线粒体通透性转换孔对红景天苷减轻心肌缺血再灌注损伤的作用
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摘要
目的探讨线粒体通透性转换孔(mPTP)在红景天苷减轻大鼠心肌缺血再灌注损伤(MIRI)中的作用和机制。方法使用Langendorff装置建立大鼠离体心脏缺血/再灌注损伤模型。将60只SPF级健康雄性成年SD大鼠随机分为假手术组(Sham组)、缺血/再灌注组(I/R组)、红景天苷组(Sal组)和环孢菌素A组(CsA组)。用Western Blot法检测细胞色素C(Cyt-c)分别在细胞浆和线粒体中的表达情况;用免疫组化法检测活化型半胱天冬酶-9(cleaved caspase-9)和活化型半胱天冬酶-3(cleaved caspase-3)在各组心肌组织的表达水平;用线粒体肿胀实验法评估各组心肌的mPTP开放程度;酶联免疫分析(Elisa)法测定各组大鼠心肌组织中线粒体细胞色素C氧化酶(COX)和琥珀酸脱氢酶(SDH)的活性。结果与Sham组比较,I/R组心肌组织中大量Cyt-c由线粒体释放到细胞浆中,cleaved caspase-9和cleaved caspase-3的表达水平均显著上升,线粒体吸光度下降率明显升高,COX和SDH活性降低(P<0.05);与I/R组比较,Sal组和CsA组Cyt-c释放和线粒体吸光度下降均受抑制,cleaved caspase-9和cleaved caspase-3的表达水平均下降,COX和SDH活性均升高(P<0.05)。结论红景天苷可通过抑制mPTP开放、保护线粒体功能来发挥心肌保护作用,其具体机制与减少Cyt-c的释放、抑制caspase-9和caspase-3的激活及提高COX和SDH的活性有关。
Objective To study the effect of mitochondrial permeablity transition pore(mPTP)on cardio-protection of salidroside against ischemia reperfusion injury.Methods The device of Langendorff was used to establish the isolated rat heart ischemia reperfusion model.60 SD male rats of SPF grade were divided into Sham group,I/R group,Sal group and CsA group.Western Blot analysis was used to determine the expression of Cyt-c in cytoplasm and mitochondria respectively.Immunohistochemistry was used to detect the expression levels of cleaved caspase-9 and cleaved caspase-3.Mitochondrial swelling experiment was used to estimate the degree of mPTP opening.Elisa was used to determine the activity of COX and SDH.Results Compared with the Sham group,I/R can induce the release of Cyt-c from the mitochondria,increase the expression level of cleaved caspase-9 and cleaved caspase-3,increase the extent of mitochondrial swelling and reduce the activity of COX and SDH(P<0.05).Compared with I/R group,the Cyt-c release and mitochondrial absorbance decrease in Sal group and CsA group were both inhibited,the expression levels of cleaved caspase-9 and cleaved caspase-3 were decreased,and COX and SDH activities are alleviated(P<0.05).Conclusion Salidroside can inhibit the myocardial ischemia reperfusion injury by inhibiting mPTP opening and protecting mitochondrial function.The specific mechanism is related to reducing the release of Cyt-c,inhibiting the activation of caspase-9 and caspase-3,and increasing the activity of COX and SDH.
Objective To study the effect of mitochondrial permeablity transition pore(mPTP)on cardio-protection of salidroside against ischemia reperfusion injury.Methods The device of Langendorff was used to establish the isolated rat heart ischemia reperfusion model.60 SD male rats of SPF grade were divided into Sham group,I/R group,Sal group and CsA group.Western Blot analysis was used to determine the expression of Cyt-c in cytoplasm and mitochondria respectively.Immunohistochemistry was used to detect the expression levels of cleaved caspase-9 and cleaved caspase-3.Mitochondrial swelling experiment was used to estimate the degree of mPTP opening.Elisa was used to determine the activity of COX and SDH.Results Compared with the Sham group,I/R can induce the release of Cyt-c from the mitochondria,increase the expression level of cleaved caspase-9 and cleaved caspase-3,increase the extent of mitochondrial swelling and reduce the activity of COX and SDH(P<0.05).Compared with I/R group,the Cyt-c release and mitochondrial absorbance decrease in Sal group and CsA group were both inhibited,the expression levels of cleaved caspase-9 and cleaved caspase-3 were decreased,and COX and SDH activities are alleviated(P<0.05).Conclusion Salidroside can inhibit the myocardial ischemia reperfusion injury by inhibiting mPTP opening and protecting mitochondrial function.The specific mechanism is related to reducing the release of Cyt-c,inhibiting the activation of caspase-9 and caspase-3,and increasing the activity of COX and SDH.
引文
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[18]程斌,杨峰,李锋涛,等.人参皂苷Rb1通过减轻线粒体损伤对大鼠脊髓缺血再灌注损伤的保护作用[J].中国康复理论与实践,2018,24(6):629-633.
[19]Li Q,Shen Li,Wang Z,et al.TanshinoneⅡA protects against myocardial ischemia reperfusion injury by activating the PI3K/Akt/mTOR signaling pathway[J].Biomed Pharmacother,2016,84:106-114.
[2]史志辉,刘洋,肖会敏,等.心宁片对小鼠心肌缺血再灌注损伤的保护作用及其机制研究[J].西北药学杂志,2017,32(5):614-618.
[3]Carley A N,Taglieri D M,Bi J,et al.Metabolic efficiency promotes protection from pressure overload in hearts expressing slow skeletal troponin I[J].Circ Heart Fail,2014,8(1):119-127.
[4]Bulon V V,Krylova I B,Selina E N,et al.Antiarrhythmic effect of uridine and uridine-5′-monophosphate in acute myocardial ischemia[J].Bull Exp Biol Med,2014,157(6):728-731.
[5]Yang S,Li H,Tang L,et a1.Apelin-13 protects the heart against ischem ia-reperfasion injury through the RISK-GSK-3β-mPTP pathway[J].Arch Med Sci,2015,11(5):1065-1073.
[6]姚伟锋,黑子清.红景天及其有效成分防治器官缺血再灌注损伤的研究进展[J].实用医学杂志,2015,31(3):498-501.
[7]Mohammad A H,John M D,Manuel G,et al.The mPTPstatus during early reoxygenation is critical for cardioprotection[J].J Surg Res,2012,174(1):62-72.
[8]Penna C,Perrelli M G,Pagliaro P.Mitochondrial pathways,permeability transition pore,and redox signaling in cardioprotection:therapeutic implications[J].Antioxid Redox Signal,2013,18(5):556-599.
[9]Xu M C,Shi H M,Gao X F,et al.Salidroside attenuates myocardial ischemia-reperfusion injury via PI3K/Akt signaling pathway[J].J Asian Nat Prod Res,2013,15(3):244-252.
[10]Lai M C,Lin J G,Pai P Y,et al.Protective effect of salidroside on cardiac apoptosis in mice with chronic intermittent hypoxia[J].Int J Cardiol,2014,174(3):565-573.
[11]李建明,张杰,史忠良,等.大株红景天注射液联合低分子肝素治疗不稳定型心绞痛的效果观察[J].中国医药,2015,10(5):623-625.
[12]罗银利,黄晓松,谭李红,等.红景天苷对大鼠脑缺血再灌注损伤后PI3-K/AKT信号通路的影响[J].中国医师杂志,2014,16(6):734-738.
[13]雷尚芳,郭俐宏,鲍升娟,等.红景天苷对大鼠急性心肌缺血/再灌注损伤离体心脏心功能的影响[J].实用药物与临床,2015,18(11):1289-1292.
[14]尹巧香,王恒,裴志勇,等.环孢菌素A拮抗心肌缺血/再灌注损伤的作用[J].心脏杂志,2014,26(1):15-20.
[15]李有富,李辉,韩涛,等.杨梅素诱导人宫颈癌HeLa细胞凋亡及凋亡通路的研究[J].西北药学杂志,2016,31(3):270-274.
[16]Han B J,Li W,Jing G B,et al.Effects of daidzein in regards to cytotoxicity in vitro apoptosis,reactive oxygen species level,cell cycle arrest and the expression of caspase and Bcl-2family proteins[J].Oncol Rep,2015,34(3):1115-1120.
[17]Liu J,Yao Y,Ding H,et al.Oxymatrine triggers apoptosis by regulating Bcl-2family proteins and activating caspase-3/caspase-9pathway in human leukemia HL-60cells[J].Tumour Biol,2014,35(6):5409-5415.
[18]程斌,杨峰,李锋涛,等.人参皂苷Rb1通过减轻线粒体损伤对大鼠脊髓缺血再灌注损伤的保护作用[J].中国康复理论与实践,2018,24(6):629-633.
[19]Li Q,Shen Li,Wang Z,et al.TanshinoneⅡA protects against myocardial ischemia reperfusion injury by activating the PI3K/Akt/mTOR signaling pathway[J].Biomed Pharmacother,2016,84:106-114.