脑心通胶囊肠吸收液保护过氧化氢致H9c2心肌细胞损伤的作用机制及有效药味筛选
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摘要
目的:以肠吸收液为药物载体,利用拆方重组法筛选脑心通胶囊组方中发挥体外心肌保护作用的单味或多味中药,并丰富脑心通胶囊心肌保护作用的分子机制。方法:应用外翻肠囊法制备肠吸收液,体外构建过氧化氢(H2O2)损伤大鼠胚胎心肌细胞株(H9c2)模型,分为正常组、模型组、脑心通胶囊肠吸收液低、中、高剂量组(15.63,31.25,62.50 mg·L-1),每组3个复孔,实验重复3次。噻唑蓝(MTT)比色法检测心肌细胞的存活率,AnnexinⅤ-FITC/PI双染结合流式细胞仪检测心肌细胞凋亡率,蛋白免疫印迹法(Western blot)检测水通道蛋白1(AQP1)蛋白的表达;同时拆方或构建重组方,MTT比色法定位脑心通胶囊组方中发挥心肌保护作用的单味或多味中药。结果:与正常组比较,模型组AQP1的表达升高(P<0.01),心肌细胞凋亡数量增多(P<0.05)。与模型组比较,脑心通胶囊肠吸收液中、高剂量组均可下调AQP1的表达(P<0.05),脑心通胶囊肠吸收液高剂量组可抑制心肌细胞凋亡(P<0.05)。与脑心通全方组比较,丹参、地龙和全蝎这3种单味药心肌保护作用与脑心通全方组相当,此3味药重组方高剂量组心肌保护作用强于脑心通全方组(P<0.05),去3味药重组方心肌保护作用弱于脑心通全方组(P<0.05)。结论:脑心通胶囊通过抑制AQP1表达和细胞凋亡,发挥心肌保护作用;丹参、地龙和全蝎是组方中发挥心肌保护作用的主要药味。
Objective:Intestinal absorption liquid as medicine carriers,one or more kinds of traditional Chinese medicine protecting cardiac injury in Naoxintong capsule will be found through separation and combination of prescription.This study also can expand protecting mechanisms of Naoxintong capsule.Method:Naoxintong intestinal absorption liquids of single,combination and total prescription were prepared.H9c2 cell line exposed to H_2O_2 was established.Cell survival rate was determined with methyl thiazolyl tetrazolium(MTT).Cell apoptosis rate was examined by Annexin Ⅴ-FITC/PI staining with a flow cytometer.Aquaporin1(AQP1) expression was detected by Western blot.One or more kinds of traditional Chinese medicine in Naoxintong capsule which exerted protective effect from cardiac injury were screened through separation and combination of prescription.Result:As compared with control group,the protein expression level of AQP1 was significantly increased(P<0.01),and the apoptotic H9c2 cells increased(P<0.05).Compared with model group,Naoxintong intestinal absorption liquid low,medium and high dose group decreased AQP1 expression(P<0.05),and naoxintong intestinal absorption liquid high dose group inhibited cardiac cells apoptosis(P<0.05),thus protected H9c2 cells from oxidative damages.Salviae Miltiorrhizae Radix et Rhizoma,Pheretima,Scorpio are main constituents which had protective actions in total prescription.Protective effect of those three combination prescription in high dose group was the best(P<0.05),and effect of combination prescription without those three was the second(P<0.05).Conclusion:Naoxintong capsule acts as a protective role in myocardial injury through decreasing AQP1 expression and inhibiting cell apoptosis.Salviae Miltiorrhizae Radix et Rhizoma,Pheretima,Scorpio are important components in Naoxintong capsule.
Objective:Intestinal absorption liquid as medicine carriers,one or more kinds of traditional Chinese medicine protecting cardiac injury in Naoxintong capsule will be found through separation and combination of prescription.This study also can expand protecting mechanisms of Naoxintong capsule.Method:Naoxintong intestinal absorption liquids of single,combination and total prescription were prepared.H9c2 cell line exposed to H_2O_2 was established.Cell survival rate was determined with methyl thiazolyl tetrazolium(MTT).Cell apoptosis rate was examined by Annexin Ⅴ-FITC/PI staining with a flow cytometer.Aquaporin1(AQP1) expression was detected by Western blot.One or more kinds of traditional Chinese medicine in Naoxintong capsule which exerted protective effect from cardiac injury were screened through separation and combination of prescription.Result:As compared with control group,the protein expression level of AQP1 was significantly increased(P<0.01),and the apoptotic H9c2 cells increased(P<0.05).Compared with model group,Naoxintong intestinal absorption liquid low,medium and high dose group decreased AQP1 expression(P<0.05),and naoxintong intestinal absorption liquid high dose group inhibited cardiac cells apoptosis(P<0.05),thus protected H9c2 cells from oxidative damages.Salviae Miltiorrhizae Radix et Rhizoma,Pheretima,Scorpio are main constituents which had protective actions in total prescription.Protective effect of those three combination prescription in high dose group was the best(P<0.05),and effect of combination prescription without those three was the second(P<0.05).Conclusion:Naoxintong capsule acts as a protective role in myocardial injury through decreasing AQP1 expression and inhibiting cell apoptosis.Salviae Miltiorrhizae Radix et Rhizoma,Pheretima,Scorpio are important components in Naoxintong capsule.
引文
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[7]岳燕林,刘剑平,刘青松,等.乙酰唑胺对类风湿关节炎滑膜细胞水通道蛋白1,3表达的影响[J].细胞与分子免疫学杂志,2013,29(11):1177-1180.
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[12]TIE L,WANG D,SHI Y,et al.Aquaporins in cardiovascular system[J].Adv Exp Med Biol,2017,969:105-113.
[13]李雷兵,张琦.中医药关于水通道蛋白表达的研究进展[J].中国中医基础医学杂志,2015,21(9):1194-1195.
[14]孙维建,胡丹虹,卢明东,等.胃癌组织中水通道蛋白1的表达与细胞凋亡、血管生成及预后的关系[J].浙江医学,2012,34(6):405-407.
[15]杨志霞,林谦,马利.丹参对心血管疾病药理作用的文献研究[J].世界中西医结合杂志,2012,7(2):93-96,114.
[16]FU J,HUANG H,LIU J,et al.TanshinoneⅡAprotects cardiac myocytes against oxidative stresstriggered damage and apoptosis[J].Eur J Pharmacol,2007,568(1/3):213-221.
[17]袁宝萍,吕嵘,章忱,等.复方丹参滴丸对急性心肌缺血大鼠模型的影响[J].中国实验方剂学杂志,2012,18(22):222-226.
[18]黄庆,李志武,马志国,等.地龙的研究进展[J].中国实验方剂学杂志,2018,24(13):220-226.
[19]史磊,张天锡,杜聪颖,等.中药全蝎活性成分、药理作用及临床应用研究进展[J].辽宁中医药大学学报,2015,17(4):89-91.
[20]耿亚,杨洪军,马月香,等.基于“肠外翻-心肌细胞”联用模型的益气活血方药效学作用评价及机制探讨[J].中国中药杂志,2016,41(20):3821-3827.
[21]陈强,安娜,庄曾渊,等.补肾养血明目方含药肠吸收液对ARPE-19细胞氧化损伤的保护作用[J].中国实验方剂学杂志,2017,8(17):1433-1436.
[22]许照,肖雄,黄刚,等.基于含药肠吸收液的麻黄-杏仁药对物质基础与作用机制分析[J].中国实验方剂学杂志,2018,24(6):1-6.
[23]吴林霖,李梅,巩仔鹏,等.基于大鼠离体外翻肠囊模型考察杜仲提取物在正常和自发性高血压状态下的肠吸收特性差异[J].中国实验方剂学杂志,2018,24(9):15-21.
[24]张旻昱,龚慕辛,杨洪军.含药肠吸收液:一种新的中药体外药理实验方法[J].中草药,2018,49(15):3457-3462.
[2]WANG Y,YAN X,MI S,et al.Naoxintong attenuates ischaemia/reperfusion injury through inhibiting NLRP3inflammasome activation[J].J Cell Mol Med,2017,21(1):4-12.
[3]陈立,李志尚,黎丽娴,等.步长脑心通胶囊对急性心肌梗死兔心功能及梗死面积的影响[J].实验研究,2012,53(15):1315-1317.
[4]刘振权,徐秋萍,张文生,等.脑心通胶囊对脑缺血再灌注损伤大鼠脑水肿作用及机制研究[J].北京中医药大学学报,2007,30(4):235-238.
[5]王乔悦,郭虹,赵步长,等.脑心通胶囊对脑缺血再灌注损伤大鼠血脑屏障的影响[J].中国临床药理学杂志,2016,32(10):890-893.
[6]ZHANG F B,HUANG B,ZHAO Y,et al.BNCprotects H9c2 cardiomyoblasts from H2O2-induced oxidative injury through ERK1/2 signaling pathway[J].Evid Based Complement Alternat Med,2013,doi:10.1155/2013/802784.
[7]岳燕林,刘剑平,刘青松,等.乙酰唑胺对类风湿关节炎滑膜细胞水通道蛋白1,3表达的影响[J].细胞与分子免疫学杂志,2013,29(11):1177-1180.
[8]Brown D I,Griendling K K.Regulation of signal transduction by reactive oxygen species in the cardiovascular system[J].Circ Res,2015,116(3):531-549.
[9]Vakifahmetoglu-Norberg H,Ouchida A T,Norberg E.The role of mitochondria in metabolism and cell death[J].Biochem Biophys Res Commun,2017,482(3):426-431.
[10]Dedkova E N,Blatter L A.Measuring mitochondrial function in intact cardiac myocytes[J].J Mol Cell Cardiol,2012,52(1):48-61.
[11]DornⅡG W.Apoptotic and non-apoptotic programmed cardiomyocyte death in ventricular remodeling[J].Cardiovasc Res,2009,81(3):465-473.
[12]TIE L,WANG D,SHI Y,et al.Aquaporins in cardiovascular system[J].Adv Exp Med Biol,2017,969:105-113.
[13]李雷兵,张琦.中医药关于水通道蛋白表达的研究进展[J].中国中医基础医学杂志,2015,21(9):1194-1195.
[14]孙维建,胡丹虹,卢明东,等.胃癌组织中水通道蛋白1的表达与细胞凋亡、血管生成及预后的关系[J].浙江医学,2012,34(6):405-407.
[15]杨志霞,林谦,马利.丹参对心血管疾病药理作用的文献研究[J].世界中西医结合杂志,2012,7(2):93-96,114.
[16]FU J,HUANG H,LIU J,et al.TanshinoneⅡAprotects cardiac myocytes against oxidative stresstriggered damage and apoptosis[J].Eur J Pharmacol,2007,568(1/3):213-221.
[17]袁宝萍,吕嵘,章忱,等.复方丹参滴丸对急性心肌缺血大鼠模型的影响[J].中国实验方剂学杂志,2012,18(22):222-226.
[18]黄庆,李志武,马志国,等.地龙的研究进展[J].中国实验方剂学杂志,2018,24(13):220-226.
[19]史磊,张天锡,杜聪颖,等.中药全蝎活性成分、药理作用及临床应用研究进展[J].辽宁中医药大学学报,2015,17(4):89-91.
[20]耿亚,杨洪军,马月香,等.基于“肠外翻-心肌细胞”联用模型的益气活血方药效学作用评价及机制探讨[J].中国中药杂志,2016,41(20):3821-3827.
[21]陈强,安娜,庄曾渊,等.补肾养血明目方含药肠吸收液对ARPE-19细胞氧化损伤的保护作用[J].中国实验方剂学杂志,2017,8(17):1433-1436.
[22]许照,肖雄,黄刚,等.基于含药肠吸收液的麻黄-杏仁药对物质基础与作用机制分析[J].中国实验方剂学杂志,2018,24(6):1-6.
[23]吴林霖,李梅,巩仔鹏,等.基于大鼠离体外翻肠囊模型考察杜仲提取物在正常和自发性高血压状态下的肠吸收特性差异[J].中国实验方剂学杂志,2018,24(9):15-21.
[24]张旻昱,龚慕辛,杨洪军.含药肠吸收液:一种新的中药体外药理实验方法[J].中草药,2018,49(15):3457-3462.
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