅰ假手术组;Ⅱ缺血组;Ⅲ缺血+NaHS低剂量组;Ⅳ缺血+NaHS中剂量组;Ⅴ缺血+NaHS高剂量组。用10%的水合氯醛350mg/kg麻醉大鼠,结扎左冠状动脉前降支,记录心电图的变化, NaHS低、中、高剂量组分别于缺血3h时腹腔注射0.78 mg/kg、1.56mg/kg和3.12 mg/kg的NaHS,缺血组注射等容量的生理盐水,假手术组只穿线不结扎。各组大鼠均于缺血6h时经颈总动脉取血迅速开胸取心脏,采用去蛋白的方法检测血浆中H2S含量,亚甲基蓝法检测心肌组织中CSE的活性,差速离心法提取心肌线粒体,测定线粒体活性、膜肿胀度,以及线粒体总ATP酶、谷胱甘肽过氧化物酶(GSH-PX)、超氧化物岐化酶(SOD)和丙二醛(MDA)含量,流式细胞术(FCM)检测心肌细胞凋亡率,免疫组化法测定Bcl-2、Bax蛋白的表达,HE染色观察心肌组织学变化。
结果:
1与假手术组大鼠比较,缺血组大鼠血浆中H2S含量和心肌组织中CSE活性明显降低(P<0.05)。与缺血组大鼠比较,缺血+NaHS低、中、高剂量组大鼠血浆中H2S含量和心肌组织中CSE活性明显升高(P<0.05或P<0.01)。
2与假手术组大鼠比较,缺血组大鼠心肌线粒体总ATP酶、GSH-PX活性、SOD活性明显降低(P<0.01),MDA含量明显升高(P<0.01),心肌线粒体膜肿胀、线粒体活力下降,表现为540nm和570nm处吸光度值下降(P<0.01),与缺血组比较,缺血+NaHS低、中、高剂量组大鼠心肌线粒体总ATP酶、GSH-PX活性、SOD活性升高,MDA含量降低,心肌线粒体膜肿胀、线粒体活力明显改善(P<0.05或P<0.01)。
3与假手术组大鼠比较,缺血组大鼠心肌细胞凋亡率、Bax表达阳性细胞数百分率明显升高(P<0.01),缺血+NaHS低、中、高剂量组大鼠心肌细胞凋亡率、Bax表达阳性细胞数百分率明显低于缺血组,Bcl-2表达阳性细胞数百分率明显升高(P<0.05或P<0.01)。
4 HE染色光镜观察心肌组织形态学变化:假手术组大鼠心肌细胞排列整齐,着色均匀。缺血组大鼠部分区域心肌纤维横纹不齐或消失,核偏移甚至裂解消失,有炎性因子渗出,NaHS低、中、高剂量组大鼠也有心肌细胞变性的改变,但程度较缺血组轻。
结论:硫化氢对大鼠急性心肌缺血具有一定的保护作用,改善线粒体功能,上调凋亡抑制蛋白Bcl-2的表达,下调促凋亡蛋白Bax表达,抑制细胞凋亡可能是其保护作用机制之一。
Myocardial ischemia is the reduction of hemoperfusion and the myocardial energy metabolism is not normal, which can not support the heart to work.Several pathogenic mechanisms have been proposed to explain the myocardial damage that occurs during myocardial ischemia injury. In recent years, oxidative stress has been shown to play a pivotal role in the pathogenesis of ischemia injury. Apoptosis is the cellular basis in oxidative stress induced by ischemia injury. Mitochondria as oxidative stress sensor and death signal integrator plays a key role in initiating and mediating apoptosis of cardiomyocytes suffered from ischemia damage.
Hydrogen sulfide (H2S) is a colorless,water soluble,flammable gas that has the characteristic smell of rotten eggs. Like other members of the gasotransmitter family (nitric oxide and carbon monoxide), H2S has traditionally been considered to be a highly toxic gas and environmental hazard. However, much like for nitric oxide and carbon monoxide, the initial negative perception of H2S has evolved with the discovery that H2S is produced enzymatically in mammals under normal conditions. Endogenous H2S is mainly producted from cysteine and other sulfur-containing amino acids by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). It has been shown that H2S plays an important physiological role in diastolic blood vessels, inhibition vascular remodeling and protection of the myocardium. It has also been found that H2S plays a very important role in myocardial ischemia-reperfusion and has a protective effect. The role of H2S in myocardial ischemia has not been previously studied. We therefore investigated the effect of H2S in a rat model of myocardial ischemia in vivo.
Part 1 Change of endogenous hydrogen sulfide/cystathionine-γ-lyase system in acute myocardial ischemia rats
Objective: To observe the changes of H2S/CSE system in acute myocardial ischemia rats and to investigate the effect of H2S on acute myocardial ischemia rats.
Methods: Eighty male rats were randomly divided into six groups.①sham group;②ischemia 1h group;③ischemia 3h group;④ischemia 6h group;⑤ischemia 9h group;⑥ischemia 12h group. All rats were subjected to openning chest then the left anterior desending coronaries(LADC) were ligated, while the LADC was not ligated in sham group rats. The electrocardiogram(ECG) was recorded under anesthetized condition.The cardiac function indexes such as the left ventricular systolic pressure(LVSP), the left ventricular developed pressure (LVDP), the left ventricular end diastolic pressure (LVEDP),±dp/dtmax were respectively recorded at 1,3,6,9 or 12 hour after ligation.The content of H2S in plasma and the activity of CSE in myocardium were respectively detected. The pathological changes of myocardium were obersved by electron microscope.
Results:
1 In sham group , the LVEDP, LVDP,±dp/dtmax were not altered from 1 hour to 12 hour after ligating LADC. The LVEDP, LVDP,±dp/dtmax were not altered at 1 hour after ischemia compared with those of sham group(P > 0.05) and the LVDP,±dp/dtmax were significantly decreased and the LVEDP was markedly increased at 3 hour after ischemia compared with those of sham group(P﹤0.05, P﹤0.01), during 6 hour to 12 hour after ligating LADC the cardiac function parameters were significantly altered compared with those of sham group (P < 0.05, P < 0.01).
2 In sham group, the content of H2S in plasma and the activity of CSE in myocardium were not altered from 1 hour to 12 hour. The content of H2S in plasma and the activity of CSE in myocardium were not altered at 1 hour after ischemia compared with those of sham group rats ( P > 0.05). The content of H2S in plasma and the activity of CSE in myocardium were significantly decreased at 3 hour after ischemia compared with those of sham group ( P < 0.01). During 6 hour to 12 hour after ischemia, the content of H2S in plasma and the activity of CSE in myocardium were significantly decreased compared with those of sham group (P < 0.05, P < 0.01).
3 In sham group, the structure of myocardium were intact. The structure of myocardium were not altered at 1 hour after ischemia compared with that of sham group rats. In ischemia 3 h, 6 h, 9 h and 12 h groups the myocardium were injuried, such as perinuclear cytoplasmic edema, nuclear membrane disappearance myofibrillar fragmentation, mitochondrial cristae and membrane dissolution, disappearance.
Conclusion:The content of H2S in plasma and the activity of CSE in myocardium and the structure of myocardium were not altered at 1 hour after ischemia. During 3 hour to 12 hour after myocardial ischemia the content of H2S in plasma and the activity of CSE in myocardium were markedly decreased .It could be concluded that H2S/CSE system may be play a role in the physiopathologic process of acute myocardial ischemia.
Part 2 Effects of hydrogen sulfide on the function of mitochondria and apoptosis in acute myocardial ischemia rats
Objective: To study the effect of H2S on acute myocardial ischemia and explore its possible molecular mechanisms from the function of mitochondria and apoptosis in rats.
Methods: Fourty male rats were randomly divided into five groups (n=8).①sham group;②ischemia group;③ischemia+NaHS Low dose group;④ischemia +NaHS Middle dose group;⑤ischemia+NaHS High dose group. The left anterior descending coronary was ligatded in ischemia group rats while it was not ligated in sham group rats. In ischemia + NaHS Low (0.78mg/kg),Middle(1.56mg/kg) and High(3.12mg/kg) dose groups rats sodium hydrosulfide (NaHS) were respectively administrated at 3 hour after ischemia. The rats were respectively killed at 6 hour after ischemia. The content of H2S in plasma and the activity of CSE in myocardium were respectively detected. The mitochondria were prepared by differential centrifugation.The swelling and activity of mitochondria were determined. The activities of SOD, GSH-PX and ATPase, and the contents of malondialdehyde (MDA) in myocardial mitochondria were measured. The apoptotic rate of cardiomyocytes was evaluated by Flow Cytometry. The positive expressions of Bcl-2 and Bax in cardiomyocytes were respectively detected by immunohistochemistry.
Results:
1 The content of H2S in plasma and the activity of CSE in myocardium were significantly decreased in ischemia group rats compared with those of sham group (P < 0.05). Compared with those of ischemia group, the content of H2S in plasma and the activity of CSE in myocardium were significantly increased in ischemia+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01).
2 In ischemia group, the swelling of mitochondria and the activity of mitochondria were markedly decreased (P﹤0.01) and the activities of SOD, GSH-PX and ATPase were significantly decreased and the content of malondialdehyde (MDA) in myocardial mitochondria was significantly increased(P﹤0.01) compared with those of sham group. In ischemia+NaHS Low, Middle and High dose groups , the activities of SOD, GSH-Px and ATPase and the activity of myocardium mitochondria were significantly enhanced.The swelling of mitochondria was markedly ameliorated and the content of MDA was significantly decreased compared with those of ischemia groups(P < 0.05 or P < 0.01).
3 The apoptotic rate of cardiomyocytes and the expression of Bax in ischemia group were significantly increased compared with those of sham group (P<0.01). Compared with those of ischemia group, the apoptotic rates of cardiomyocytes and the expression of Bax were markedly decreased and the expression of Bcl-2 was markedly increased in ischemia +NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01).
4 In sham group , myocardial cells arranged in neat rows and colored evenly. Myocardial fiber striped or disappeared, the nuclear migrated and even cracking disappeared and mang inflammatory cells infiltrates in ischemia group compared with that of sham group. The injury of the myocardium was decreased in NaHS Low, Middle and High dose groups compared with that of ischemia group .
Conclusion:It could be concluded that H2S has a beneficial myocardial protection against the ischemia injury in rats by ameliorating the function of myocardium mitochondria, upregulating the expression of Bcl-2,downreg- ulating the expression of Bax.
作为继一氧化氮(nitric oxide,NO)和一氧化碳(carbon monoxide,CO)之后的第三种重要化学递质和细胞内信号分子—硫化氢(hydrogen sulfide,H2S)一直倍受医学界的关注。内源性H2S主要由半胱氨酸等含硫氨基酸在胱硫醚-β-合成酶(cystathionine-β-synthase,CBS)和胱硫醚-γ-裂解酶(cystathionine-γ-lyase,CSE)作用下生成的,人们对其在体内各种生理和病理过程的调节作用进行了大量研究,发现其在舒张血管、抑制血管重构和保护心肌等方面具有重要的生理作用,并且与多种心血管疾病相关。近年来研究发现,H2S在心肌缺血再灌注损伤中起着十分重要的作用,对缺血再灌注损伤心肌具有保护作用,但关于H2S在心肌缺血中的作用,尚未见报道。因此,本实验通过建立心肌缺血动物模型,首先观察在心肌缺血过程中H2S和CSE的时程性变化,在此基础上给予H2S供体硫氢化钠(sodium hydrosulfide,NaHS),观察其对心肌损伤的改善作用,并探讨其心肌保护作用机制,为其临床应用提供实验依据。
第一部分急性心肌缺血大鼠内源性硫化氢/胱硫醚-γ-裂解酶体系的变化
目的:观察大鼠急性心肌缺血过程中不同时间内源性H2S/CSE体系的变化。
方法:健康雄性SD大鼠(270±20g)共80只,随机分为6组①假手术组;②缺血1 h组;③缺血3 h组;④缺血6h组;⑤缺血9h组;⑥缺血12h组,假手术组共40只(不同时间点各8只)。大鼠麻醉后连接心电图电极,记录术前Ⅱ导联心电图,消毒手术区域,结扎左冠状动脉前降支2分钟后再次记录心电图的变化,以S-T段较术前抬高0.15mv以上作为模型成功,假手术组只穿线不结扎。分别于1 h、3 h、6 h、9 h和12 h后Powerlab/8s多导生理仪记录各组大鼠缺血末左室收缩峰压(LVSP)、左室舒张末压(LVEDP)、左室压力变化速率(±dp/dtmax)、计算左室发展压(LVDP),经颈总动脉取血并迅速开胸取心脏,采用去蛋白法检测血浆中H2S含量,亚甲基蓝法检测心肌组织中CSE的活性,电镜观察心肌组织结构的变化。
结果:
1假手术组大鼠LVEDP、±dp/dtma、LVDP在缺血后1h～12h之间无明显变化。与相应的假手术组比较,缺血1h时大鼠上述各项指标均无明显变化,缺血3h时±dp/dtmax、LVDP明显降低(P<0.05或P<0.01),LVEDP明显升高(P<0.01),随着时间的延长,缺血6h、9h和12h时LVDP、±dp/dtmax明显降低,LVEDP明显升高(P<0.05或P<0.01)。
2假手术组大鼠血浆中H2S含量和心肌组织CSE活性在缺血后1h～12h之间无明显变化。与相应的假手术组比较,缺血1h时大鼠血浆中H2S含量和心肌组织CSE活性无明显变化,随着时间的延长,缺血3h、6h、9h和12h大鼠血浆中H2S含量和心肌组织CSE活性均明显降低(P<0.05或P<0.01)。
3电镜下观察心肌组织的超微结构,假手术组大鼠心肌细胞结构清晰,肌原纤维排列整齐,线粒体膜完整,嵴清晰。缺血1h组大鼠心肌组织的超微结构基本正常。缺血3h、6h、9h和12h组大鼠心肌组织超微结构明显受损,核周胞质有水肿,核膜消失,肌原纤维断裂,线粒体嵴和膜溶解、消失,胞浆内糖原包涵体增多,提示代谢障碍。甚至有的线粒体髄样化,呈同心圆排列,电子密度增高。
结论:大鼠缺血1h时心肌组织未出现明显损伤,缺血3h至12h时均存在组织损伤,内源性H2S含量和CSE活性均明显降低。提示H2S/CSE体系可能参与急性心肌缺血的病理生理过程。
第二部分硫化氢对心肌缺血大鼠线粒体功能及细胞凋亡的影响
目的:观察H2S对大鼠急性心肌缺血的影响并从线粒体功能及细胞凋亡方面探讨其作用机制。
方法:健康成年雄性SD大鼠(270±20g)共40只,随机分为5组,每组8只,分别为Ⅰ假手术组;Ⅱ缺血组;Ⅲ缺血+NaHS低剂量组;Ⅳ缺血+NaHS中剂量组;Ⅴ缺血+NaHS高剂量组。用10%的水合氯醛350mg/kg麻醉大鼠,结扎左冠状动脉前降支,记录心电图的变化, NaHS低、中、高剂量组分别于缺血3h时腹腔注射0.78 mg/kg、1.56mg/kg和3.12 mg/kg的NaHS,缺血组注射等容量的生理盐水,假手术组只穿线不结扎。各组大鼠均于缺血6h时经颈总动脉取血迅速开胸取心脏,采用去蛋白的方法检测血浆中H2S含量,亚甲基蓝法检测心肌组织中CSE的活性,差速离心法提取心肌线粒体,测定线粒体活性、膜肿胀度,以及线粒体总ATP酶、谷胱甘肽过氧化物酶(GSH-PX)、超氧化物岐化酶(SOD)和丙二醛(MDA)含量,流式细胞术(FCM)检测心肌细胞凋亡率,免疫组化法测定Bcl-2、Bax蛋白的表达,HE染色观察心肌组织学变化。
结果:
1与假手术组大鼠比较,缺血组大鼠血浆中H2S含量和心肌组织中CSE活性明显降低(P<0.05)。与缺血组大鼠比较,缺血+NaHS低、中、高剂量组大鼠血浆中H2S含量和心肌组织中CSE活性明显升高(P<0.05或P<0.01)。
2与假手术组大鼠比较,缺血组大鼠心肌线粒体总ATP酶、GSH-PX活性、SOD活性明显降低(P<0.01),MDA含量明显升高(P<0.01),心肌线粒体膜肿胀、线粒体活力下降,表现为540nm和570nm处吸光度值下降(P<0.01),与缺血组比较,缺血+NaHS低、中、高剂量组大鼠心肌线粒体总ATP酶、GSH-PX活性、SOD活性升高,MDA含量降低,心肌线粒体膜肿胀、线粒体活力明显改善(P<0.05或P<0.01)。
3与假手术组大鼠比较,缺血组大鼠心肌细胞凋亡率、Bax表达阳性细胞数百分率明显升高(P<0.01),缺血+NaHS低、中、高剂量组大鼠心肌细胞凋亡率、Bax表达阳性细胞数百分率明显低于缺血组,Bcl-2表达阳性细胞数百分率明显升高(P<0.05或P<0.01)。
4 HE染色光镜观察心肌组织形态学变化:假手术组大鼠心肌细胞排列整齐,着色均匀。缺血组大鼠部分区域心肌纤维横纹不齐或消失,核偏移甚至裂解消失,有炎性因子渗出,NaHS低、中、高剂量组大鼠也有心肌细胞变性的改变,但程度较缺血组轻。
结论:硫化氢对大鼠急性心肌缺血具有一定的保护作用,改善线粒体功能,上调凋亡抑制蛋白Bcl-2的表达,下调促凋亡蛋白Bax表达,抑制细胞凋亡可能是其保护作用机制之一。
Myocardial ischemia is the reduction of hemoperfusion and the myocardial energy metabolism is not normal, which can not support the heart to work.Several pathogenic mechanisms have been proposed to explain the myocardial damage that occurs during myocardial ischemia injury. In recent years, oxidative stress has been shown to play a pivotal role in the pathogenesis of ischemia injury. Apoptosis is the cellular basis in oxidative stress induced by ischemia injury. Mitochondria as oxidative stress sensor and death signal integrator plays a key role in initiating and mediating apoptosis of cardiomyocytes suffered from ischemia damage.
Hydrogen sulfide (H2S) is a colorless,water soluble,flammable gas that has the characteristic smell of rotten eggs. Like other members of the gasotransmitter family (nitric oxide and carbon monoxide), H2S has traditionally been considered to be a highly toxic gas and environmental hazard. However, much like for nitric oxide and carbon monoxide, the initial negative perception of H2S has evolved with the discovery that H2S is produced enzymatically in mammals under normal conditions. Endogenous H2S is mainly producted from cysteine and other sulfur-containing amino acids by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). It has been shown that H2S plays an important physiological role in diastolic blood vessels, inhibition vascular remodeling and protection of the myocardium. It has also been found that H2S plays a very important role in myocardial ischemia-reperfusion and has a protective effect. The role of H2S in myocardial ischemia has not been previously studied. We therefore investigated the effect of H2S in a rat model of myocardial ischemia in vivo.
Part 1 Change of endogenous hydrogen sulfide/cystathionine-γ-lyase system in acute myocardial ischemia rats
Objective: To observe the changes of H2S/CSE system in acute myocardial ischemia rats and to investigate the effect of H2S on acute myocardial ischemia rats.
Methods: Eighty male rats were randomly divided into six groups.①sham group;②ischemia 1h group;③ischemia 3h group;④ischemia 6h group;⑤ischemia 9h group;⑥ischemia 12h group. All rats were subjected to openning chest then the left anterior desending coronaries(LADC) were ligated, while the LADC was not ligated in sham group rats. The electrocardiogram(ECG) was recorded under anesthetized condition.The cardiac function indexes such as the left ventricular systolic pressure(LVSP), the left ventricular developed pressure (LVDP), the left ventricular end diastolic pressure (LVEDP),±dp/dtmax were respectively recorded at 1,3,6,9 or 12 hour after ligation.The content of H2S in plasma and the activity of CSE in myocardium were respectively detected. The pathological changes of myocardium were obersved by electron microscope.
Results:
1 In sham group , the LVEDP, LVDP,±dp/dtmax were not altered from 1 hour to 12 hour after ligating LADC. The LVEDP, LVDP,±dp/dtmax were not altered at 1 hour after ischemia compared with those of sham group(P > 0.05) and the LVDP,±dp/dtmax were significantly decreased and the LVEDP was markedly increased at 3 hour after ischemia compared with those of sham group(P﹤0.05, P﹤0.01), during 6 hour to 12 hour after ligating LADC the cardiac function parameters were significantly altered compared with those of sham group (P < 0.05, P < 0.01).
2 In sham group, the content of H2S in plasma and the activity of CSE in myocardium were not altered from 1 hour to 12 hour. The content of H2S in plasma and the activity of CSE in myocardium were not altered at 1 hour after ischemia compared with those of sham group rats ( P > 0.05). The content of H2S in plasma and the activity of CSE in myocardium were significantly decreased at 3 hour after ischemia compared with those of sham group ( P < 0.01). During 6 hour to 12 hour after ischemia, the content of H2S in plasma and the activity of CSE in myocardium were significantly decreased compared with those of sham group (P < 0.05, P < 0.01).
3 In sham group, the structure of myocardium were intact. The structure of myocardium were not altered at 1 hour after ischemia compared with that of sham group rats. In ischemia 3 h, 6 h, 9 h and 12 h groups the myocardium were injuried, such as perinuclear cytoplasmic edema, nuclear membrane disappearance myofibrillar fragmentation, mitochondrial cristae and membrane dissolution, disappearance.
Conclusion:The content of H2S in plasma and the activity of CSE in myocardium and the structure of myocardium were not altered at 1 hour after ischemia. During 3 hour to 12 hour after myocardial ischemia the content of H2S in plasma and the activity of CSE in myocardium were markedly decreased .It could be concluded that H2S/CSE system may be play a role in the physiopathologic process of acute myocardial ischemia.
Part 2 Effects of hydrogen sulfide on the function of mitochondria and apoptosis in acute myocardial ischemia rats
Objective: To study the effect of H2S on acute myocardial ischemia and explore its possible molecular mechanisms from the function of mitochondria and apoptosis in rats.
Methods: Fourty male rats were randomly divided into five groups (n=8).①sham group;②ischemia group;③ischemia+NaHS Low dose group;④ischemia +NaHS Middle dose group;⑤ischemia+NaHS High dose group. The left anterior descending coronary was ligatded in ischemia group rats while it was not ligated in sham group rats. In ischemia + NaHS Low (0.78mg/kg),Middle(1.56mg/kg) and High(3.12mg/kg) dose groups rats sodium hydrosulfide (NaHS) were respectively administrated at 3 hour after ischemia. The rats were respectively killed at 6 hour after ischemia. The content of H2S in plasma and the activity of CSE in myocardium were respectively detected. The mitochondria were prepared by differential centrifugation.The swelling and activity of mitochondria were determined. The activities of SOD, GSH-PX and ATPase, and the contents of malondialdehyde (MDA) in myocardial mitochondria were measured. The apoptotic rate of cardiomyocytes was evaluated by Flow Cytometry. The positive expressions of Bcl-2 and Bax in cardiomyocytes were respectively detected by immunohistochemistry.
Results:
1 The content of H2S in plasma and the activity of CSE in myocardium were significantly decreased in ischemia group rats compared with those of sham group (P < 0.05). Compared with those of ischemia group, the content of H2S in plasma and the activity of CSE in myocardium were significantly increased in ischemia+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01).
2 In ischemia group, the swelling of mitochondria and the activity of mitochondria were markedly decreased (P﹤0.01) and the activities of SOD, GSH-PX and ATPase were significantly decreased and the content of malondialdehyde (MDA) in myocardial mitochondria was significantly increased(P﹤0.01) compared with those of sham group. In ischemia+NaHS Low, Middle and High dose groups , the activities of SOD, GSH-Px and ATPase and the activity of myocardium mitochondria were significantly enhanced.The swelling of mitochondria was markedly ameliorated and the content of MDA was significantly decreased compared with those of ischemia groups(P < 0.05 or P < 0.01).
3 The apoptotic rate of cardiomyocytes and the expression of Bax in ischemia group were significantly increased compared with those of sham group (P<0.01). Compared with those of ischemia group, the apoptotic rates of cardiomyocytes and the expression of Bax were markedly decreased and the expression of Bcl-2 was markedly increased in ischemia +NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01).
4 In sham group , myocardial cells arranged in neat rows and colored evenly. Myocardial fiber striped or disappeared, the nuclear migrated and even cracking disappeared and mang inflammatory cells infiltrates in ischemia group compared with that of sham group. The injury of the myocardium was decreased in NaHS Low, Middle and High dose groups compared with that of ischemia group .
Conclusion:It could be concluded that H2S has a beneficial myocardial protection against the ischemia injury in rats by ameliorating the function of myocardium mitochondria, upregulating the expression of Bcl-2,downreg- ulating the expression of Bax.
引文
1 Wang R. Two′s company, three′s a crowd can H2S be the third endogenous gaseous tranamitter?[J]. FASEB J,2002,16(13): 1792-1798
2 Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator[J]. J Neurosci,1996,6(3): 1066-1071
3 Johns T,Olson B. Experimental myocardial infarction:A method of coronary occlusion in small animals [J]. Ann surg,1954,140(4): 675-682
4 Hosoki R, MatsukiN, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergywith nitric oxide[J].Biochem Biophys Res Commun, 1997,237(3): 527-531
5 Wang R. Two′s company, three′s a crowd can H2S be the third endogenous gaseous tranamitter? [J].FASEB J, 2002,16 (13): 1792-1798
6 Zhao W, Zhang J, Lu Y, et al. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener [J]. EMBO J,2001,20(21): 6008 - 6016
7 Eto K, Kimura H. The production of hydrogen sulfide is regulated by testosterone and S-adenosyl-L-methionine in mouse brain[J]. Neurochem,2002,83(1): 80 - 86
8 Eto K, OgasawaraM, Umemura K, et al. Hydrogen sulfide is produced in response to neuronal excitation [J]. Neurosci,2002,22(9): 3386 - 3391
9 Eto K, Asada T, Arima K, et al. Brain hydrogen sulfide is severely decreased in Alzheimer,s disease[J]. Biochem Biophys Res Commun 1997,237(3): 527-531
10 Geng B, Chang, Pan C , et al. Endogenous hydrogen sulfideregulation of myocardial injury induced by isoproterenol[J]. Biochem Biophys Res.Commun,2004,318 (3): 756-763
11 Zhao W,Wang R. H(2)S induced vasorelaxation and underling cellular and molecular mechanisms[J]. AmJ physiol Heart Circ physiol,2002,283 (2): 474-480
12 ChunyuZ,JunbaoD,DingfangB,et al. The regulatory effect of hydrogen sulfide on hypoxic hypertension in rats[J]. Biochem Biophys Res common,2003,302 (4): 810-816
1 Olivetti G, Quaini F , Sala R , et al . Acute myocardial iinfarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart[J]. J Mol Cell Cardiol,1996,28(9): 2005-2016
2 Piriou V, Chiari P, Gateau-Roesch O, et al. Desflurane-induced preconditioning alters calcium-induced mitochondrial permeability transition[J]. Anesthesiology,2004,100(3): 581-588
3 Oddis CV, Finkel MS. Cytokine-stimulated nitric oxide production inhibits mitochondrial activity in cardiac- myocytes[J]. Biochem Biophys Res Commun,1995,213(3): 1002-1009
4弓景波,钱令嘉.分光光度法检测心肌线粒体渗透性转换[J].中国应用生理学杂志, 2001,17(1): 97-99
5尹瑞兴,卡扎里,刘唐威,等.普罗布考对缺血再灌注心肌细胞凋亡的作用[J].中国临床康复,2004,8(24): 5202-5204
6 Stipanuk MH, Beck PW. Characterization of enzymatic capacity for cysteine desulphhydration in liver and kindney of the rat[J]. Biochem J, 1982,206(2): 267-277
7 Hosoki R, Matsuki N, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide[J]. Biochem Biophys Res Commun,1997,237(3): 527-531
8徐建兴.呼吸链的电子漏路径和线粒体的超氧自由基代谢及其生物学意义[J].基础医学与临床,2001,21(5): 389-394
9谷天祥,张显清,谷春久,等.心肌缺血再灌注损伤亚细胞Ca2+反常与ATP酶泵功能抑制[J].中华心血管病杂志,2001,29(7): 420-423
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2 Hosoki R, MatsukiN, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergywith nitric oxide[J]. Biochem Biophys Res Commun,1997,237(3): 527-531
3 StipanukMH, Beck PW. Characterization of the enzymatic capacity for cysteine desulphhydration in liver and kidneyof the rat[J]. Biochem J, 1982,206(2): 267-277
4 Wang R. The gasotransmitter role of hydroge sulfide[J]. Antioxid Redox Signal,2003,5(4): 493-501
5 Eto K, Kimura H. The production of hydrogen sulfide is regulated by testosterone and S-adenosyl-L-methionine in mouse brain[J].JNeurochem,2002,83(1): 80-86
6 Eto K, OgasawaraM, Umemura K, et al. Hydrogen sulfide is produced in response to neuronal excitation[J]. J Neurosci,2002,22(9): 3386 - 3391
7 ZhaoW, Zhang J, Lu Y, et al. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener[J]. EMBO J,2001,20(21): 6008-6016
8 Wang R. Two′s company, three′s a crowd can H2S be the third endogenous gaseous tranamitter?[J]. FASEB J,2002,16(13): 1792-1798
9 Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator[J]. J Neurosci,1996,6(3): 1066-1071
10 StipanukMH, Beck PW. Characterization of the enzymatic enzymatic capacity for cysteine desulphhydration in liver and kidneyof the rat[J]. Biochem J,1982,206(2): 267 - 277
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2 Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator[J]. J Neurosci,1996,6(3): 1066-1071
3 Johns T,Olson B. Experimental myocardial infarction:A method of coronary occlusion in small animals [J]. Ann surg,1954,140(4): 675-682
4 Hosoki R, MatsukiN, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergywith nitric oxide[J].Biochem Biophys Res Commun, 1997,237(3): 527-531
5 Wang R. Two′s company, three′s a crowd can H2S be the third endogenous gaseous tranamitter? [J].FASEB J, 2002,16 (13): 1792-1798
6 Zhao W, Zhang J, Lu Y, et al. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener [J]. EMBO J,2001,20(21): 6008 - 6016
7 Eto K, Kimura H. The production of hydrogen sulfide is regulated by testosterone and S-adenosyl-L-methionine in mouse brain[J]. Neurochem,2002,83(1): 80 - 86
8 Eto K, OgasawaraM, Umemura K, et al. Hydrogen sulfide is produced in response to neuronal excitation [J]. Neurosci,2002,22(9): 3386 - 3391
9 Eto K, Asada T, Arima K, et al. Brain hydrogen sulfide is severely decreased in Alzheimer,s disease[J]. Biochem Biophys Res Commun 1997,237(3): 527-531
10 Geng B, Chang, Pan C , et al. Endogenous hydrogen sulfideregulation of myocardial injury induced by isoproterenol[J]. Biochem Biophys Res.Commun,2004,318 (3): 756-763
11 Zhao W,Wang R. H(2)S induced vasorelaxation and underling cellular and molecular mechanisms[J]. AmJ physiol Heart Circ physiol,2002,283 (2): 474-480
12 ChunyuZ,JunbaoD,DingfangB,et al. The regulatory effect of hydrogen sulfide on hypoxic hypertension in rats[J]. Biochem Biophys Res common,2003,302 (4): 810-816
1 Olivetti G, Quaini F , Sala R , et al . Acute myocardial iinfarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart[J]. J Mol Cell Cardiol,1996,28(9): 2005-2016
2 Piriou V, Chiari P, Gateau-Roesch O, et al. Desflurane-induced preconditioning alters calcium-induced mitochondrial permeability transition[J]. Anesthesiology,2004,100(3): 581-588
3 Oddis CV, Finkel MS. Cytokine-stimulated nitric oxide production inhibits mitochondrial activity in cardiac- myocytes[J]. Biochem Biophys Res Commun,1995,213(3): 1002-1009
4弓景波,钱令嘉.分光光度法检测心肌线粒体渗透性转换[J].中国应用生理学杂志, 2001,17(1): 97-99
5尹瑞兴,卡扎里,刘唐威,等.普罗布考对缺血再灌注心肌细胞凋亡的作用[J].中国临床康复,2004,8(24): 5202-5204
6 Stipanuk MH, Beck PW. Characterization of enzymatic capacity for cysteine desulphhydration in liver and kindney of the rat[J]. Biochem J, 1982,206(2): 267-277
7 Hosoki R, Matsuki N, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide[J]. Biochem Biophys Res Commun,1997,237(3): 527-531
8徐建兴.呼吸链的电子漏路径和线粒体的超氧自由基代谢及其生物学意义[J].基础医学与临床,2001,21(5): 389-394
9谷天祥,张显清,谷春久,等.心肌缺血再灌注损伤亚细胞Ca2+反常与ATP酶泵功能抑制[J].中华心血管病杂志,2001,29(7): 420-423
1 Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator.[J]. J Neurosci,1996,16(3): 1066-1071
2 Hosoki R, MatsukiN, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergywith nitric oxide[J]. Biochem Biophys Res Commun,1997,237(3): 527-531
3 StipanukMH, Beck PW. Characterization of the enzymatic capacity for cysteine desulphhydration in liver and kidneyof the rat[J]. Biochem J, 1982,206(2): 267-277
4 Wang R. The gasotransmitter role of hydroge sulfide[J]. Antioxid Redox Signal,2003,5(4): 493-501
5 Eto K, Kimura H. The production of hydrogen sulfide is regulated by testosterone and S-adenosyl-L-methionine in mouse brain[J].JNeurochem,2002,83(1): 80-86
6 Eto K, OgasawaraM, Umemura K, et al. Hydrogen sulfide is produced in response to neuronal excitation[J]. J Neurosci,2002,22(9): 3386 - 3391
7 ZhaoW, Zhang J, Lu Y, et al. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener[J]. EMBO J,2001,20(21): 6008-6016
8 Wang R. Two′s company, three′s a crowd can H2S be the third endogenous gaseous tranamitter?[J]. FASEB J,2002,16(13): 1792-1798
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