基于自噬基因Beclin-1与LC3探讨丹参素对H/R大鼠心肌细胞ATP5G1表达的影响
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摘要
目的探讨丹参素对缺氧/复氧(H/R)大鼠心肌细胞的保护作用,并揭示其作用机制。方法将培养的心肌细胞分为3组,正常组(N组),模型组(M组),丹参素组(D组)。正常组以完全培养基37℃、5%CO2条件下持续培养7 h;模型组、丹参素组更换无糖培养基,转移至缺氧培养箱中培养(5%CO2+95%N2),进行缺氧刺激4 h后,丹参素组更换含有浓度为10μM丹参素的完全培养基;模型组更换不含有丹参素的完全培养基,然后将模型组、丹参素组细胞恢复正常培养3 h(37℃、5%CO2)。采用CCK-8法检测大鼠心肌细胞活力;采用RTPCR法检测Bcl-2、Bax、LC3、Beclin-1、ATP5G1的基因表达。结果与正常组比较,模型组大鼠心肌细胞活力下降,抑制凋亡基因Bcl-2的mRNA表达下调,促进凋亡基因Bax上调,自噬基因LC3、Beclin-1表达上调,ATP5G1基因表达上调,差异具有统计学意义(P <0. 01);与模型组相比较,丹参素组大鼠心肌细胞活力升高,抑制凋亡基因Bcl-2的mRNA表达上调,促进凋亡基因Bax下调,自噬基因LC3、Beclin-1表达下调,ATP5G1基因表达下调,差异具有统计学意义(P <0. 05,P <0. 01)。结论丹参素具有提升H/R大鼠心肌细胞活力、抑制凋亡的作用,这可能与丹参素调节自噬、下调ATP5G1的基因表达有关。
Objective To explore the protective effects of tanshinol on cardiomyocytes in the rats with hypoxia and reoxygenation( H/R) and its effect mechanism. Methods The cultured cardiomyocytes were divided into 3 groups,a normal group,a model group and a tanshinol group. In the normal group,the cells were cultured continuously with complete medium 5% CO2,at 37 ℃,for 7 h. In the model group and the tanshinol group,the sugar-free medium was used and the cells were shifted to hypoxia box( 5% CO2+ 95% N2). 4 h later with hypoxia stimulation,in the tanshinol group,the complete medium with tanshinol 10 μM was adopted. In the model group,the complete medium without tanshinol was replaced.Afterwards,the cells in the model group and the tanshinol group were cultured regularly for 3 h( 5% CO2,at 37 ℃). CCK-5 assay was adopted to determine the activity of cardiomyocytes. RT-PCR method was used to determine the gene expressions of Bcl-2,Bax,LC3,Beclin-1 and ATP5 G1. Results Compared with the normal group,the activity of cardiomyocytes was reduced,the apoptosis inhibiting gene,Bcl-2 mRNA expression down-regulated,the apoptosis promoting gene,Bax promoted up-regulated,the expressions of autophagy genes,LC3 and Beclin-1 were up-regulated and ATP5 G1 up-regulated in the model group,indicating the significant differences( P < 0. 01). Compared with the model group,in the tanshinol group,the activity of cardiomyocytes was increased,the Bcl-2 mRNA expression was up-regulated,the Bax down-regulated,the expressions of autophagy genes,LC3 and Beclin-1 were down-regulated and ATP5 G1 down-regulated,indicating the significant differences( P < 0. 05,P < 0. 01). Conclusion The tanshinol increases the activity of cardiomyocytes in H/R rats and inhibits the apoptosis,which is probably related to the autophagy regulation and the down-regulation of ATP5 G1.
Objective To explore the protective effects of tanshinol on cardiomyocytes in the rats with hypoxia and reoxygenation( H/R) and its effect mechanism. Methods The cultured cardiomyocytes were divided into 3 groups,a normal group,a model group and a tanshinol group. In the normal group,the cells were cultured continuously with complete medium 5% CO2,at 37 ℃,for 7 h. In the model group and the tanshinol group,the sugar-free medium was used and the cells were shifted to hypoxia box( 5% CO2+ 95% N2). 4 h later with hypoxia stimulation,in the tanshinol group,the complete medium with tanshinol 10 μM was adopted. In the model group,the complete medium without tanshinol was replaced.Afterwards,the cells in the model group and the tanshinol group were cultured regularly for 3 h( 5% CO2,at 37 ℃). CCK-5 assay was adopted to determine the activity of cardiomyocytes. RT-PCR method was used to determine the gene expressions of Bcl-2,Bax,LC3,Beclin-1 and ATP5 G1. Results Compared with the normal group,the activity of cardiomyocytes was reduced,the apoptosis inhibiting gene,Bcl-2 mRNA expression down-regulated,the apoptosis promoting gene,Bax promoted up-regulated,the expressions of autophagy genes,LC3 and Beclin-1 were up-regulated and ATP5 G1 up-regulated in the model group,indicating the significant differences( P < 0. 01). Compared with the model group,in the tanshinol group,the activity of cardiomyocytes was increased,the Bcl-2 mRNA expression was up-regulated,the Bax down-regulated,the expressions of autophagy genes,LC3 and Beclin-1 were down-regulated and ATP5 G1 down-regulated,indicating the significant differences( P < 0. 05,P < 0. 01). Conclusion The tanshinol increases the activity of cardiomyocytes in H/R rats and inhibits the apoptosis,which is probably related to the autophagy regulation and the down-regulation of ATP5 G1.
引文
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[13]于海,刘清华,肖培伦,等. Beclin1、LC3和m TOR在肝癌中的表达及临床意义[J].安徽医药,2018,22(2):246-249.
[14]孙瑶,张婵,简洁.九龙藤总黄酮调控自噬对抗心肌缺血/再灌注损伤的实验研究[J].中国药理学通报,2015,31(2):232-236.
[15]Min Xie,Yongli Kong,Wei Tan,et al. HDAC Inhibition Blunts Ischemia/Reperfusion Injury by Inducing Cardiomyocyte Autophagy[J]. Circulation,2014,129(10):1139-1151.
[16]朱文叶,王肖龙,高俊杰,等.中医药在防治急性心肌缺血/再灌注损伤中的研究进展[J].中国医药导报,2015,12(9):31-34.
[17]陈路,李玮玮,刘春慧.心肌缺血再灌注损伤的中医药研究进展及其模型的制备[J].中国生化药物杂志,2015,35(9):174-176.
[2]陈福晖,刘达兴,容松.心肌缺血再灌注损伤发生机制的研究进展[J].安徽医药,2017,21(12):2145-2148.
[3]张金明,陈建军,张艳红.丹参多酚酸盐对缺血再灌注损伤大鼠心肌的保护作用及机制[J].中华实用诊断与治疗杂志,2017,31(11):1055-1058.
[4]郝艳玲,董薇.银杏内酯A、B联合改善缺血/再灌注损伤老年大鼠心功能[J].齐齐哈尔医学院学报,2017,38(16):1863-1865.
[5]王博,李德芳,韩吉春,等.杨梅素对离体大鼠心肌缺血再灌注损伤保护作用的研究[J].中国新药杂志,2017,26(12):1455-1460.
[6]Massimo Bonora,Angela Bononi,Elena De Marchi,et al. Role of the c subunit of the F0 ATP synthase in mitochondrial permeability transition[J]. Cell Cycle,2013,12(4):674-683.
[7]Guanwei Fan,Jiahui Yu,Patrick Fordjour Asare,et al. Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of m TOR signalling[J]. Cell Mol Med,2016,20(10):1908-1919.
[8]张琳,程亮星,陈文超.原花青素预处理对大鼠心肌缺血再灌注损伤的作用[J].中医学报,2017,32(11):2140-2143.
[9]张登沈,梁贵友,刘达兴,等.阿卡地新对体外循环心肌缺血再灌注损伤模型犬心肌能量代谢的影响[J].中国药房,2017,28(28):3918-3923.
[10]Athanassios Vassilopoulos,J. Daniel Pennington,Thorkell Andresson,et al. SIRT3 Deacetylates ATP Synthase F1 Complex Proteins in Response to Nutrient-and Exercise-Induced Stress[J]. Antioxid Redox Signal,2014,21(4):551-564.
[11]Jiuya He,Holly C. Ford,Joe Carroll,et al. Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase[J]. Proc Natl Acad Sci USA,2017,114(13):3409-3414.
[12]白桦,卢圣锋,陈婉莹,等.麦粒灸预处理对大鼠心肌缺血再灌注损伤不同时期保护效应及Beclin1表达的影响[J].针刺研究,2017,42(6):471-476.
[13]于海,刘清华,肖培伦,等. Beclin1、LC3和m TOR在肝癌中的表达及临床意义[J].安徽医药,2018,22(2):246-249.
[14]孙瑶,张婵,简洁.九龙藤总黄酮调控自噬对抗心肌缺血/再灌注损伤的实验研究[J].中国药理学通报,2015,31(2):232-236.
[15]Min Xie,Yongli Kong,Wei Tan,et al. HDAC Inhibition Blunts Ischemia/Reperfusion Injury by Inducing Cardiomyocyte Autophagy[J]. Circulation,2014,129(10):1139-1151.
[16]朱文叶,王肖龙,高俊杰,等.中医药在防治急性心肌缺血/再灌注损伤中的研究进展[J].中国医药导报,2015,12(9):31-34.
[17]陈路,李玮玮,刘春慧.心肌缺血再灌注损伤的中医药研究进展及其模型的制备[J].中国生化药物杂志,2015,35(9):174-176.