Cardiovascular Department, First Affiliated Hospital, Guangdong Pharmaceutical University, Guangzhou, Guangdong, China.
Braz J Med Biol Res. 2020;53(10):e9849. doi: 10.1590/1414-431X20209849. Epub 2020 Sep 7.
Testosterone has been demonstrated to antagonize doxorubicin-induced cardiomyocyte senescence. However, whether testosterone prevents the paraquat-induced cardiomyocyte senescence is largely unknown. The detection of SA-β-gal activity was performed using senescence β-gal staining kit and the reactive oxygen species levels were determined by reactive oxygen species assay kit. The plasmids for insulin-like growth factor 1 shRNA (sh-mIGF-1), sirtuin-1 shRNA (sh-SIRT1), scramble shRNA (sh-NC), overexpressing mIGF-1 (mIGF-1), overexpressing SIRT1 (SIRT1), and negative controls (NC) were obtained for this study. The expression of target genes was detected using quantitative real-time PCR, immunolabeling, and western blot. We found that testosterone significantly delayed the paraquat-induced HL-1 cardiomyocyte senescence as evidenced by decreasing senescence-associated β-galactosidase activity and reactive oxygen species generation, which were accompanied by the up-regulated expression of mIGF-1 and SIRT1. RNA interference to reduce mIGF-1 and SIRT1 expression showed that testosterone prevented paraquat-induced HL-1 senescence via the mIGF-1/SIRT1 signaling pathway. Furthermore, myocardial contraction was evaluated by expression of genes of the contractile proteins/enzymes, such as α-myosin heavy chain 6 (MHC6), α-myosin heavy chain 7 (MHC7), α-skeletal actin (ACTA-1), and sarco/endoplasmic reticulum calcium ATPase-2 (SERCA2). Testosterone adjusted the above four gene expressions and the adjustment was blocked by mIGF-1 or SIRT1 inhibition. Our findings suggested that the mIGF-1/SIRT1 signaling pathway mediated the protective function of testosterone against the HL-1 cardiomyocyte senescence by paraquat, which provided new clues for the mechanisms underlying the anti-aging role of testosterone in cardiomyocytes.
睾酮已被证实可拮抗阿霉素诱导的心肌细胞衰老。然而，睾酮是否能预防百草枯诱导的心肌细胞衰老还知之甚少。使用衰老β-半乳糖苷酶染色试剂盒检测 SA-β-半乳糖苷酶活性，使用活性氧检测试剂盒测定活性氧水平。本研究获得了胰岛素样生长因子 1 shRNA（sh-mIGF-1）、沉默信息调节因子 1 shRNA（sh-SIRT1）、 scramble shRNA（sh-NC）、过表达 mIGF-1（mIGF-1）、过表达 SIRT1（SIRT1）和阴性对照（NC）的质粒。使用定量实时 PCR、免疫标记和 Western blot 检测靶基因的表达。我们发现，睾酮可显著延缓百草枯诱导的 HL-1 心肌细胞衰老，表现为衰老相关β-半乳糖苷酶活性和活性氧生成减少，同时 mIGF-1 和 SIRT1 表达上调。用 RNA 干扰降低 mIGF-1 和 SIRT1 的表达表明，睾酮通过 mIGF-1/SIRT1 信号通路预防百草枯诱导的 HL-1 衰老。此外，通过收缩蛋白/酶的基因表达来评估心肌收缩，如α-肌球蛋白重链 6（MHC6）、α-肌球蛋白重链 7（MHC7）、α-骨骼肌肌动蛋白（ACTA-1）和肌浆/内质网钙 ATP 酶-2（SERCA2）。睾酮调节了上述四个基因的表达，而 mIGF-1 或 SIRT1 的抑制阻断了这种调节作用。我们的研究结果表明，mIGF-1/SIRT1 信号通路介导了睾酮对百草枯诱导的 HL-1 心肌细胞衰老的保护作用，为睾酮在心肌细胞中抗衰老作用的机制提供了新的线索。
