内质网应激在二十碳五烯酸抵抗棕榈酸诱导的心肌细胞凋亡中的作用

Effect of endoplasmic reticulum stress on eicosapentaenoic acid resisting myocardial apoptosis induced by palimitate

目的:研究内质网应激(endoplasmic reticulum stress,ERS)在二十碳五烯酸(eicosapentaenoic acid,E PA)抵抗棕榈酸(palimitate,PA L)诱导的心肌细胞凋亡中的作用。方法:将培养的心肌细胞随机分为对照组、PAL处理组、EPA+PAL组、毒胡萝卜素(thapsigargin)+EPA+PAL组、thapsigargin组及thapsigargin+EPA组。采用CCK-8检测细胞活力、Tunel染色检测细胞凋亡率、Western印迹检测葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)和钙网蛋白(calreticulin,CRT),以及ERS促凋亡蛋白C/EBP同源蛋白(C/EBP homologous protein,CHOP)、JNK和半胱氨酸天冬氨酸蛋白酶12(caspase-12)的表达。结果:与对照组比较,PAL处理可显著降低心肌细胞活力,促进细胞凋亡,激活ERS应激相关GRP78和CRT,以及ERS促凋亡蛋白CHOP,p-JNK及活化的caspase-12(cleaved caspase-12)的表达(P<0.0 5)。相比于PA L组,E PA+PA L预处理可使PA L诱导的心肌细胞活力显著升高,GRP78,CRT,CHOP,cleaved caspase-12及p-JNK蛋白表达明显降低(P<0.05),且使细胞凋亡率由43.9%降低至24.07%(P<0.05)。添加ER S特异性激活剂thapsigarg in激活ERS后,与EPA+PAL组相比,thapsigargin+EPA+PAL组ERS应激相关蛋白GRP78和CRT蛋白表达比率显著升高,ERS促凋亡蛋白CHOP,JNK及caspase-12上调,心肌细胞活力下降,凋亡率增加。此外,与对照组比较,单独添加thapsigargin激活ERS,显著降低细胞活性,促进细胞凋亡,而EPA+thapsigargin组却明显逆转了thapsigargin对心肌细胞的促凋亡效应。结论:EPA可有效抑制PAL诱导的大鼠心肌细胞凋亡,其保护机制可能与抑制PAL诱导的心肌细胞ERS激活,进而抑制caspase依赖的级联凋亡反应有关。

Objective： To investigate the functional role of endoplasmic reticulum stress （ERS） in eicosapentaenoic acid （EPA） attenuating myocardial apoptosis induced by palimitate （PAL）. Methods： Cells were divided randomly into six groups： the blank control group, the PAL stimulated group, the EPA ＋ PAL stimulated group, the thapsigargin ＋ EPA ＋ PAL-stimulated group, thapsigargin group, thapsigargin ＋ EPA stimulated group. CCK-8 was used to test the cell viability, TUNEL staining was used to detect the apoptosis, and Western blot method was used to detect the expression of glucose-regulated protein 78 （GRP78）, calreticulin （CRT）, C/EBP homologous protein （CHOP）, JNK and caspase-12. Results： PAL treatment resulted in a decrease of cell viability and an increase of cell apoptotic rates and led to a significant up-regulation of the expression of GRP78, CRT, CHOP, p-JNK and cleaved caspase-12 proteins. Compared with PAL treatment, pretreatment with EPA ＋ PAL resulted in a significant decrease of the expression of GRP78, CRT, cleaved caspase-12, p-JNK and CHOP proteins （P〈0.05）, and decreased PAL-induced cardiomyocytes apoptosis from 43.9% to 24.07% （P〈0.05）. Cells were treated with thapsigargin, a specific activator of ERS; compared with EPA＋PAL treatment, thapsigargin ＋ EPA ＋ PAL showed increased significantly of the expression of GRP78, cleaved caspase-12, p-JNK and CHOP proteins （P〈0.05）, down-regulated of cell viability, and enhanced apoptosis of cardiomyocyte cell. Furthermore, thapsigargin pretreatment obviously decreased cell viability and increase cell apoptosis compared with the control group, pretreatment with thapsigargin ＋ EPA decreased thapsigargin-induced cardiomyocytes apoptosis. Conclusion： EPA could prevent from PAL-induced cardiomyocytes apoptosis through a restraining ERS and caspase dependent apoptotic pathway.