瓜蒌皮提取物对缺氧/复氧损伤H9c2心肌细胞的保护作用及其机制研究
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摘要
目的:研究瓜蒌皮提取物对缺氧/复氧损伤H9c2心肌细胞的保护作用及其机制。方法:以采用连二亚硫酸钠(Na_2S_2O_4)为化学缺氧剂,建立大鼠H9c2心肌细胞缺氧/复氧损伤模型,筛选不同浓度Na_2S_2O_4(0.625～10 mmol/L)和不同缺氧时间(10～60min)、复氧时间(2～8 h)的造模条件,以及瓜蒌皮提取物给药浓度(12.5～400μg/mL)。将细胞分为正常组、模型组、瓜蒌皮提取物不同剂量组(即TPE低、中、高剂量组,25、50、100μg/mL)和阳性对照组(槲皮素,25μmol/L),加入相应药物进行预处理24 h后,再建立缺氧/复氧损伤模型。采用MTT法检测各组细胞存活率;采用酶联免疫吸附法检测细胞中乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平;采用流式细胞术观察细胞凋亡情况;采用Western blotting法检测细胞中Bax、Bcl-2的蛋白表达水平。结果:心肌细胞缺氧/复氧损伤造模条件为Na_2S_2O_4造模浓度2.5 mmol/L,缺氧时间30 min,复氧时间4 h;12.5～400μg/mL的瓜蒌皮提取物对细胞均无毒性。分组处理后结果显示,与空白组比较,模型组细胞存活率显著降低、凋亡率显著升高,细胞中CK-MB、LDH、MDA水平均显著升高,SOD水平显著降低,Bax的蛋白表达水平及Bax/Bcl-2比值均显著升高,Bcl-2的蛋白表达水平均显著降低(P<0.05或P<0.01);与模型组比较,瓜蒌皮提取物各剂量组细胞上述变化均被逆转(P<0.05或P<0.01)。结论:瓜蒌皮提取物对缺氧/复氧损伤心肌细胞具有一定的保护作用;其机制可能与抑制细胞中脂质过氧化物的增加、提高细胞清除活性氧自由基的能力、上调Bcl-2/下调Bax蛋白表达等,从而抑制细胞凋亡有关。
OBJECTIVE:To study the protective effects of Pericarpium Trichosanthis extract(TPE)on H9c2 myocardial cells injured by hypoxia/reoxygenation(H/R). METHODS:H/R injury model of H9c2 myocardial cells was established by using sodium dithionite(Na_2 S_2 O_4)as chemical hypoxia agent. The modeling conditions of different concentrations of Na_2S_2O_4(0.625-10 mmol/L)and different time of hypoxia(10-60 min)and reoxygenation(2-8 h),as well as the concentration of TPE(12.5-400 μg/mL)were screened. The cultured H9 c2 myocardial cells were randomly divided into normal group,model group,TPE different dose groups(TPE low-dose,medium-dose and high-dose groups,25,50,100 μg/mL)and positive control group(quercetin,25 μmol/L).They were pre-treated with relevant medicine for 24 h,and then H/R injury model was established. Cell viability was measured by MTT assay. The levels of LDH,CK-MB,SOD and MDA in cells were tested by ELISA. Apoptosis of H9c2 myocardial cells were observed by flow cytometry. Western blotting was used to detect the protein expression of Bax and Bcl-2 in cells. RESULTS:The condition of H/R injury modeling included modeling concentration of Na_2S_2O_4 2.5 mmol/L,hypoxia time 30 min,reoxygenation time 4 h. 12.5-400 μg/mL TPE showed no toxicity to H9c2 myocardial cells. After treatment,compared with blank group,survival rate and apoptotic rate of H9c2 myocardial cells in model group were increased significantly;the levels of CK-MB,LDH and MDA were increased significantly,while SOD level was decreased significantly;protein expression of Bax and Bax/Bcl-2 ratio were increased significantly,while that of Bcl-2 was decreased significantly(P<0.05 or P<0.01). Compared with model group,above changes of H9c2 myocardial cells were reversed in all dose groups of TPE(P<0.05 or P<0.01). CONCLUSIONS:TPE can protect H9c2 myocardial cells against H/R injury. Its mechanism may be associated with inhibiting the increase of lipid peroxide,improving the ability of scavenging reactive oxygen free radicals,up-regulating the protein expression of Bcl-2 or down-regulating protein expression of Bax,so as to inhibit the cell apoptosis.
OBJECTIVE:To study the protective effects of Pericarpium Trichosanthis extract(TPE)on H9c2 myocardial cells injured by hypoxia/reoxygenation(H/R). METHODS:H/R injury model of H9c2 myocardial cells was established by using sodium dithionite(Na_2 S_2 O_4)as chemical hypoxia agent. The modeling conditions of different concentrations of Na_2S_2O_4(0.625-10 mmol/L)and different time of hypoxia(10-60 min)and reoxygenation(2-8 h),as well as the concentration of TPE(12.5-400 μg/mL)were screened. The cultured H9 c2 myocardial cells were randomly divided into normal group,model group,TPE different dose groups(TPE low-dose,medium-dose and high-dose groups,25,50,100 μg/mL)and positive control group(quercetin,25 μmol/L).They were pre-treated with relevant medicine for 24 h,and then H/R injury model was established. Cell viability was measured by MTT assay. The levels of LDH,CK-MB,SOD and MDA in cells were tested by ELISA. Apoptosis of H9c2 myocardial cells were observed by flow cytometry. Western blotting was used to detect the protein expression of Bax and Bcl-2 in cells. RESULTS:The condition of H/R injury modeling included modeling concentration of Na_2S_2O_4 2.5 mmol/L,hypoxia time 30 min,reoxygenation time 4 h. 12.5-400 μg/mL TPE showed no toxicity to H9c2 myocardial cells. After treatment,compared with blank group,survival rate and apoptotic rate of H9c2 myocardial cells in model group were increased significantly;the levels of CK-MB,LDH and MDA were increased significantly,while SOD level was decreased significantly;protein expression of Bax and Bax/Bcl-2 ratio were increased significantly,while that of Bcl-2 was decreased significantly(P<0.05 or P<0.01). Compared with model group,above changes of H9c2 myocardial cells were reversed in all dose groups of TPE(P<0.05 or P<0.01). CONCLUSIONS:TPE can protect H9c2 myocardial cells against H/R injury. Its mechanism may be associated with inhibiting the increase of lipid peroxide,improving the ability of scavenging reactive oxygen free radicals,up-regulating the protein expression of Bcl-2 or down-regulating protein expression of Bax,so as to inhibit the cell apoptosis.
引文
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[22]石瑞丽,胡金凤,孔令雷,等.瓜子金皂苷己对连二亚硫酸钠致氧糖剥夺/复供诱导PC12细胞凋亡的抑制作用[J].中国药理学通报,2013,29(3):333-336.
[23]DEL PG,VENDITTI A,DEL PM,et al.Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia(AML)[J].Blood,2003,101(6):2125-2131.(收稿日期:2018-11-04修回日期:2019-03-11)
[2]张幼怡,吴立玲.心血管病理生理学[M].北京:北京大学医学出版社,2009:1-3.
[3]陈伟伟,高润霖,刘力生,等.《中国心血管病报告2017》概要[J].中国循环杂志,2018,33(1):1-8.
[4]国家药典委员会.中华人民共和国药典:一部[S].2015年版.北京:中国医药科技出版社,2015:114.
[5]王姗姗.瓜蒌皮的药理作用及其临床应用[J].山西医药杂志,2009,38(1):67-68.
[6]杨丽,杨玲.瓜蒌皮对冠心病的药理作用及其机制研究[J].临床医药文献电子杂志,2016,3(37):7495-7496.
[7]陈昌喆,段磊,王贤,等.瓜蒌皮提取液对大鼠缺血心肌的保护作用[J].中国老年学杂志,2014,34(23):6723-6725.
[8]单红燕,高兆慧,伏瑶,等.瓜蒌皮不同提取物干预急性心肌缺血的药效学研究[J].山东中医杂志,2017,36(5):414-418.
[9]操全霞,丁旵东.α-硫辛酸对H9c2心肌细胞低氧及低氧/复氧损伤的保护作用及其机制探讨[J].安徽医科大学学报,2015,50(7):1024-1028.
[10]许蜀闽,王培勇,马红英.连二亚硫酸钠在建立培养细胞的无氧环境中的应用[J].第三军医大学学报,2005,27(4):359-360.
[11]ZHANG XQ,EYZAGUIRRE C.Effects of hypoxia induced by Na2S2O4on intracellular calcium and resting potential of mouse glomus cells[J].Brain Res,1999,818(1):118-126.
[12]WANG J,JI SY,LIU SZ,et al.Cardioprotective effect of breviscapine:inhibition of apoptosis in H9c2 cardiomyocytes via the PI3K/Akt/eNOS pathway following simulated ischemia/reperfusion injury[J].Pharmazie,2015,70(9):593-597.
[13]芦丽莉,王冬梅.瓜蒌皮提取液对实验性高脂血症大鼠血清NO,SOD,MDA的影响[J].北华大学学报(自然科学版),2008,9(5):423-425.
[14]LIAO P,SUN G,ZHANG C,et al.Bauhinia championii flavone attenuates hypoxia-reoxygenation induced apoptosis in H9c2 cardiomyocytes by improving mitochondrial dysfunction[J].Molecules,2016,21(11):1469-1480.
[15]李立美,智光.心血管病理生理学[M].5版.北京:人民军医出版社,2013:122-144.
[16]郑显杰,庞力智,寇俊萍,等.中药抗心肌缺血再灌注损伤的信号通路研究进展[J].药学进展,2015,39(6):425-436.
[17]高兆慧.瓜蒌皮干预大鼠急性心肌缺血药效学研究[D].济南:山东中医药大学,2015.
[18]孙娟.瓜蒌皮干预急性心肌梗死的药效与机制研究[D].济南:山东中医药大学,2013.
[19]汤蕾.槲皮素抗心肌缺血再灌注损伤的作用机制研究[D].南昌:南昌大学,2013.
[20]李小兰,方方,朱伟飞.不同状态下连二亚硫酸钠的稳定性研究[J].纺织科技进展,2014,40(2):71-73.
[21]YU MF,GORENNE I,SU X,et al.Sodium hydrosulfite contractions of smooth muscle are calcium and myosin phosphorylation independent[J].Am J Physiol,1998,275(5 Pt 1):L976-L982.
[22]石瑞丽,胡金凤,孔令雷,等.瓜子金皂苷己对连二亚硫酸钠致氧糖剥夺/复供诱导PC12细胞凋亡的抑制作用[J].中国药理学通报,2013,29(3):333-336.
[23]DEL PG,VENDITTI A,DEL PM,et al.Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia(AML)[J].Blood,2003,101(6):2125-2131.(收稿日期:2018-11-04修回日期:2019-03-11)