分泌型丛生蛋白通过抑制线粒体自噬缓解H2O2诱导的心肌细胞损伤 #br#

目的 探讨分泌型丛生蛋白（sCLU）对心肌细胞氧化损伤的影响及其机制。方法 采用大鼠心肌细胞 H9C2。实验1分为Control组（仅更换培养基）和H2O2组（100 µmol/L H2O2处理）。实验2分为Control组、pcDNA3.1组 （转染 pcDNA3.1 对照空质粒）、pcDNA3.1-sCLU 组（转染 pcDNA3.1-sCLU 过表达质粒）、H2O2组（100 µmol/L H2O2处 理）、H2O2+pcDNA3.1 组（pcDNA3.1 对照空质粒转染 48 h 后加入 100 µmol/L H2O2处理）、H2O2+pcDNA3.1-sCLU 组 （pcDNA3.1-sCLU 过表达质粒转染 48 h 后加入 100 µmol/L H2O2处理）。实验 3 分为 DMSO 组（加入 1% 体积分数 的 DMSO）、Mdivi-1 组（10 µmol/L 的 Mdivi-1 处理）、H2O2+DMSO 组（加入 1% 体积分数的 DMSO 处理 30 min 后加入 100 µmol/L的H2O2处理）、H2O2+Mdivi-1组（10 µmol/L的Mdivi-1处理30 min后加入100 µmol/L的H2O2处理）、H2O2+ pcDNA3.1-sCLU 组（pcDNA3.1-sCLU 过表达质粒转染 48 h 后加入 100 µmol/L H2O2处理）、H2O2+pcDNA3.1-sCLU+ Mdivi-1组（pcDNA3.1-sCLU过表达质粒转染48 h后加入10 µmol/L的Mdivi-1处理30 min，再加入100 µmol/L H2O2 处理）。采用MTT法检测细胞活力，TUNEL法检测细胞凋亡，试剂盒检测细胞中超氧化物歧化酶（SOD）活力和丙二 醛（MDA）水平。DFCH-DA荧光探针测定细胞活性氧（ROS）水平。实时定量聚合酶链式反应和酶联免疫吸附试验 检测细胞中sCLU表达，Western blot法检测CLU、PINK1、Parkin和LC3蛋白表达水平。结果 （1）实验1。与Control 组比较，H2O2组细胞中sCLU mRNA、细胞培养上清中sCLU蛋白及细胞中CLU蛋白相对表达水平均降低（P＜0.01）。 （2）实验2。与Control组比较，H2O2组细胞活力、SOD水平、LC3-Ⅱ/LC3-Ⅰ比值均降低，细胞凋亡率、ROS水平、MDA 水平、PINK1和Parkin蛋白表达水平均升高（P＜0.05）；与H2O2组比较，H2O2+pcDNA3.1-sCLU组细胞活力、SOD水平、 LC3-Ⅱ/LC3-Ⅰ比值均升高，细胞凋亡率、ROS水平、MDA水平、PINK1和Parkin蛋白表达水平均降低（P＜0.05）。（3） 实验3。与DMSO组比较，Mdivi-1组细胞活力与细胞凋亡率差异无统计学意义；H2O2+DMSO组细胞活力下降，细胞 凋亡率升高（P＜0.05）。与H2O2+DMSO组比较，H2O2+Mdivi-1组、H2O2+pcDNA3.1-sCLU组和H2O2+pcDNA3.1-sCLU+ Mdivi-1 组细胞活力均升高，细胞凋亡率均下降（P＜0.05）。与 H2O2+Mdivi-1 组比较，H2O2+pcDNA3.1-sCLU 组和 H 2O2+pcDNA3.1-sCLU+Mdivi-1组细胞活力差异无统计学意义，细胞凋亡率降低（P＜0.05）。结论 sCLU对氧化应 激条下的心肌细胞具有保护作用，其机制可能与线粒体自噬的抑制有关。

Secretory clusterin attenuated hydrogen peroxide induced cardiomyocyte injury by inhibiting mitophagy #br#

Objective To investigate the effects of secretory clusterin (sCLU) on oxidative-induced cardiomyocyte injury and the underlying mechanism. Methods Rat cardiomyocytes H9C2 were used in this experiment. Experiment 1 was divided into the control group (only medium change) and the H2O2 group (treated with 100 µmol/L H2O2). Experiment 2 was divided into the control group, the pcDNA3.1 group (transfected with pcDNA3.1 empty plasmids), the pcDNA3.1-sCLU group (transfected with pcDNA3.1-sCLU overexpressed plasmids), the H2O2 group and the H2O2+pcDNA3.1 group (transfected with pcDNA3.1 empty plasmids for 48 h, and then treated with 100 µmol/L H2O2). The H2O2+pcDNA3.1-sCLU was transfected with pcDNA3.1-sCLU over-expressed plasmids for 48 h, and then 100 µmol/L H2O2. Experiment 3 was divided into the DMSO group (adding 1% volume of DMSO), the Mdivi-1 group (treated with 10 µmol/L Mdivi-1), the H 2O2+DMSO group (treated with 1% volume of DMSO for 30 min, and then added 100 µmol/L H2O2), the H2O2+Mdivi-1 group (treated with 100 µmol/L Mdivi-1 for 30 min, and then treated with 100 µmol/L H2O2), the H2O2+pcDNA3.1-sCLU group and the H2O2+pcDNA3.1-sCLU+Mdivi-1 group (transfected with pcDNA3.1-sCLU overexpressed plasmids for 48 h, and then treated with 10 µmol/L Mdivi-1 for 30 min, and then added 100 µmol/L H2O2). MTT assay was used to detect cell viability. TUNEL assay was used to detect cell apoptosis. The corresponding Kit was used to detect the activity of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA). The expression of sCLU in cells was detected by qRT-PCR and ELISA. The protein expression levels of CLU, mitochondrial autophagy related proteins PINK1, Parkin and LC3 were detected by Western blot assay. Results In experiment 1, compared with the control group, the relative expression levels of sCLU mRNA, sCLU protein levels in cell culture supernatant and total CLU protein levels in cells were decreased in the H 2O2 group (P＜0.05). In experiment 2, compared with the control group, cell viability, SOD levels and LC3-II/LC3-I ratio were all decreased in the H 2O2 group (P＜0.05). Apoptosis rate, ROS and MDA levels, PINK1 and Parkin protein expression levels were all increased in the H2O2 group (P＜0.05). Compared with H2O2 group, cell viability rate, SOD levels and LC3-Ⅱ/LC3-Ⅰ ratio were increased in H2O2+pcDNA3.1-sCLU group (P＜0.05). Apoptosis rate, ROS and MDA levels, PINK1 and Parkin protein expression levels were all decreased in H2O2+pcDNA3.1-sCLU group (P＜0.05). In experiment 3, compared with DMSO group, mdivi-1 group showed no significant difference in cell viability and apoptosis rate. Cell viability was decreased and apoptosis rate was increased in H2O2+DMSO group (P＜0.05). Compared with the H2O2+DMSO group, cell viability was increased and cell apoptosis rate was decreased in the H2O2+ Mdivi-1 group and H2O2+pcDNA3.1-sCLU group (P＜0.05). There were no significant differences in cell viability and the cell apoptosis rate between the DMSO group and the Mdivi-1 group. Compared with the DMSO group, the cell viability decreased and the cell apoptosis rate increased in the H2O2+DMSO group (P＜0.05). Compared with the H2O2+DMSO group, cell viability increased and cell apoptosis rate decreased in the H2O2+Mdivi-1 group, the H2O2+PCDNA3.1-sCLU group and the H2O2+PCDNA3.1-SCLU+Mdivi-1 group (P＜0.05). There was no significant difference in cell viability between the H 2O2+Mdivi-1 group, H2O2+pcDNA3.1-sCLU group and the H2O2+pcDNA3.1-sCLU+Mdivi-1 group (P＞ 0.05). Conclusion sCLU can protect myocardial cells under oxidative stress, and the mechanism may be related to the inhibition of mitophagy.