胡黄连苦苷Ⅱ通过下调microRNA-1表达抑制H_2O_2诱导的心肌细胞凋亡
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摘要
目的:研究胡黄连苦苷Ⅱ(picrosideⅡ)对过氧化氢(H_2O_2)诱导的大鼠H9c2心肌细胞凋亡中微小RNA-1(microRNA-1,miR-1)及下游靶基因抗凋亡因子B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)表达的影响,探讨胡黄连苦苷Ⅱ的心肌保护作用及其机制。方法:将H9c2心肌细胞随机分为正常组,模型组(H_2O_2,200μmol·L~(-1)),胡黄连苦苷Ⅱ(50,100,200μmol·L~(-1))+H_2O_2(200μmol·L~(-1))组,胡黄连苦苷Ⅱ200μmol·L~(-1)组。胡黄连苦苷Ⅱ与心肌细胞孵育6 h后,加入H_2O_2继续培养2 h。药物处理结束后,噻唑蓝(methyl thiazolyl tetrazolium,MTT)比色法检测细胞存活率,比色法测定细胞培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)活性,4',6-二脒基-2-苯基吲哚(destination access point identifier,DAPI)染色和细胞凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶-3 (cysteinyl aspartate specific proteinase-3,Caspase-3)检测评估细胞凋亡,实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞中Caspase-3,Bcl-2,miR-1 mRNA的表达,蛋白免疫印迹法(Western blot)检测细胞抗凋亡因子Bcl-2蛋白的表达。结果:与正常组相比,H_2O_2能显著降低细胞生存率,增加细胞凋亡率,且Caspase-3活性和mRNA表达均增加(P <0. 01),同时伴有miR-1 mRNA表达水平的上调及其下游靶基因抗凋亡因子Bcl-2 mRNA表达的下调(P <0. 01)。与模型组比较,胡黄连苦苷Ⅱ预处理可明显升高细胞存活率,明显降低LDH活性,降低细胞凋亡率,明显降低Caspase-3活性和mRNA表达(P <0. 05,P <0. 01),并使Bcl-2 mRNA表达升高(P <0. 05,P <0. 01),Western blot结果表明,胡黄连苦苷Ⅱ能明显下调其下游靶基因抗凋亡因子Bcl-2蛋白表达(P <0. 05,P <0. 01)。结论:胡黄连苦苷Ⅱ对H_2O_2诱导的H9c2心肌细胞凋亡具有保护作用,其机制与下调miR-1的表达进而上调其靶基因Bcl-2的表达有关。
Objective: To study the effect of picroside Ⅱ on the expression of microRNA-1( miR-1) in the H_2O_2-induced H9 c2 cardiomyocytes damage,in order to explore the mechanism of picroside Ⅱ in protecting H9 c2 cardiomyocytes from oxidative stress. Method: H9 c2 cardiomyocytes were divided into 6 groups: control group,model group( H_2O_2200 μmol·L~(-1)),picroside Ⅱ( 50,100,200 μmol·L~(-1)) + H_2O_2( 200 μmol·L~(-1))group and picroside Ⅱ( 200 μmol·L~(-1)) group. Picroside Ⅱ group was incubated with picroside Ⅱ for 6 h and then cultured with H_2O_2 for 2 h. At the end of drugs treatment,the cell viability and the cellular damage of cardiomyocytes were respectively assessed by 3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide( MTT) and lactate dehydrogenase( LDH) assays. 4',6-diamidino-2-phenylindole( DAPI) staining and cysteinyl aspartate specific proteinase-3( Caspase-3) test were used to evaluate cell apoptosis. The mRNA expressions of Caspase-3,B-cell lymphoma-2( Bcl-2) and miR-1 were measured by Real-time polymerase chain reaction( Realtime PCR). The protein expression of Bcl-2 was detected by Western blot. Result: Compared with the control group,H_2O_2 could significantly decrease the cell viability and increase the rate of apoptosis,up-regulate mRNA expression of Caspase-3 and miR-1,and down-regulate expression of Bcl-2 in H9 c2 cells( P < 0. 01). Compared with the model group,the cell survival rate was significantly increased by pretreating with Picroside Ⅱ for 6 h( P < 0. 05,P < 0. 01). Picroside Ⅱ not only decreased the rate of apoptosis and up-regulated mRNA expression of Bcl-2,but also down-regulated mRNA expressions of Caspase-3 and miR-1( P < 0. 05,P < 0. 01). Western blot showed that picroside Ⅱ can significantly enhance the expression of Bcl-2( P < 0. 05, P < 0. 01).Conclusion: Picroside Ⅱ has a protective effect on H9 c2 cells from H_2O_2-induced cardiomyocyte injury by downregulating mRNA-1 expression and up-regulating the expression of the downstream Bcl-2.
Objective: To study the effect of picroside Ⅱ on the expression of microRNA-1( miR-1) in the H_2O_2-induced H9 c2 cardiomyocytes damage,in order to explore the mechanism of picroside Ⅱ in protecting H9 c2 cardiomyocytes from oxidative stress. Method: H9 c2 cardiomyocytes were divided into 6 groups: control group,model group( H_2O_2200 μmol·L~(-1)),picroside Ⅱ( 50,100,200 μmol·L~(-1)) + H_2O_2( 200 μmol·L~(-1))group and picroside Ⅱ( 200 μmol·L~(-1)) group. Picroside Ⅱ group was incubated with picroside Ⅱ for 6 h and then cultured with H_2O_2 for 2 h. At the end of drugs treatment,the cell viability and the cellular damage of cardiomyocytes were respectively assessed by 3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide( MTT) and lactate dehydrogenase( LDH) assays. 4',6-diamidino-2-phenylindole( DAPI) staining and cysteinyl aspartate specific proteinase-3( Caspase-3) test were used to evaluate cell apoptosis. The mRNA expressions of Caspase-3,B-cell lymphoma-2( Bcl-2) and miR-1 were measured by Real-time polymerase chain reaction( Realtime PCR). The protein expression of Bcl-2 was detected by Western blot. Result: Compared with the control group,H_2O_2 could significantly decrease the cell viability and increase the rate of apoptosis,up-regulate mRNA expression of Caspase-3 and miR-1,and down-regulate expression of Bcl-2 in H9 c2 cells( P < 0. 01). Compared with the model group,the cell survival rate was significantly increased by pretreating with Picroside Ⅱ for 6 h( P < 0. 05,P < 0. 01). Picroside Ⅱ not only decreased the rate of apoptosis and up-regulated mRNA expression of Bcl-2,but also down-regulated mRNA expressions of Caspase-3 and miR-1( P < 0. 05,P < 0. 01). Western blot showed that picroside Ⅱ can significantly enhance the expression of Bcl-2( P < 0. 05, P < 0. 01).Conclusion: Picroside Ⅱ has a protective effect on H9 c2 cells from H_2O_2-induced cardiomyocyte injury by downregulating mRNA-1 expression and up-regulating the expression of the downstream Bcl-2.
引文
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[13]王全伟,凡文博,王智昊,等.氧化应激与心血管疾病关系的研究进展[J].中国老年学杂志,2014,34(1):270-273.
[14] JI L,FU F,ZHANG L,et al. Insulin attenuates myocardial ischemia/reperfusion injury via reducing oxidative/nitrative stress[J]. Am J Physiol Endocrinol Metab,2010,298(4):E871-E880.
[15]成军.现代细胞凋亡分子生物学[M]. 2版.北京:科学出版社,2012:24-169.
[16] LI Q,SONG X W,ZOU J,et al. Attenuation of microRNA-1 derepresses the cytoskeleton regulatory protein twinfilin-1 to provoke cardiac hypertrophy[J]. J Cell Sci,2010,123(Pt 14):2444-2452.
[17] ZHAO Y,Ransom J F,LI A,et al. Dysregulation of cardiogenesis,cardiac conduction,and cell cycle in mice lacking miRNA-1-2[J]. Cell,2007,129(2):303-317.
[18] TANG Y,ZHENG J,SUN Y,et al. MicroRNA-1regulates cardiomyocyte apoptosis by targeting Bcl-2[J]. Int Heart J,2009,50(3):377-387.
[19] MENG F J,JIAO S M,YU B. PicrosideⅡprotects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis by activating the PI3K/Akt and CREB pathways[J]. Int J Mol Med,2012,30(2):263-270.
[2] Cannell I G,KONG Y W, Bushell M. How do microRNAs regulate gene expression?[J]. Biochem Soc Trans,2008,36(Pt 6):1224-1231.
[3] Chistiakov D A,Orekhov A N,Bobryshev Y V. Cardiacspecific miRNA in cardiogenesis,heart function,and cardiac pathology(with focus on myocardial infarction)[J]. J Mol Cell Cardiol,2016,94:107-121.
[4] ZHU H,HAO J,CHEN H,et al. Nanovesicles system for rapid-onset sublingual delivery containing sodium tanshinoneⅡA sulfonate:in vitro and in vivo evaluation[J]. J Pharm Sci,2013,102(7):2332-2340.
[5] Schmelzer C,Kitano M,Rimbach G,et al. Effects of ubiquinol-10 on microRNA-146a expression in vitro and in vivo[J]. Mediators Inflamm,2009,415-437.
[6] Simone N L,Soule B P,Ly D,et al. Ionizing radiationinduced oxidative stress alters miRNA expression[J].PLo S One,2009,4(7):e6337.
[7]黄林清,张恩娟,王军.胡黄连的研究进展[J].中国药业,2007,16(7):1-2.
[8] CAO Y,LIU J W,YU Y J,et al. Synergistic protective effect of picrosideⅡand NGF on PC12 cells against oxidative stress induced by H2O2[J]. Pharmacol Rep,2007,59(5):573-579.
[9] HE L J,LIANG M,HOU F F,et al. Ethanol extraction of picrorhiza scrophulariiflora prevents renal injury in experimental diabetes via anti-inflammation action[J]. J Endocrinol,2009,200(3):347-355.
[10] LI J Z,XIE M Q,MO D,et al. PicrosideⅡprotects myocardium from ischemia/reperfusion-induced injury through inhibition of the inflammatory response[J]. Exp Ther Med,2016,12(6):3507-3514.
[11]申静,刘冲,陈文.芳香新塔花总黄酮对大鼠心肌缺血再灌注损伤的保护作用及机制[J].中国实验方剂学杂志,2018,24(14):115-121.
[12]范鹤馨,张志强.葛花总黄酮对大鼠心肌缺血再灌注动物损伤模型的保护作用[J].中国实验方剂学杂志,2017,23(12):119-125.
[13]王全伟,凡文博,王智昊,等.氧化应激与心血管疾病关系的研究进展[J].中国老年学杂志,2014,34(1):270-273.
[14] JI L,FU F,ZHANG L,et al. Insulin attenuates myocardial ischemia/reperfusion injury via reducing oxidative/nitrative stress[J]. Am J Physiol Endocrinol Metab,2010,298(4):E871-E880.
[15]成军.现代细胞凋亡分子生物学[M]. 2版.北京:科学出版社,2012:24-169.
[16] LI Q,SONG X W,ZOU J,et al. Attenuation of microRNA-1 derepresses the cytoskeleton regulatory protein twinfilin-1 to provoke cardiac hypertrophy[J]. J Cell Sci,2010,123(Pt 14):2444-2452.
[17] ZHAO Y,Ransom J F,LI A,et al. Dysregulation of cardiogenesis,cardiac conduction,and cell cycle in mice lacking miRNA-1-2[J]. Cell,2007,129(2):303-317.
[18] TANG Y,ZHENG J,SUN Y,et al. MicroRNA-1regulates cardiomyocyte apoptosis by targeting Bcl-2[J]. Int Heart J,2009,50(3):377-387.
[19] MENG F J,JIAO S M,YU B. PicrosideⅡprotects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis by activating the PI3K/Akt and CREB pathways[J]. Int J Mol Med,2012,30(2):263-270.