苓桂术甘汤含药血清对过氧化氢诱导的乳鼠原代心肌细胞氧化应激损伤及细胞凋亡的影响
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摘要
目的观察苓桂术甘汤含药血清对H_2O_2诱导的心肌细胞氧化应激损伤及细胞凋亡的影响。方法建立H_2O_2诱导的心肌细胞氧化应激损伤模型,将心肌细胞随机分为正常对照组、H_2O_2模型组、空白大鼠血清(20%)组、苓桂术甘汤含药血清(5%、10%、20%)组,用苓桂术甘汤含药血清(5%、10%、20%),分别预处理心肌细胞12 h后,加入100μmol/L H_2O_2继续培养6 h,用MTT法测定心肌细胞存活率,比色法检测乳酸脱氢酶(lactate dehydrogenase,LDH)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化酶(glutathione peroxidase,GSH-Px)活性及丙二醛(malonic dialdehyde,MDA)水平,用荧光核染色法观察细胞凋亡形态学变化,流式细胞术(AnnexinⅤ-FITC/PI)检测细胞凋亡率,荧光探针法测定氧自由基水平。结果与正常组比较,模型组心肌细胞活力明显降低(P<0.05),氧自由基、LDH活性及MDA水平均显著升高(P<0.05),SOD及GSH-Px活性均显著降低(P<0.05),细胞核浓染数目增加,细胞凋亡率明显升高(P<0.05);与模型组比较,苓桂术甘汤含药血清组心肌细胞活力明显升高(P<0.05),GSH-Px、SOD活性均显著升高(P<0.05),LDH活性,氧自由基、MDA水平均显著降低(P<0.05),心肌细胞核浓染减少,细胞凋亡率明显降低(P<0.05)。结论苓桂术甘汤能够清除细胞内氧自由基,抑制心肌细胞凋亡,增强细胞活性,对心肌细胞氧化应激损伤具有保护作用。
Objective To investigate the effect of serum containing Linggui Zhugan Decoction on hydrogen peroxide(H_2O_2)-induced oxidative stress injury and apoptosis of primary cardiomyocytes. Methods Cardiomyocytes were randomly divided into normal group, H_2O_2 model group, blank rat serum(20%) group, and serum containing Linggui Zhugan Decoction groups(5%, 10%, and 20%). All cardiomyocytes except those in the normal group were used to establish a model of H_2O_2-induced oxidative stress injury, and the cardiomyocytes in the serum containing Linggui Zhugan Decoction groups were pretreated with serum containing Linggui Zhugan Decoction(5%, 10%, and 20%) for 12 hours and then cultured with 100 μmol/L H_2O_2 for 6 hours. MTT assay was used to measure the survival rate of cardiomyocytes; colorimetry was used to measure the activities of lactate dehydrogenase(LDH), superoxide dismutase(SOD), and glutathione peroxidase(GSH-Px) and the level of malondialdehyde(MDA); fluorescence nuclear staining was used to observe the apoptosis and morphological changes of cardiomyocytes; flow cytometry(Annexin Ⅴ-FITC/PI) was used to measure the apoptosis rate of cardiomyocytes; the fluorescent probe method was used to measure the level of oxygen free radicals. Results Compared with the normal group, the model group had a significant reduction in cardiomyocyte viability(P<0.05), significant increases in oxygen free radicals, LDH activity, and MDA level(P<0.05), significant reductions in the activities of SOD and GSH-Px(P<0.05), and significant increases in the number of densely stained nuclei and the apoptosis rate of cardiomyocytes(P<0.05). Compared with the model group, the serum containing Linggui Zhugan Decoction groups had a significant increase in cardiomyocyte viability(P<0.05), significant increases in the activities of SOD and GSH-Px(P<0.05), significant reductions in LDH activity, oxygen free radicals, and MDA level(P<0.05), and significant reductions in the number of densely stained nuclei and the apoptosis rate of cardiomyocytes(P<0.05). Conclusion Linggui Zhugan Decoction can clear oxygen free radicals in cardiomyocytes, inhibit cardiomyocyte apoptosis, enhance cardiomyocyte activity, and protect cardiomyocytes against oxidative stress injury.
Objective To investigate the effect of serum containing Linggui Zhugan Decoction on hydrogen peroxide(H_2O_2)-induced oxidative stress injury and apoptosis of primary cardiomyocytes. Methods Cardiomyocytes were randomly divided into normal group, H_2O_2 model group, blank rat serum(20%) group, and serum containing Linggui Zhugan Decoction groups(5%, 10%, and 20%). All cardiomyocytes except those in the normal group were used to establish a model of H_2O_2-induced oxidative stress injury, and the cardiomyocytes in the serum containing Linggui Zhugan Decoction groups were pretreated with serum containing Linggui Zhugan Decoction(5%, 10%, and 20%) for 12 hours and then cultured with 100 μmol/L H_2O_2 for 6 hours. MTT assay was used to measure the survival rate of cardiomyocytes; colorimetry was used to measure the activities of lactate dehydrogenase(LDH), superoxide dismutase(SOD), and glutathione peroxidase(GSH-Px) and the level of malondialdehyde(MDA); fluorescence nuclear staining was used to observe the apoptosis and morphological changes of cardiomyocytes; flow cytometry(Annexin Ⅴ-FITC/PI) was used to measure the apoptosis rate of cardiomyocytes; the fluorescent probe method was used to measure the level of oxygen free radicals. Results Compared with the normal group, the model group had a significant reduction in cardiomyocyte viability(P<0.05), significant increases in oxygen free radicals, LDH activity, and MDA level(P<0.05), significant reductions in the activities of SOD and GSH-Px(P<0.05), and significant increases in the number of densely stained nuclei and the apoptosis rate of cardiomyocytes(P<0.05). Compared with the model group, the serum containing Linggui Zhugan Decoction groups had a significant increase in cardiomyocyte viability(P<0.05), significant increases in the activities of SOD and GSH-Px(P<0.05), significant reductions in LDH activity, oxygen free radicals, and MDA level(P<0.05), and significant reductions in the number of densely stained nuclei and the apoptosis rate of cardiomyocytes(P<0.05). Conclusion Linggui Zhugan Decoction can clear oxygen free radicals in cardiomyocytes, inhibit cardiomyocyte apoptosis, enhance cardiomyocyte activity, and protect cardiomyocytes against oxidative stress injury.
引文
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[15] Q1AO H,CHEN H,DONG Y,et al.Polydatin attenuates H2O2-induced oxidative stress via PKC pathway[J].Oxid Med Cell Longev,2016,2016(3):5139458.
[2] ZHOU X,LI C,XU W,et al.Trimetazidine protects against smoking-induced left ventricular remodeling via attenuating oxidative stress,apoptosis,and inflammation[J].Plos One,2012,7(7):e40424.
[3] LAW S K,LEUNG C S,YAU K L,et al.Regulation of multiple transcription factors by reactive oxygen species and effects of pro-inflammatory cytokines released during myocardial infarction on cardiac differentiation of embryonic stem cells[J].International Journal of Cardiology,2013,168(4):3458-3472.
[4] 黄金玲,桑方方,王桐生,等.苓桂术甘汤对充血性心衰竭大鼠心脏指数与血流动力学的影响[J].安徽中医学院学报,2009,28(5):58-61.
[5] 王靓,侯晓燕,保永亮,等.苓桂术甘汤对急性心肌梗死心室重构模型大鼠TNF-α、IL-6和ET-1的影响[J].山西中医学院学报,2012,13(4):23-25.
[6] 黄金玲,王慧慧,陈慧芳,等.正交试验法优选苓桂术甘汤的提取工艺[J].安徽中医学院学报,2011,30(6):65-68.
[7] 陈希,许瑞,蒋易楠,等.密度梯度离心法同时分离新生大鼠原代心肌细胞与成纤维细胞[J].生理学报,2015,67(4):423-430.
[8] 司徒镇强,吴军正.细胞培养 [M].2版.北京:世界图书出版公司,2007.
[9] LEMARIé J,HUTTIN O,GIRERD N,et al.Usefulness of speckle-tracking imaging for right ventricular assessment after acute myocardial Infarction:a magnetic resonance imaging/echocardiographic comparison within the relation between aldosterone and cardiac remodeling after myocardial infarction study[J].J Am Soc Echocardiogr,2015,28(7):818-827.
[10] TSUTSUI H,KINUGAWA S,MATSUSHIMA S.Oxidative stress and heart fallure[J].Am J Physiolheart Circ Physiol,2011,301(6):H2181-2190.
[11] QUEISSER N,SCHUPP N.Aldosterone,oxidative stress,and NF-κB activation in hypertension-related cardiovascular and renal diseases[J].Free Radical Biology & Medicine,2012,53(2):314-327.
[12] LARSEN M,WEBB G,KENNINGTON S,et al.Mannitol in cardioplegia as an oxygen free radical scavenger measured by malondialdehyde[J].Perfusion,2002,17(1):51-55.
[13] DIVATE R D,WANG P M,WANG C C,et al.Protective effect of medicinal fungus Xylaria nigripes mycelia extracts against hydrogen peroxide-induced apoptosis in PC12 cells[J].Immunopathol Pharmacol,2017,30(1):105-112.
[14] ZHANG T,ZHAO Q,YE F,et al.Alda-1,an ALDH2 activator,protects against hepatic ischemia/reperfusion injury in rats via inhibition of oxidative stress[J].Free Radic Res,2018,52(6):629-638.
[15] Q1AO H,CHEN H,DONG Y,et al.Polydatin attenuates H2O2-induced oxidative stress via PKC pathway[J].Oxid Med Cell Longev,2016,2016(3):5139458.