阿霉素对心肌细胞ACADSB表达和自噬的影响

Effect of doxorubicin on the expression of ACADSB and autophagy in cardiomyocytes

目的探讨短支链酰基辅酶A脱氢酶(acyl-CoA dehydrogenase short/branched chain,ACADSB)和细胞自噬在阿霉素(doxorubicin,DOX)诱导的心肌细胞氧化损伤过程中的变化。方法细胞水平构建心肌细胞氧化损伤模型,DOX诱导H9c2心肌细胞24 h,实验共分两组,对照组和诱导组,诱导组添加0.5μmol/L的DOX,对照组添加等量的PBS。细胞增殖实验(cell counting kit-8,CCK-8)检测H9c2心肌细胞活性,丙二醛试剂盒检测H9c2细胞中丙二醛(malondialdehyde,MDA)含量,RT-PCR检测SOD 2、Catalase、ACADSB、Atg 5、Atg 7、Beclin 1和ULK1的mRNA变化,蛋白质印迹法和免疫荧光检测ACADSB蛋白的表达,以及检测自噬相关蛋白LC3Ⅱ/LC3Ⅰ的变化。结果DOX显著抑制H9c2心肌细胞的活性,增加丙二醛的含量,促进SOD 2和Catalase的mRNA表达,以及抑制ACADSB mRNA和蛋白的表达。DOX诱导自噬相关基因Beclin 1 mRNA表达显著增加,Atg 7 mRNA表达明显降低,而ULK 1和Atg 5的mRNA表达没有变化,LC3Ⅱ/LC3Ⅰ蛋白表达显著升高。结论DOX诱导H9c2心肌细胞氧化损伤并且抑制ACADSB的转录活性和蛋白表达,增强自噬蛋白LC3Ⅱ/LC3Ⅰ的表达,提示ACADSB可作为心肌病治疗的靶点。

Objective To investigate the changes of acyl-CoA dehydrogenase short branched-chain(ACADSB)and autophagy in the process of doxorubicin(DOX)-induced oxidative damage in cardiomyocytes.Methods The oxidative damage model of cardiomyocytes was established at the cell level,and H9c2 cardiomyocytes were induced by DOX for 24 hours.The experiment was divided into 2 groups:a control group and an induction group.The induction group was treated with 0.5μmol/L DOX,and the control group was added with the same amount of PBS.Cell counting kit-8(CCK8)was used to detect the activity of H9c2 cardiomyocytes.Kit were used to detect the content of malondialdehyde(MDA)in H9c2 cells.RT-PCR was used to detect the mRNA changes of SOD 2,Catalase,ACADSB,Atg 5,Atg 7,Beclin 1,and ULK 1.Western blotting and immunofluorescence were used to detect the expression of ACADSB protein,and western blotting was used to detect the changes of autophagy related protein LC3Ⅱ/Ⅰ.Results DOX significantly inhibited the activity of H9c2 cardiomyocytes,increased the content of malondialdehyde,and promoted the mRNA expression of SOD 2 and Catalase,as well as suppressed the expression of ACADSB mRNA and protein.DOX significantly increased the mRNA expression of autophagy-related gene Beclin 1,and decreased the mRNA expression of Atg 7.While the mRNA expression of ULK 1 and Atg 5 remained unchanged,and the protein expression of LC3Ⅱ/Ⅰwas significantly improved.Conclusion DOX induces oxidative damage in H9c2 cardiomyocytes,inhibites the transcriptional activity and protein expression of ACADSB,and enhancs the expression of autophagy protein LC3Ⅱ/Ⅰ,suggesting that ACADSB may be a target in the treatment of cardiomyopathy.