摘要
【目的】探讨丹参酮ⅡA对棕榈酸诱导心肌细胞脂毒性损伤模型内质网应激凋亡的影响。【方法】将对数期H9c2细胞分6组,即对照组,棕榈酸(400μmol/L)组,丹参酮ⅡA 20μmol/L组和棕榈酸+丹参酮ⅡA(5、10、20μmol/L)组,另外引入棕榈酸+CCT020312组与棕榈酸+CCT020312+丹参酮ⅡA 20μmol/L组考察通路激活状态。细胞计数试剂盒8(CCK-8)法测定H9c2细胞活力;流式细胞术测定细胞凋亡情况;实时定量聚合酶链反应(RT-qPCR)法检测细胞内质网应激相关分子葡萄糖调控蛋白78(GRP78)、转录激活因子4(ATF4)和CCAAT/增强子结合蛋白同源蛋白(CHOP)的mRNA表达水平;蛋白免疫印迹(Western Blot)法检测细胞GRP78、ATF4、CHOP、Caspase-3、Caspase-9、Caspase-12、蛋白激酶RNA样内质网激酶(PERK)、磷酸化PERK(p-PERK)、真核生物翻译起始因子2α(eIF2α)、磷酸化eIF2α(p-eIF2α)的蛋白表达水平。【结果】与对照组比较,棕榈酸组细胞存活率显著降低(P <0.01),GRP78、ATF4、CHOP m RNA和蛋白表达水平均显著升高(P <0.01),H9c2细胞的凋亡率升高,p-PERK/PERK和p-eIF2α/eIF2α比例也显著增大(P <0.01),凋亡相关分子Caspase-3、Caspase-9和Caspase-12蛋白表达水平均显著升高(P <0.01)。不同浓度丹参酮ⅡA作用后,与棕榈酸组比较,棕榈酸+丹参酮ⅡA(5、10、20μmol/L)组细胞存活率显著升高(P <0.01),GRP78、ATF4、CHOP mRNA和蛋白表达水平均显著降低(P <0.01),H9c2细胞的凋亡率降低,p-PERK/PERK和p-eIF2α/eIF2α比例也显著减小(P <0.01),Caspase-3、Caspase-9和Caspase-12蛋白表达水平显著降低(P <0.01),均具有剂量依赖性,且棕榈酸+丹参酮ⅡA 10μmol/L组(P <0.05)和棕榈酸+丹参酮ⅡA 20μmol/L组(P <0.05)变化显著。加入PERK通路激活剂CCT020312作用后,与棕榈酸组比较,棕榈酸+CCT020312组的p-PERK/PERK和p-eIF2α/eIF2α比例,细胞凋亡率,GRP78、ATF4、CHOP的mRNA和蛋白表达水平均显著升高(P <0.05);再经过丹参酮ⅡA处理,与棕榈�
Objective To explore the effects of tanshinone Ⅱ A on endoplasmic reticulum stress-apoptosis in palmitic acid induced myocardial cell injury model. Methods The H9 c2 cells at logarithmic phase were divided into control group,palmitic acid(400 μmol/L)group, tanshinone ⅡA 20 μmol/L group, and palmitic acid plus tanshinone ⅡA(5,10,20 μmol/L)groups,moreover palmitic acid plus CCT020312 group and palmitic acid plus CCT020312 plus tanshinone Ⅱ A group were added for exploring the activation situation of pathway. The viability of H9 c2 cells was detected by cell count kit 8(CCK-8)assay. The apoptosis of H9 c2 cells was detected by flow cytometry. The m RNA expression levels of endoplasmic reticulum stress-related molecule glucose-regulated protein 78(GRP78), activating transcription factor 4(ATF4), CCAAT/enhancer binding protein homologues protein(CHOP)were determined by real-time quantitative polymerase chain reaction(RT-qPCR). The protein expression levels of molecules GRP78,ATF4,CHOP,Caspase-3,Caspase-9,Caspase-12,protein kinase R-like endoplasmic reticulum kinase(PERK), phosphorylated PERK(p-PERK)、 eukaryotic translation initiation factor alpha two(e IF2α),phosphorylated eIF2α(p-eIF2α)were determined by Western blotting assay.Results Compared with the control group, H9 c2 cell survival rate of palmitic acid group was decreased significantly(P < 0.01),m RNA and protein expression levels of GRP78,ATF4 and CHOP in H9 c2 cells were significantly increased(P < 0.01),H9 c2 cell apoptosis rate was increased significantly(P < 0.01),p-PERK/PERK and p-eIF2α/eIF2α ratios also were increased significantly(P < 0.01),and protein expression levels of Caspase-3,Caspase-9 and Caspase-12 were significantly increased(P < 0.01). After treatment with tanshinoneⅡA at different concentrations,compared with the palmitic acid group,the H9 c2 cell survival rate of the palmitic acid plus tanshinone ⅡA(5,10,20 μmol/L)groups were increased,mRNA and protein expression levels of GRP78,ATF4 and CHOP were decreased,apoptosis
作者
王亚丹
艾景雪
高瑞
李运丽
张海波
WANG Ya-Dan;AI Jing-Xue;GAO Rui;LI Yun-Li;ZHANG Hai-Bo(Dept.3 of Cardiology,the First Affiliated Hospital of Henan University,Kaifeng 475000 Henan,China)
出处
《广州中医药大学学报》
CAS
2021年第8期1685-1692,共8页
Journal of Guangzhou University of Traditional Chinese Medicine
基金
河南省医学科技攻关计划联合共建项目(编号:2018020313)。
关键词
丹参酮ⅡA
脂毒性损伤
凋亡
内质网应激
PERK信号通路
棕榈酸
心肌细胞
tanshinoneⅡA
lipotoxic injury
apoptosis
endoplasmic reticulum(ER)stress
PERK signaling pathway
palmitic acid
myocardial cells
