自噬下降对β1受体自身抗体诱导的H9c2心肌细胞凋亡的作用  

Decreased autophagy participate in the apoptosis induced by β1 adrenergic receptor autoantibodies in H9c2 cells

作　　者：李杨[1] 乔爱秀[2] 徐云云[2] 王晓晖[2] 王丽[2] 刘慧荣[3] 

机构地区：[1]山西医科大学基础医学院生理学系细胞生理学山西省重点实验室,山西省太原市030001 [2]山西医科大学基础医学院病理教研室,山西省太原市030001 [3]首都医科大学基础医学院生理学与病理生理学教研室

出　　处：《中国心血管病研究》2017年第3期279-283,I0003,I0004,共7页Chinese Journal of Cardiovascular Research

基　　金：国家自然科学基金（项目编号：31401006）;山西省应用基础研究项目（项目编号：201601D021146）;山西医科大学博士启动基金（项目编号：B03201202）

摘　　要：目的探讨自噬下降对β1肾上腺素受体自身抗体（β1-AA）诱导的H9c2心肌细胞凋亡的作用。方法使用1μM的β1-AA对H9c2心肌细胞进行处理，Realtime—PCR检测自噬相关基因LC3mRNA表达的改变，Westernblot检测LC3、Beclin1以及P62蛋白表达水平的变化；使用流式细胞术、Caspase-3活性检测及Hoechst33258染色反映细胞凋亡情况；使用经典的自噬抑制剂3-甲基腺嘌呤（3-MA）预处理30min，以及mTOR通路抑制剂雷帕霉素（RAPA）预处理1h后，再给予细胞β1-AA处理，检测此时细胞凋亡的变化情况。结果与对照组相比，1μM的β1-AA干预H9c2心肌细胞6h可以明显降低细胞存活率[（83．46±12．87）％比（96．45±6．99）％]。与对照组（1．01±0．14）相比，β1-AA干预细胞6h、12h、24h，LC3 mRNA水平依次降低[6h（0．85±0．11），12h（0．71±0．10），24h（0．62±0．07）]，LC3Ⅱ和Beclin 1蛋白水平依次降低，P62蛋白水平逐渐增加，表明β1-AA可以引起心肌细胞自噬水平逐渐下降。同时发现，与对照组（1．13±0．24）相比，β1-AA干预细胞6h、12h，细胞凋亡水平明显升高，分别达到3．21±0．89和2．14±0．24。使用经典自噬抑制剂3-MA预处理心肌细胞30min抑制自噬后，H9c2心肌细胞凋亡水平较β1-AA单独处理组显著增加[（41．45±6．37）％比（29．55±4．32）％]；而使用经典mTOR通路抑制剂RAPA预处理心肌细胞1h上调自噬后，心肌细胞凋亡水平较β1-AA单独处理组明显下降[（17．36±2．98）％比（29．55±4．32）％]，说明自噬水平改变可以影响心肌细胞凋亡水平。结论自噬下调能够增加β1-AA诱导的H9e2心肌细胞凋亡。Objective To investigate the role of autophagy in the regulation of apoptosis induced by β1 adrenergic receptor autoantibodies（β1-AA） in H9c2 ceils. Methods H9c2 myocardial cells treated with 1 μM β1- AA were processed to detect the changes of autophagy related gene LC3 mRNA expression by Real time-PCR; using western blot to test the varieties of Beclin 1, P62 and LC3 protein expression level; flow cytometry, Caspase-3 activity detection and Hoechst 33258 staining were used to assess cell apoptosis; using classical autophagy inhibitor 3-Methyladenine（3-MA ） pretreated ceils for 30 min or pretreated with mTOR signaling pathway inhibitor rapamyein （RAPA） for 1 h, then used β1 -AA, detecting the changes of apoptosis. Results Compared with the control group （96.45±6.99）%, after 1 μM β1-AA acting on H9c2 myocardial ceils 6 h, the survival rate of cells significantly reduced to（83.46±12.87）%; as compared to control（ 1.01 ±0.14）, β1-AA intervened cells for 6 h, 12 h and 24 h, LC3 mRNA level was fallen to 0.85±0.11, 0.71±0.10, and 0.62±0.07, respectively; also, LC3 Ⅱ and Beclin 1 protein level was descended, nevertheless P62 protein level increased gradually, showed that the level of autophagy decreased gradually; at the same time, compared to controls （1.13±0.24）, β1-AA acting to cardiomyocytes for 6 h and 12 h, the level of cardiomyocytes apoptosis severally increased to 3.21±0.89 and 2.14±0.24; after preusing classical autophagy inhibitor 3-MA for 30 min to inhibit autophagy, apoptosis of H9c2 myocardial cells increased significantly than only β1-AA treatment group [ （41.45±6.37）% vs （29.55±4.32）% 1, in contrast, after using mTOR signaling pathway inhibitor RAPA for 1 h to upregulate autophagy, apoptosis of H9c2 cardiomyocytes decreased[ （17.36±2.98）% vs （29.55±4.32）% ], indicated that changes in autophagy can affect H9c2 ceils apoptosis. Conclusion Reduced autophagy could increase apoptosis induced by β1-AA in H9c2 cells.

关 键 词：自噬 β1肾上腺素受体自身抗体 H9C2 凋亡 

分 类 号：R542.2[医药卫生—心血管疾病]