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1960年,Jenning首次提出了心肌再灌注损伤的概念[1]。心肌缺血/再灌注损伤的主要原因包括氧自由基增多、细胞内钙超载及微血管损伤等[2-3],缺血组织细胞恢复灌注后发生的再灌注损伤(MIRI)在恢复血流的过程中常会引起心肌细胞的氧化应激损伤[4-5],往往造成患者预后不佳 [6]。
升陷汤出自张锡纯《医学衷中参西录》,该方由黄芪、柴胡、升麻、桔梗、知母组成,主治大气下陷之证,临床广泛用来防治心肌缺血性疾病[7]、治疗老年慢性充血性心力衰竭[8]、治疗不稳定型心绞痛[9]、治疗青少年病毒性心肌炎[10-11]。课题组前期考察了升陷汤及各单味药对阿霉素致心肌细胞的保护作用[12],尚未见升陷汤对心肌细胞缺氧/复氧损伤模型保护作用的报道。本实验基于经典的心肌细胞缺氧/复氧损伤模型[13],从细胞凋亡的角度初步探讨升陷汤及单味药对心肌缺氧/复氧的作用机制。
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本实验根据临床给药剂量及预实验结果,在药物试验浓度分别为2.5、5.0、10.0、20.0、40.0、80.0、160.0和320.0 μg/ml的药物浸膏时,研究全方及各单味药对心肌细胞缺氧/复氧后细胞活力的影响。MTT结果显示:升陷汤、黄芪、知母、桔梗、升麻在20 μg/ml时,细胞活力最佳,因此确定上述提取物浓度组均为20 μg/ml;由于20 μg/ml柴胡提取物对心肌细胞有一定损伤,经试验最终确定药物浓度为0.3 μg/ml时对缺氧/复氧后细胞活力无明显影响(表1)。
表 1 升陷汤各单味药对缺氧/复氧造成的心肌细胞活力的影响
组别 终浓度(μg/ml) MTT增殖率(%) 全方 20 18.745 黄芪 20 14.300 知母 20 11.199 桔梗 20 9.244 升麻 20 10.977 柴胡 0.3 26.512 -
实验结果表明:心肌细胞在缺氧/复氧条件下,细胞内活性氧含量明显增加,升高至对照组的2.49倍(P<0.01,图1),表明缺氧/复氧引起细胞内自由基损伤;给药后,除柴胡外,升陷汤全方及各单味药均能明显降低心肌缺氧/复氧致心肌损伤模型的细胞内ROS的荧光强度,升陷汤全方、黄芪、知母、桔梗和升麻处理组分别为对照组的1.46、1.40、1.79、1.52和1.83倍(P<0.01,图1)。其中,升陷汤与黄芪作用最强,两者无明显统计学差异。
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实验结果表明:心肌细胞在常氧下(对照组),Ca2+荧光强度较低,缺氧/复氧后,Ca2+荧光强度增加(P<0.01,图2),表明心肌细胞低氧/复氧时存在Ca2+超载。升陷汤全方和黄芪、知母药物干预后,各组细胞内荧光强度与模型组相比,分别降低了15.20%、23.98%和15.79%(P<0.05,图2),表明其对低氧/复氧时心肌细胞内Ca2+ 超载有抑制作用。桔梗、升麻药物处理后,上述值虽然有降低,但差异不明显。
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采用Annexin V-FITC/Pl双染流式细胞术检测药物对缺氧/复氧损伤后心肌细胞凋亡情况。从检测结果发现,心肌细胞在常氧下(对照组),细胞凋亡较低;缺氧/复氧后,细胞凋亡增加;给药组中升陷汤全方、黄芪、桔梗可以降低细胞的凋亡率(P<0.05,图3)。
Protective effect of Shengxian decoction and the decoction of single herb component against myocardial injury induced by hypoxia/reoxygenation
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摘要:
目的 通过观察升陷汤及单味药材水提物对离体培养的大鼠心肌细胞缺氧/复氧损伤的影响,并对其作用机制进行初步探讨。 方法 培养H9C2大鼠心肌细胞,共分成8组:空白对照组,缺氧/复氧组(模型组),缺氧复氧损伤后药物干预组(升陷汤全方及5个单味药材水提物组)。分别对心肌细胞凋亡率、心肌细胞的活力、细胞内活性氧(ROS)活性、细胞内钙离子浓度(Ca2+)等指标进行检测。 结果 升陷汤全方及黄芪、知母等药材干预能明显降低细胞凋亡率、细胞内ROS活性和Ca2+浓度(P<0.05),其中,全方的作用最强。与缺氧/复氧组细胞内ROS活性和Ca2+浓度增加至空白对照组的2.49倍及1.71倍相比,全方能使细胞内ROS活性和Ca2+浓度增加率降至缺氧/复氧组的41.37%和15.20%。 结论 升陷汤及单味药材对缺氧/复氧致心肌损伤具有保护作用,该作用的机制可能通过抑制细胞凋亡、降低细胞内ROS以及Ca2+的浓度所致。 Abstract:Objective To study the protective effect of Shengxian decoction and the single herb decoction against myocardial injury induced by hypoxia/reoxygenation. Methods The H9c2 cells were cultured to establish hypoxia/reoxygenation model. Rats were divided into 8 groups: normal control group, hypoxia/reoxygenation group (model group) and treated groups (Shengxian decoction and the single herb decoction). The apoptotic rate of cardiomyocytes, the activity of reactive oxygen species (ROS) and intracellular calcium concentration (Ca2+) were measured. Results Compared with hypoxia/reoxygenation group, the apoptosis rate, ROS activity and intracellular Ca2+ concentration were significantly lower in all treated groups (P<0.05). The ROS activity and intracellular Ca2+ concentration was decreased by 41.37% and 15.20% in Shengxian decoction group compared to the model group. Conclusion Shengxian decoction and the single herb decoction had protective effect on myocardial injury induced by hypoxia/reoxygenation. -
Key words:
- Shengxian decoction /
- hypoxia/reoxygenation injury /
- cardiomyocyte /
- protection effect
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表 1 升陷汤各单味药对缺氧/复氧造成的心肌细胞活力的影响
组别 终浓度(μg/ml) MTT增殖率(%) 全方 20 18.745 黄芪 20 14.300 知母 20 11.199 桔梗 20 9.244 升麻 20 10.977 柴胡 0.3 26.512 -
[1] SEWELL W H, KOTH D R, HUGGINS C E. Ventricular fibrillation in dogs after sudden return of flow to the coronary artery[J]. Surgery,1955,38(6):1050-1053. [2] RANA A, GOYAL N, AHLAWAT A, et al. Mechanisms involved in attenuated cardio-protective role of ischemic preconditioning in metabolic disorders[J]. Perfusion,2015,30(2):94-105. doi: 10.1177/0267659114536760 [3] 陈福晖, 刘达兴, 容松. 心肌缺血再灌注损伤发生机制的研究进展[J]. 安徽医药, 2017, 21(12):2145-2148. doi: 10.3969/j.issn.1009-6469.2017.12.003 [4] CHANG G L, ZHANG D Y, YU H, et al. Cardioprotective effects of exenatide against oxidative stress-induced injury[J]. Int J Mol Med,2013,32(5):1011-1020. doi: 10.3892/ijmm.2013.1475 [5] 宋旭辉. Decorin在脑缺血防治中的功能及机制研究[D]. 上海: 第二军医大学, 2010. [6] 吴立玲, 张幼怡. 心血管病理生理学[M]. 北京: 北京大学医学出版社, 2009: 8. [7] 康红钰, 张福华, 刘喜民, 等. 升陷汤对大鼠急性心肌缺血作用机制的探讨[J]. 中国医院药学杂志, 2007, 27(5):617-619. doi: 10.3321/j.issn:1001-5213.2007.05.018 [8] 张万义, 邱云卿, 张万芬. 升补宗气法治疗老年慢性充血性心力衰竭疗效观察[J]. 中国中西医结合杂志, 2004, 24(1):43. doi: 10.3321/j.issn:1003-5370.2004.01.035 [9] 裴天仙, 徐长庆, 李滨, 等. 槲皮素对阿霉素致小鼠心肌损伤的保护作用及其机制[J]. 药学学报, 2007, 42(10):1029-1033. doi: 10.3321/j.issn:0513-4870.2007.10.004 [10] 曹洪欣, 朱海燕. 大气下陷证与病毒性心肌炎相关性机理的理论探讨[J]. 陕西中医, 2002, 23(2):141. doi: 10.3969/j.issn.1000-7369.2002.02.035 [11] 曹洪欣, 朱海燕. 益气升陷法治疗病毒性心肌炎的辨治要点[J]. 中医药学报, 2002, 30(5):18-19. doi: 10.3969/j.issn.1002-2392.2002.05.010 [12] 满缓, 张凤, 黄豆豆, 等. 升陷汤及各单味药对阿霉素致心肌细胞损伤的保护作用[J]. 药学实践杂志, 2019, 37(4):304-308. doi: 10.3969/j.issn.1006-0111.2019.04.004 [13] THANDROYEN F T, BELLOTTO D, KATAYAMA A, et al. Subcellular electrolyte alterations during progressive hypoxia and following reoxygenation in isolated neonatal rat ventricular myocytes[J]. Circ Res,1992,71(1):106-119. doi: 10.1161/01.RES.71.1.106 [14] MIMURO S, KATOH T, SUZUKI A, et al. Deterioration of myocardial injury due to dexmedetomidine administration after myocardial ischaemia[J]. Resuscitation,2010,81(12):1714-1717. doi: 10.1016/j.resuscitation.2010.07.021 [15] MIYAMAE M, CAMACHO S A, WEINER M W, et al. Attenuation of postischemic reperfusion injury is related to prevention of [Ca2+]m overload in rat hearts[J]. Am J Physiol,1996,271(5 Pt 2):H2145-H2153. [16] HAUSENLOY D J, YELLON D M. Myocardial ischemia-reperfusion injury: a neglected therapeutic target[J]. J Clin Invest,2013,123(1):92-100. doi: 10.1172/JCI62874 [17] JIANG J T, YUAN X, WANG T, et al. Antioxidative and cardioprotective effects of total flavonoids extracted from Dracocephalum moldavica L. against acute ischemia/reperfusion-induced myocardial injury in isolated rat heart[J]. Cardiovasc Toxicol,2014,14(1):74-82. doi: 10.1007/s12012-013-9221-3 [18] ZHOU M J, REN H H, HAN J C, et al. Protective effects of kaempferol against myocardial ischemia/reperfusion injury in isolated rat heart via antioxidant activity and inhibition of glycogen synthase kinase-3β[J]. Oxid Med Cell Longev,2015,2015:481405. [19] MOENS A L, CLAEYS M J, TIMMERMANS J P, et al. Myocardial ischemia/reperfusion-injury, a clinical view on a complex pathophysiological process[J]. Int J Cardiol,2005,100(2):179-190. doi: 10.1016/j.ijcard.2004.04.013 [20] MOZAFFARI M S, LIU J Y, ABEBE W, et al. Mechanisms of load dependency of myocardial ischemia reperfusion injury[J]. Am J Cardiovasc Dis,2013,3(4):180-196. [21] NEUHOF C, NEUHOF H. Calpain system and its involvement in myocardial ischemia and reperfusion injury[J]. World J Cardiol,2014,6(7):638-652. doi: 10.4330/wjc.v6.i7.638 [22] ZHANG F, ZHAN Q, DONG X, et al. Shengxian decoction in chronic heart failure treatment and synergistic property of Platycodonis Radix: a metabolomic approach and its application[J]. Mol Biosyst,2014,10(8):2055-2063. doi: 10.1039/C4MB00055B [23] ZHANG F, ZHAN Q, GAO S H, et al. Chemical profile- and pharmacokinetics-based investigation of the synergistic property of platycodonis Radix in traditional Chinese medicine For Mula Shengxian decoction[J]. J Ethnopharmacol,2014,152(3):497-507. doi: 10.1016/j.jep.2014.01.033 [24] 杨承芝, 朱爱华. 浅析张锡纯大气下陷证与升陷汤[J]. 中国中医药现代远程教育, 2014, 12(14):116-117. doi: 10.3969/j.issn.1672-2779.2014.14.072