刺五加皂苷B对大鼠心肌缺血再灌注和过氧化氢损伤心肌细胞凋亡的影响及机制
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摘要
刺五加叶皂苷(Acanthopanax senticosus saponins, ASS)是从刺五加叶中分离得到的含有16种活性成分的总皂苷,其中刺五加皂苷B(Ciwujianoside B, C-B)是一种新发现的主要单体成分。本论文研究C-B对心肌细胞过氧化氢(H_2O_2)损伤和大鼠心肌缺血再灌注损伤的影响,探讨C-B对H_2O_2和心肌缺血再灌注诱导的心肌细胞凋亡的作用及机制。主要研究内容如下:
1.原代培养乳鼠心肌细胞,利用不同浓度的H_2O_2引起心肌细胞损伤,结果显示: C-B可增加H_2O_2 50、100μmol·L~(-1)损伤后的心肌细胞存活率,提高抗氧化能力,减轻氧化损伤。
2.原代培养乳鼠心肌细胞,通过H_2O_2 200μmol·L~(-1)诱导心肌细胞凋亡,采用流式细胞术及蛋白免疫印迹等方法,证实:C-B可减轻氧化损伤,降低Caspase-3和Caspase-9的蛋白表达及活性,调节Bcl-2和Bax蛋白表达,阻止线粒体膜电位降低,抑制线粒体释放Cyt c、Smac和AIF,通过调控线粒体途径相关的凋亡蛋白表达抑制细胞凋亡。
3.通过结扎大鼠左冠状动脉前降支30 min、再灌注4 h建立心肌缺血再灌注损伤模型,结果表明:C-B可改善大鼠心脏功能及心肌组织病理改变,增加GSH含量及GSH-Px、SOD活性,降低MDA含量及MPO活性,通过抗氧化机理对心肌缺血再灌注损伤起保护作用。
4.通过大鼠在体心肌缺血再灌注诱导心肌细胞凋亡,采用TUNEL原位标记细胞凋亡检测法、免疫组化、蛋白免疫印迹及实时荧光定量PCR等方法,结果表明:C-B可提高Bcl-2/Bax比值,抑制线粒体释放Cyt c、Smac和AIF,减少心肌组织Caspase-3、Caspase-9蛋白表达和活性,减弱PARP降解,抑制心肌细胞凋亡,其保护心肌的作用可能与P38MAPK、JNK及PI3K/AKT信号转导通路的激活有关。
综上所述,C-B对乳鼠心肌细胞过氧化氢损伤及大鼠心肌缺血再灌注损伤有保护作用,可抑制其所诱导的心肌细胞凋亡。
Myocardial ischemia is one kind of cardiovascular diseases of high disease incidence, and it can endanger human health. When myocardial ischemia happened, the restoration of blood supply can not improve cardiac function, but aggravate the original myocardial injury induced by ischemic, and induce the incidence of arrhythmias, increased myocardial infarct size, and even cause heart failure. This phenomenon is called myocardial ischemia reperfusion injury (MIRI). Cardiomyocyte apoptosis plays a important role in the process of MIRI. Therefore, to find drugs which can treat MIRI, inhibit cardiomyocyte apoptosis and improve cardiac fuction has very important clinical significance.
Acanthopanax senticosus saponins (ASS) is extracted from the leaves of Acanthopanax. Ciwujianoside B(C-B) is one of a monomer component of ASS. The previous studies shown that ASS has protective effect on myocardial ischemia and MIRI. However, the study about the effect of C-B on MIRI has not been reported.
In the thesis, we establish the model of oxidative injury and apoptosis in cardiomyocytes by hydrogen peroxide(H_2O_2) of different concentration to study the effect of C-B on cardiomy-ocytes oxidative injury and apoptosis,and we establish the MIRI model of rats in vivo to probe the effect of C-B on MIRI and apoptosis.
1. Effect of C-B on oxidative injury of neonatal rat cardiomyocytes induced by H_2O_2 The cardiomyocytes were primary cultured for 72h. And the cardiomyocytes were subjected with H_2O_2(25,50,100,200μmol·L~(-1))for 60 or 120min, and the cell morphology change and the rate of survival cell was observed. The cardiomyocytes were cultured and subjected to H_2O_2 50,100μmol·L~(-1) for 60 min to induce oxidative injury. The cardiomyocytes were incubated with C-B 200 and 400μg·mL~(-1) for 24 h before exposed to H_2O_2. The cell morphology change, the rate of survival cardiomyocytes, the activity of LDH in medium, the content of maleic dialdehyde(MDA) and glutathione(GSH) in cardiomyocytes, the activity of superoxide dismutase(SOD), glutathione peroxidase(GSH-Px) and catalase(CAT) in cardiomyocytes was observed.
The results shown that the cardiomyocytes morphology change in the groups of H_2O_2 50,100,200μmol·L~(-1) was obvious. With the increase of the concentration of H_2O_2, the oxidative injury was more serious. The rate of survival cells in groups of H_2O_2 50, 100, 200μmol·L~(-1) was decreased significantly. C-B of 200 and 400μg·mL~(-1) decreased the activity of LDH in medium and the content of MDA significantly, increased the content of GSH, the activity of SOD, GSH-Px and CAT of cardiomyocytes injuried by H_2O_2 of 50 and 100μmol·L~(-1),which indicated that C-B can extenuate oxidative injury and increase the antioxidant capacity of cardiomyocytes.
2. Effect of C-B on cardiomyocytes apoptosis induced by H_2O_2 and its mechanism
The cardiomyocytes were incubated with C-B 200,400μg·mL~(-1) for 24 h, then were subjected with H_2O_2 200μmol·L~(-1) for 120 min to induce apoptosis. The cell morphology, the rate of survival cells, the activity of LDH in medium, the content of MDA and the activity of SOD in cardiomyocytes were assayed. The apoptosis was observed by staining of Hoechst 33342 and AO/EB. The rate of apoptosis was assayed by AnnexinⅤ-FITC kit. The mitochondrial membrane potential was assayed by staining with Rhodamine123 and detected by flow cytometry. The activity of Caspase-3, Caspase-9 were assayed by spectrophotometric detection. The protein expression of Cyt c, Smac, AIF, Caspase-3, Caspase-9, PARP, Bcl-2 and Bax were dertermined with the method of Western Blot.
The results demonstrated that C-B 200 and 400μg·mL~(-1) can reduce the changes of oxidative injury induced by H_2O_2 200μmol·L~(-1) for 120min, increase the cardiomyocytes survival rate, and decrease the apoptosis rate. C-B can decrease the activity of Caspase-3 and Caspase-9. C-B can decrease the expression of Cyt c, Smac, AIF, Bax, Caspase-3, Caspase-9 protein and PARP degradation, increase the expression of Bcl-2 protein. C-B can inhibit apoptosis in cardiomyocytes by regulating the expression of apoptosis-related proteins about the mitochondrial route .
3. The effect of C-B on myocardial ischemia reperfusion injury in rats
The myocardial ischemia reperfusion injury model was established by ligation of the left anterior descending coronary artery for 30min and reperfused for 4h in rats The dose of C-B used in the study are 20 and 40 mg.kg~(-1). The changes of cardiac hemodynamics, the activity of myocardial enzymes, the activity of SOD and the content of MDA in myocardial tissue and serum, the activity of MPO and GSH-Px of serum, the content of GSH in myocardial tissue, and the level of ANP, TNFαand IL-6 in plasma were detected. The myocardial infarct size, myocardial histopathology and ultrastructure changes were observed.
The results shown that C-B 20 and 40 mg.kg~(-1) can increase HR, SBP, DBP, MAP, LVSP and±dp/dtmax in MIRI rats, which manifested that C-B can improve heart function. C-B 20 and 40 mg.kg~(-1) can reduce myocardial infarct size and increase the activitIies of AST, LDH and CPK. C-B 20 and 40mg.kg~(-1) can increase the level of SOD, GSH-Px and GSH , reduce MDA content and MPO activity. C-B can increase the content of ANP and reduce the content of TNFαand IL-6. Myocardial histopathology and ultrastructural observation results shown that C-B can reduce myocardial pathological changes. It has a protective effect on MIRI in rat, and it mechnism may be related to its antioxidant effects.
4. Effect of C-B on apoptosis induced by MIRI and its mechanism
The MIRI model was established by ligation of the left anterior descending coronary artery for 30min and then reperfused for 4h. The dose of C-B used were 20 and 40 mg.kg~(-1). The apoptosis rate was assayed by TUNEL kit. The protein expression of Caspase-3, Cyt c, Bcl-2 and Bax were detected by immunohistochemistry. The protein expression of Caspase-3, Caspase-9, PARP, Bax, Bcl-2 and Bax were detected by Western Blot. The activities of Caspase-3 and Caspase-9 were detected by spectrophotometric detection. The level of Bax and Bcl-2 gene were determined by RT-PCR. The level of P38MAPK, JNK and AKT and its phosphorylation state were analysed by Western Blot.
The results demonstrated that C-B can decrease the number of positive stained cells by TUNEL. The immunohistochemical results shown that C-B can decrease the expression of Cyt c, Caspase-3 and Bax, increase Bcl-2 expression, increase the ratio of Bcl-2/Bax of MIRI rats. C-B can decrease the activity of Caspase-3 and Caspase-9, lower the expression of Caspase-3 and Caspase-9 and PARP degradation. C-B can up-regulate Bcl-2 protein and gene level, and reduced Bax protein and gene level. C-B can reduce the phosphorylation of P38MAPK and JNK and increase AKT phosphorylation. The results above manifested that C-B can inhibit apoptosis by regulating the expression of apoptosis-related proteins about the mitochondrial route. The mechanism of its protective effect may be related to the activition of pathway of P38MAPK, JNK and AKT.
In summary, C-B can extenuate oxidative injury of cardiomyocytes induced by H_2O_2 and has protective effect on the myocardial ischemia reperfusion injury in rats. C-B can inhibit cardiomyocytes apoptosis induced by H_2O_2 and myocardial ischemia reperfusion injury in rats by regulating the expression of apoptosis-related proteins about the mitochondrial route.
1.原代培养乳鼠心肌细胞,利用不同浓度的H_2O_2引起心肌细胞损伤,结果显示: C-B可增加H_2O_2 50、100μmol·L~(-1)损伤后的心肌细胞存活率,提高抗氧化能力,减轻氧化损伤。
2.原代培养乳鼠心肌细胞,通过H_2O_2 200μmol·L~(-1)诱导心肌细胞凋亡,采用流式细胞术及蛋白免疫印迹等方法,证实:C-B可减轻氧化损伤,降低Caspase-3和Caspase-9的蛋白表达及活性,调节Bcl-2和Bax蛋白表达,阻止线粒体膜电位降低,抑制线粒体释放Cyt c、Smac和AIF,通过调控线粒体途径相关的凋亡蛋白表达抑制细胞凋亡。
3.通过结扎大鼠左冠状动脉前降支30 min、再灌注4 h建立心肌缺血再灌注损伤模型,结果表明:C-B可改善大鼠心脏功能及心肌组织病理改变,增加GSH含量及GSH-Px、SOD活性,降低MDA含量及MPO活性,通过抗氧化机理对心肌缺血再灌注损伤起保护作用。
4.通过大鼠在体心肌缺血再灌注诱导心肌细胞凋亡,采用TUNEL原位标记细胞凋亡检测法、免疫组化、蛋白免疫印迹及实时荧光定量PCR等方法,结果表明:C-B可提高Bcl-2/Bax比值,抑制线粒体释放Cyt c、Smac和AIF,减少心肌组织Caspase-3、Caspase-9蛋白表达和活性,减弱PARP降解,抑制心肌细胞凋亡,其保护心肌的作用可能与P38MAPK、JNK及PI3K/AKT信号转导通路的激活有关。
综上所述,C-B对乳鼠心肌细胞过氧化氢损伤及大鼠心肌缺血再灌注损伤有保护作用,可抑制其所诱导的心肌细胞凋亡。
Myocardial ischemia is one kind of cardiovascular diseases of high disease incidence, and it can endanger human health. When myocardial ischemia happened, the restoration of blood supply can not improve cardiac function, but aggravate the original myocardial injury induced by ischemic, and induce the incidence of arrhythmias, increased myocardial infarct size, and even cause heart failure. This phenomenon is called myocardial ischemia reperfusion injury (MIRI). Cardiomyocyte apoptosis plays a important role in the process of MIRI. Therefore, to find drugs which can treat MIRI, inhibit cardiomyocyte apoptosis and improve cardiac fuction has very important clinical significance.
Acanthopanax senticosus saponins (ASS) is extracted from the leaves of Acanthopanax. Ciwujianoside B(C-B) is one of a monomer component of ASS. The previous studies shown that ASS has protective effect on myocardial ischemia and MIRI. However, the study about the effect of C-B on MIRI has not been reported.
In the thesis, we establish the model of oxidative injury and apoptosis in cardiomyocytes by hydrogen peroxide(H_2O_2) of different concentration to study the effect of C-B on cardiomy-ocytes oxidative injury and apoptosis,and we establish the MIRI model of rats in vivo to probe the effect of C-B on MIRI and apoptosis.
1. Effect of C-B on oxidative injury of neonatal rat cardiomyocytes induced by H_2O_2 The cardiomyocytes were primary cultured for 72h. And the cardiomyocytes were subjected with H_2O_2(25,50,100,200μmol·L~(-1))for 60 or 120min, and the cell morphology change and the rate of survival cell was observed. The cardiomyocytes were cultured and subjected to H_2O_2 50,100μmol·L~(-1) for 60 min to induce oxidative injury. The cardiomyocytes were incubated with C-B 200 and 400μg·mL~(-1) for 24 h before exposed to H_2O_2. The cell morphology change, the rate of survival cardiomyocytes, the activity of LDH in medium, the content of maleic dialdehyde(MDA) and glutathione(GSH) in cardiomyocytes, the activity of superoxide dismutase(SOD), glutathione peroxidase(GSH-Px) and catalase(CAT) in cardiomyocytes was observed.
The results shown that the cardiomyocytes morphology change in the groups of H_2O_2 50,100,200μmol·L~(-1) was obvious. With the increase of the concentration of H_2O_2, the oxidative injury was more serious. The rate of survival cells in groups of H_2O_2 50, 100, 200μmol·L~(-1) was decreased significantly. C-B of 200 and 400μg·mL~(-1) decreased the activity of LDH in medium and the content of MDA significantly, increased the content of GSH, the activity of SOD, GSH-Px and CAT of cardiomyocytes injuried by H_2O_2 of 50 and 100μmol·L~(-1),which indicated that C-B can extenuate oxidative injury and increase the antioxidant capacity of cardiomyocytes.
2. Effect of C-B on cardiomyocytes apoptosis induced by H_2O_2 and its mechanism
The cardiomyocytes were incubated with C-B 200,400μg·mL~(-1) for 24 h, then were subjected with H_2O_2 200μmol·L~(-1) for 120 min to induce apoptosis. The cell morphology, the rate of survival cells, the activity of LDH in medium, the content of MDA and the activity of SOD in cardiomyocytes were assayed. The apoptosis was observed by staining of Hoechst 33342 and AO/EB. The rate of apoptosis was assayed by AnnexinⅤ-FITC kit. The mitochondrial membrane potential was assayed by staining with Rhodamine123 and detected by flow cytometry. The activity of Caspase-3, Caspase-9 were assayed by spectrophotometric detection. The protein expression of Cyt c, Smac, AIF, Caspase-3, Caspase-9, PARP, Bcl-2 and Bax were dertermined with the method of Western Blot.
The results demonstrated that C-B 200 and 400μg·mL~(-1) can reduce the changes of oxidative injury induced by H_2O_2 200μmol·L~(-1) for 120min, increase the cardiomyocytes survival rate, and decrease the apoptosis rate. C-B can decrease the activity of Caspase-3 and Caspase-9. C-B can decrease the expression of Cyt c, Smac, AIF, Bax, Caspase-3, Caspase-9 protein and PARP degradation, increase the expression of Bcl-2 protein. C-B can inhibit apoptosis in cardiomyocytes by regulating the expression of apoptosis-related proteins about the mitochondrial route .
3. The effect of C-B on myocardial ischemia reperfusion injury in rats
The myocardial ischemia reperfusion injury model was established by ligation of the left anterior descending coronary artery for 30min and reperfused for 4h in rats The dose of C-B used in the study are 20 and 40 mg.kg~(-1). The changes of cardiac hemodynamics, the activity of myocardial enzymes, the activity of SOD and the content of MDA in myocardial tissue and serum, the activity of MPO and GSH-Px of serum, the content of GSH in myocardial tissue, and the level of ANP, TNFαand IL-6 in plasma were detected. The myocardial infarct size, myocardial histopathology and ultrastructure changes were observed.
The results shown that C-B 20 and 40 mg.kg~(-1) can increase HR, SBP, DBP, MAP, LVSP and±dp/dtmax in MIRI rats, which manifested that C-B can improve heart function. C-B 20 and 40 mg.kg~(-1) can reduce myocardial infarct size and increase the activitIies of AST, LDH and CPK. C-B 20 and 40mg.kg~(-1) can increase the level of SOD, GSH-Px and GSH , reduce MDA content and MPO activity. C-B can increase the content of ANP and reduce the content of TNFαand IL-6. Myocardial histopathology and ultrastructural observation results shown that C-B can reduce myocardial pathological changes. It has a protective effect on MIRI in rat, and it mechnism may be related to its antioxidant effects.
4. Effect of C-B on apoptosis induced by MIRI and its mechanism
The MIRI model was established by ligation of the left anterior descending coronary artery for 30min and then reperfused for 4h. The dose of C-B used were 20 and 40 mg.kg~(-1). The apoptosis rate was assayed by TUNEL kit. The protein expression of Caspase-3, Cyt c, Bcl-2 and Bax were detected by immunohistochemistry. The protein expression of Caspase-3, Caspase-9, PARP, Bax, Bcl-2 and Bax were detected by Western Blot. The activities of Caspase-3 and Caspase-9 were detected by spectrophotometric detection. The level of Bax and Bcl-2 gene were determined by RT-PCR. The level of P38MAPK, JNK and AKT and its phosphorylation state were analysed by Western Blot.
The results demonstrated that C-B can decrease the number of positive stained cells by TUNEL. The immunohistochemical results shown that C-B can decrease the expression of Cyt c, Caspase-3 and Bax, increase Bcl-2 expression, increase the ratio of Bcl-2/Bax of MIRI rats. C-B can decrease the activity of Caspase-3 and Caspase-9, lower the expression of Caspase-3 and Caspase-9 and PARP degradation. C-B can up-regulate Bcl-2 protein and gene level, and reduced Bax protein and gene level. C-B can reduce the phosphorylation of P38MAPK and JNK and increase AKT phosphorylation. The results above manifested that C-B can inhibit apoptosis by regulating the expression of apoptosis-related proteins about the mitochondrial route. The mechanism of its protective effect may be related to the activition of pathway of P38MAPK, JNK and AKT.
In summary, C-B can extenuate oxidative injury of cardiomyocytes induced by H_2O_2 and has protective effect on the myocardial ischemia reperfusion injury in rats. C-B can inhibit cardiomyocytes apoptosis induced by H_2O_2 and myocardial ischemia reperfusion injury in rats by regulating the expression of apoptosis-related proteins about the mitochondrial route.
引文
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