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- Title
分泌型丛生蛋白通过抑制线粒体自噬缓解H<sub>2</sub>O<sub>2</sub>诱导的 心肌细胞损伤.
- Authors
贺静; 孙晓慧; 杨莉; 乌宇亮
- Abstract
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 H2O2+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 H2O2 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 H2O2 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 H2O2+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.
- Publication
Tianjin Medical Journal, 2022, Vol 50, Issue 2, p136
- ISSN
0253-9896
- Publication type
Article
- DOI
10.11958/20211411