人参四逆汤及其有效成分对戊巴比妥钠所致心肌细胞损伤模型的保护作用
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摘要
目的:探讨人参四逆汤及其有效成分对戊巴比妥钠所致心肌细胞损伤的保护作用及其机制。方法:H9C2细胞经传代培养后,分别给予人参皂苷Rb20. 01,0. 1,1μmol·L~(-1),Re 0. 01,0. 1,1μmol·L~(-1),异甘草素(isoliquiritigenin,ISL) 20,40,80μmol·L~(-1),甘草次酸(glycyrrhetinic acid,GA) 10,20,40μmol·L~(-1),人参四逆汤10,100,400 mg·L~(-1),作用4 h,经0. 1%的戊巴比妥钠作用30 min后,检测细胞存活率,乳酸脱氢酶(lactate dehydrogenase,LDH),脂质过氧化物丙二醛(malondialdehyde,MDA),Na+-K+-三磷酸腺苷(ATP)酶,Ca~(2+)-ATP酶活性,同时采用实时荧光定量聚合酶链式反应(Real-time PCR)检测过氧化物酶体增殖活化受体共激活因子~(-1)α(peroxisome proliferative activated receptor,PGC~(-1)α),B淋巴细胞瘤-2相关X蛋白(Bax),半胱氨酸蛋白酶-3(Caspase-3) mRNA的表达情况。结果:人参四逆汤及其有效成分对心衰细胞模型有保护作用,与正常组比较,模型组细胞存活率显著下降,LDH,MDA含量显著升高,Na+-K+-ATP酶活性升高,Ca~(2+)-ATP酶活性显著降低,PGC~(-1)αmRNA表达下调,Bax,Caspase-3 mRNA表达(P <0. 01),证明模型成立。与模型组比较,各给药组显著升高细胞存活率,降低LDH,MDA含量,抑制Na+-K+-ATP酶活性,提高Ca~(2+)-ATP酶活性,上调PGC~(-1)αmRNA表达,抑制Bax,Caspase-3 mRNA表达(P <0. 05,P <0. 01)。结论:人参四逆汤及其有效成分对心衰细胞模型有显著保护作用,且其作用机制与抗氧化,改善线粒体能量代谢,抑制线粒体凋亡途径有关。
Objective: To explore the protective effect and mechanisms of Renshen Sinitang and its active ingredients on cardiomyocyte injury induced by pentobarbital sodium. Method: H9 C2 cells were sub-cultured with ginsenoside Rb20. 01,0. 1,1 μmol·L~(-1),Re 0. 01,0. 1,1 μmol·L~(-1),isoliquiritigenin 20,40,80 μmol·L~(-1),glycyrrhetinic acid 10,20,40 μmol·L~(-1),Renshen Sinitang,10,100,400 mg·L~(-1),for 4 h. After treatment with0. 1% of sodium pentobarbital for 30 min, cell viability, lactate dehydrogenase(LDH), lipid peroxide malondialdehyde(MDA),Na+-K+-adenosine triphosphate(ATP) ase,Ca~(2+) -ATPase activity,and real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) were used to detect the expressions of peroxisome proliferative activated receptor~(-1)α(PGC~(-1)α),B-cell lymphoma-2 associated X protein(Bax) and cysteine aspartate-specific protease-3(Caspase-3) mRNA. Result: Renshen Sinitang and its active ingredients have a protective effect on heart failure cell model. Compared with the normal group,the cell survival rate of the model group decreased significantly,while the LDH and MDA contents increased significantly,and the Na+-K+-ATPase activity increased. Ca~(2+) -ATPase activity was significantly decreased,PGC~(-1)α mRNA expression was downregulated,Bax and Caspase-3 mRNA expressions indicates the modeling(P < 0. 01). Compared with the model group,each administration group showed a significantly increased cell viability,decreased LDH,MDA content,inhibited Na+-K+-ATPase activity,increased Ca~(2+) -ATPase activity,up-regulated PGC~(-1)α mRNA expression,and inhibited Bax and Caspase-3 mRNA expression(P < 0. 05,P < 0. 01). Conclusion: Renshen Sinitang and its active ingredients have a significant protective effect on heart failure cell model,and its mechanisms of action are related to anti-oxidation,improvement of mitochondrial energy metabolism and inhibition of mitochondrial apoptosis pathway.
Objective: To explore the protective effect and mechanisms of Renshen Sinitang and its active ingredients on cardiomyocyte injury induced by pentobarbital sodium. Method: H9 C2 cells were sub-cultured with ginsenoside Rb20. 01,0. 1,1 μmol·L~(-1),Re 0. 01,0. 1,1 μmol·L~(-1),isoliquiritigenin 20,40,80 μmol·L~(-1),glycyrrhetinic acid 10,20,40 μmol·L~(-1),Renshen Sinitang,10,100,400 mg·L~(-1),for 4 h. After treatment with0. 1% of sodium pentobarbital for 30 min, cell viability, lactate dehydrogenase(LDH), lipid peroxide malondialdehyde(MDA),Na+-K+-adenosine triphosphate(ATP) ase,Ca~(2+) -ATPase activity,and real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) were used to detect the expressions of peroxisome proliferative activated receptor~(-1)α(PGC~(-1)α),B-cell lymphoma-2 associated X protein(Bax) and cysteine aspartate-specific protease-3(Caspase-3) mRNA. Result: Renshen Sinitang and its active ingredients have a protective effect on heart failure cell model. Compared with the normal group,the cell survival rate of the model group decreased significantly,while the LDH and MDA contents increased significantly,and the Na+-K+-ATPase activity increased. Ca~(2+) -ATPase activity was significantly decreased,PGC~(-1)α mRNA expression was downregulated,Bax and Caspase-3 mRNA expressions indicates the modeling(P < 0. 01). Compared with the model group,each administration group showed a significantly increased cell viability,decreased LDH,MDA content,inhibited Na+-K+-ATPase activity,increased Ca~(2+) -ATPase activity,up-regulated PGC~(-1)α mRNA expression,and inhibited Bax and Caspase-3 mRNA expression(P < 0. 05,P < 0. 01). Conclusion: Renshen Sinitang and its active ingredients have a significant protective effect on heart failure cell model,and its mechanisms of action are related to anti-oxidation,improvement of mitochondrial energy metabolism and inhibition of mitochondrial apoptosis pathway.
引文
[1] Brown D A,Perry J B,Allen M E,et al. Mitochondrial function as a therapeutic target in heart failure[J]. Nat Rev Cardiol,2017,14(4):238-250.
[2]孟永梅,王伟,叶会玲.慢性心力衰竭的中医研究概况[J].中华中医药杂志,2012,27(3):670-674.
[3]蒋梅先.浅谈慢性心力衰竭防治中的中医药加载治疗[J].辽宁中医杂志,2014,41(8):1553-1555.
[4] LI X L,ZHANG J,HUANG J,et al. A multicenter randomized double-blind parallel-group placebocontrolled study of the effects of Qiliqiangxin capsules in patients with chronic heart failure[J]. J Am Coll Cardiol,2013,62(12):1065-1072.
[5]杨海润,孙建宁,张广平,等.四逆汤组方不同配伍毒效关系研究[J].中国实验方剂学杂志,2013,19(23):266-269.
[6]贺金,方艳伟,李永民.四逆汤对大鼠心肌缺血损伤的保护作用[J].中华中医药杂志,2008,2(7):638-640.
[7]翟建英,靳冉,朱晓光,等.四逆汤及其组分配伍对心力衰竭大鼠血流动力学的影响[J].中西医结合心脑血管病杂志,2013,11(12):1480-1482.
[8]缪萍,裘福荣,曾金,等.四逆汤及其不同配伍方对心力衰竭大鼠的保护作用及机制探讨[J].中国实验方剂学杂志,2015,21(5):138-142.
[9]王雪梅,刘佳,付殿斌,等.参附汤萃取液成分对阿霉素致心衰大鼠血流动力学、心肌自噬及凋亡的影响[J].陕西中医学院学报,2014,37(4):75-78.
[10]李梦婷,彭成,谢晓芳.心力衰竭小型动物模型研究进展[J].中国实验方剂学杂志,2018,24(5):213-219.
[11]谢晓芳,徐菲菲,彭成,等.人参皂苷类成分对戊巴比妥钠损伤心肌细胞ATP酶及相关离子的影响[J].中药药理与临床,2014,30(5):61-64.
[12]贺抒,谢晓芳,张雪,等.附子水溶性生物碱对心衰细胞模型的治疗作用[J].中国实验方剂学杂志,2014,20(16):127-130.
[13] LU X,XIONG Z,LI J,et al. Metabonomic study on'Kidney-Yang deficiency syndrome and intervention effects of Rhizoma Drynariae extracts in rats using ultra performance liquid chromatography coupled with mass spectrometry[J]. Talanta,2011,83(3):700-708.
[14] YANG M,CHEN J L,XU L W,et al. Navigating traditional Chinese medicine network pharmacology and computational tools[J]. Evid Based Complement Alternat Med,2013,doi:10. 1155/2013/731969.
[15] CHEN R,Moriya J,Yamakawa J,et al. Traditional Chinese medicine for chronic fatigue syndrome[J]. Evid Based Complement Alternat Med,2010,7(1):3-10.
[16]徐雅娟,赵宏峰,司云珊,等.人参四逆汤活性成分的分离和鉴定[J].中草药,2002,33(3):203-204.
[17] Tornroth-Horsefield S,Neutze R. Opening and closing the metabolite gate[J]. Proc Natl Acad Sci USA,2008,105(50):19565-19566.
[18]沈放,杨黎江,路斌,等.海枫藤提取物对Na+-K+-ATP酶活力的影响[J].时珍国医国药,2008,19(8):1959-1960.
[19]徐菲菲,彭成,谢晓芳,等.参附注射液对戊巴比妥钠致心衰模型心肌细胞膜ATP酶和相关离子的影响[J].中国实验方剂学杂志,2013,19(7):196-199.
[20]胡元会,车维新,曹雪滨,等.心复康口服液对大鼠实验心衰模型心肌细胞Ca2+-ATP酶及琥珀酸脱氢酶活性的影响[J].中国医药学报,2000,15(2):31.
[21]李超,伏圣博,刘华玲,等.细胞凋亡研究进展[J].世界科技研究与发展,2007,29(3):45-53.
[22]张晓华,刘淑荣,钱锋,等.强心康颗粒对慢性心衰大鼠心肌细胞病理形态学腺苷酸转位酶,PGC-1αmRNA表达的影响[J].中国实验方剂学杂志,2018,24(9):121-126.
[23] Arany Z,Novikov M,Chin S,et al. Transverse aortic constriction leads to accelerated heart failure in mice lacking PPAR-gamma coactivator 1alpha[J]. Proc Natl Acad Sci USA,2006,103(26):10086-10091.
[2]孟永梅,王伟,叶会玲.慢性心力衰竭的中医研究概况[J].中华中医药杂志,2012,27(3):670-674.
[3]蒋梅先.浅谈慢性心力衰竭防治中的中医药加载治疗[J].辽宁中医杂志,2014,41(8):1553-1555.
[4] LI X L,ZHANG J,HUANG J,et al. A multicenter randomized double-blind parallel-group placebocontrolled study of the effects of Qiliqiangxin capsules in patients with chronic heart failure[J]. J Am Coll Cardiol,2013,62(12):1065-1072.
[5]杨海润,孙建宁,张广平,等.四逆汤组方不同配伍毒效关系研究[J].中国实验方剂学杂志,2013,19(23):266-269.
[6]贺金,方艳伟,李永民.四逆汤对大鼠心肌缺血损伤的保护作用[J].中华中医药杂志,2008,2(7):638-640.
[7]翟建英,靳冉,朱晓光,等.四逆汤及其组分配伍对心力衰竭大鼠血流动力学的影响[J].中西医结合心脑血管病杂志,2013,11(12):1480-1482.
[8]缪萍,裘福荣,曾金,等.四逆汤及其不同配伍方对心力衰竭大鼠的保护作用及机制探讨[J].中国实验方剂学杂志,2015,21(5):138-142.
[9]王雪梅,刘佳,付殿斌,等.参附汤萃取液成分对阿霉素致心衰大鼠血流动力学、心肌自噬及凋亡的影响[J].陕西中医学院学报,2014,37(4):75-78.
[10]李梦婷,彭成,谢晓芳.心力衰竭小型动物模型研究进展[J].中国实验方剂学杂志,2018,24(5):213-219.
[11]谢晓芳,徐菲菲,彭成,等.人参皂苷类成分对戊巴比妥钠损伤心肌细胞ATP酶及相关离子的影响[J].中药药理与临床,2014,30(5):61-64.
[12]贺抒,谢晓芳,张雪,等.附子水溶性生物碱对心衰细胞模型的治疗作用[J].中国实验方剂学杂志,2014,20(16):127-130.
[13] LU X,XIONG Z,LI J,et al. Metabonomic study on'Kidney-Yang deficiency syndrome and intervention effects of Rhizoma Drynariae extracts in rats using ultra performance liquid chromatography coupled with mass spectrometry[J]. Talanta,2011,83(3):700-708.
[14] YANG M,CHEN J L,XU L W,et al. Navigating traditional Chinese medicine network pharmacology and computational tools[J]. Evid Based Complement Alternat Med,2013,doi:10. 1155/2013/731969.
[15] CHEN R,Moriya J,Yamakawa J,et al. Traditional Chinese medicine for chronic fatigue syndrome[J]. Evid Based Complement Alternat Med,2010,7(1):3-10.
[16]徐雅娟,赵宏峰,司云珊,等.人参四逆汤活性成分的分离和鉴定[J].中草药,2002,33(3):203-204.
[17] Tornroth-Horsefield S,Neutze R. Opening and closing the metabolite gate[J]. Proc Natl Acad Sci USA,2008,105(50):19565-19566.
[18]沈放,杨黎江,路斌,等.海枫藤提取物对Na+-K+-ATP酶活力的影响[J].时珍国医国药,2008,19(8):1959-1960.
[19]徐菲菲,彭成,谢晓芳,等.参附注射液对戊巴比妥钠致心衰模型心肌细胞膜ATP酶和相关离子的影响[J].中国实验方剂学杂志,2013,19(7):196-199.
[20]胡元会,车维新,曹雪滨,等.心复康口服液对大鼠实验心衰模型心肌细胞Ca2+-ATP酶及琥珀酸脱氢酶活性的影响[J].中国医药学报,2000,15(2):31.
[21]李超,伏圣博,刘华玲,等.细胞凋亡研究进展[J].世界科技研究与发展,2007,29(3):45-53.
[22]张晓华,刘淑荣,钱锋,等.强心康颗粒对慢性心衰大鼠心肌细胞病理形态学腺苷酸转位酶,PGC-1αmRNA表达的影响[J].中国实验方剂学杂志,2018,24(9):121-126.
[23] Arany Z,Novikov M,Chin S,et al. Transverse aortic constriction leads to accelerated heart failure in mice lacking PPAR-gamma coactivator 1alpha[J]. Proc Natl Acad Sci USA,2006,103(26):10086-10091.