激动核受体RXR对大鼠心肌细胞饥饿损伤中自噬和凋亡的双重调控作用  

Activating RXR regulates serum and glucose deprivation induced autophagy and apoptosis in rat cardiomyocytes

作　　者：单培仁[1] 余灵芳[2] 周昌钻 朱千里[1] 黄周青[1] 陈晨[1] SHAN Peiren;YU Lingfang;ZHOU Changzuan(Department of Cardiology,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325000,China)

机构地区：[1]温州医科大学附属第一医院心内科,325000 [2]温州医科大学育英儿童医院肾内科

出　　处：《浙江医学》2018年第22期2445-2449,共5页Zhejiang Medical Journal

基　　金：浙江省自然青年基金项目(LQ15H020005);国家自然科学青年基金项目(81600341);温州市科技局项目(Y20160307)

摘　　要：目的探讨激动维甲类X受体(RXR)对大鼠心肌细胞无糖无血清(SGD)饥饿状态下的调控作用及其机制。方法将大鼠心肌细胞株H9c2置于无糖DMEM中培养6h,建立饥饿模型。用9-顺式维甲酸(c-RA)为RXR激动剂,HX531为RXR拮抗剂,并将细胞实验按以下分组:对照组(C组)、SGD干预组(SGD组)、SGD+100nmol/L c-RA干预组(RA组)、SGD+100nmol/L c-RA+2.5μmol/L HX531干预组(HX组),MTT法检测细胞活性,流式细胞法检测细胞凋亡比例及含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-3活力,Western blot法检测凋亡相关蛋白Bcl-2、Bax及下游活性片段Caspase-9蛋白表达,以及自噬相关蛋白LC3、Beclin 1表达,透射电镜检测心肌细胞自噬小体。结果与C组比较,SGD组和HX组心肌细胞活力均下降,凋亡百分比、Bax/Bcl-2比值和Caspase-9蛋白水平均上升(均P<0.01);与SGD组比较,RA组心肌细胞活力均上升,凋亡百分比、Bax/Bcl-2比值和Caspase-9蛋白水平均下降(均P<0.01)。SGD组平均荧光强度(MFI)波峰明显右移,为C组的2.3倍(P<0.01);而RA波峰出现明显左移,MFI值仅为C组的1.3倍,明显低于SGD组(P<0.01);HX组波峰与SGD组相似,MFI值显著高于C组(P<0.01)。透射电镜下观察到C组中细胞质内细胞器完整,而SGD组和HX组自噬小体数增加,内含有被吞噬的线粒体等结构,部分呈空泡样改变,同时自噬相关蛋白LC3-Ⅱ/Ⅰ比值上升,Beclin 1表达增加(均P<0.01);RA组心肌细胞自噬小体数量明显减少,LC3-Ⅱ/Ⅰ比值下调,Beclin 1蛋白表达水平降低(均P<0.01)。结论激动核受体RXR可以保护心肌细胞SGD状态下的损伤,其机制与对凋亡和自噬通路的双重调控作用有关。Objective To determine the protective effects and mechanism of activating Retinoid X receptor （RXR） on rat cardiomyocytes H9c2 against serum and glucose deprivation （SGD） induced myocardial injury. Methods The model of SGD induced myocardial injury was established through culturing cardiomyocytes H9c2 in SGD-DMEM for 6 h. The 9-cis-retinoic acid （c-RA） was used as RXR agonist, and HX531 as RXR antagonist. Cultured cardiomyocytes were randomly divided into four groups： control group cultured with growth medium, SGD group, SGD＋ c-RA-pretreated group（100nmol/L c-RA, RA group） and SGD＋ c-RA＋ HX531-pretreated group （2.5μmol/L HX531, HX group）. The cell viability was determined by MTT, apoptosis rate of cardiomyocytes and activity of Caspase-3 by were examined by flow cytometry, apoptosis-related proteins Bcl-2, Bax and cleaved Caspase-9 were detected with Western blot, autophagic vacuoles were observed with transmission electron microscopy, and protein expressions of autophagy-related proteins LC3 and Beclin 1 were detected with Western blot. Results SGD strongly reduced the viability of H9c2 cells, induced cell apoptosis and upregulated autophagy. Dot blotting experiments showed that SGD stress disrupted mitochondria, characterized by reduction of Bcl-2 expression, increase of Bax and Caspase-9 expression,and activation of Caspase-3. Meanwhile, autophagy was significantly induced by SGD, as characterized by increase of Beclin 1 and LC3-II/LC3-I expression. Pretreatment with RXR agonist enhanced cell viability, reduced apoptosis ratio and autophagy. However, all the protective effects of activating RXR on rat cardiomyocytes against SGD induced oxidative injury were abolished when pretreated with RXR pan-antagonist HX531. Conclusion Activating RXR has the protective effects against SGD injury in rat cardiomyocytes through both anti-apoptosis and anti-autophagy effects.

关 键 词：维甲类X受体 心肌细胞 凋亡 自噬 饥饿 

分 类 号：R54[医药卫生—心血管疾病]