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we report on that astragaloside IV attenuates hypoxia-induced cardiac hypertrophy via calpain-1-mediated mTOR activation. This is significant because our findings could be applied in the clinic for that astragaloside IV is effective against hypoxia-induced cardiac hypertrophy, apoptosis and autophagy, it is likely to be of great interest to the clinicians. Astragaloside IV (AsIV), one of the main active ingredients of Astragalus membranaceus, has been reported to have cardioprotective effects. However, the effect and mechanism of AsIV on hypoxia-induced myocardial hypertrophy are unclear. The aim of this study was to evaluate the effect of AsIV on hypoxia-induced cardiac injury by focusing on calpain-1 (CAPN1)-mediated mTOR activation. Hypoxia-induced cardiac hypertrophy was carried out in CAPN1 knockout and wild-type C57BL/6 mice in vivo and in H9C2 cells in vitro. The effects of AsIV, CAPN1 inhibition and 3-MA on hypertrophy, apoptosis, autophagy, [Ca2+]i, and the expression levels of CAPN1 and mTOR were assayed in cardiac tissue and H9C2 cells. The results showed that AsIV, as well as CAPN1 knockout, attenuated cardiac hypertrophy, apoptosis, and autophagy induced by hypoxia in mice, effects that were confirmed at the H9C2 cell level. In addition, AsIV treatment, CAPN1 inhibition and 3-MA increased p-mTOR expression and decreased [Ca2+]i, accompanied by decreased CAPN1 expression. Furthermore, H9C2 cells overexpressing CAPN1 with lentivirus infection aggravated hypoxia-induced myocardial hypertrophy, apoptosis, and p-mTOR inhibition. Notably, the effects of CAPN1 overexpression on cardiac injury and p-mTOR inhibition were reversed by AsIV treatment. In summary, our data indicated that AsIV could ameliorate hypoxia-induced cardiac hypertrophy by inhibiting apoptosis and autophagy via CAPN1-mediated mTOR activation.

本研究报道了黄芪甲苷IV（astragaloside IV, AsIV）通过钙蛋白酶1（calpain-1, CAPN1）介导的雷帕霉素靶蛋白（mTOR）活化通路，缓解缺氧诱导的心肌肥厚。本研究结果具有重要临床转化价值：AsIV可有效对抗缺氧诱导的心肌肥厚、细胞凋亡与自噬，因此有望获得临床医师的广泛关注。黄芪甲苷IV（AsIV）作为黄芪（Astragalus membranaceus）的主要活性成分之一，此前已有研究报道其具有心脏保护作用，但目前关于AsIV对缺氧诱导心肌肥厚的具体作用与分子机制仍不明确。本研究旨在聚焦CAPN1介导的mTOR活化通路，探讨AsIV对缺氧诱导心肌损伤的调控作用。本研究分别在CAPN1基因敲除及野生型C57BL/6小鼠体内构建缺氧诱导心肌肥厚模型，并在体外以H9C2细胞开展对应实验。本研究分别检测了心肌组织与H9C2细胞中，AsIV、CAPN1抑制剂及3-甲基腺嘌呤（3-MA）对心肌肥厚、细胞凋亡、自噬、细胞内游离钙离子浓度（[Ca²⁺]i）以及CAPN1与mTOR表达水平的影响。实验结果显示：与CAPN1基因敲除一致，AsIV可缓解小鼠体内缺氧诱导的心肌肥厚、细胞凋亡与自噬，该效应在H9C2细胞实验中得到了验证。此外，AsIV干预、CAPN1抑制及3-MA处理均可上调磷酸化mTOR（p-mTOR）的表达水平，同时降低[Ca²⁺]i，并伴随CAPN1表达量的下调。进一步实验发现：经慢病毒感染过表达CAPN1的H9C2细胞，其缺氧诱导的心肌肥厚、细胞凋亡及p-mTOR抑制效应均出现加重。值得注意的是，AsIV干预可逆转CAPN1过表达所引发的心肌损伤与p-mTOR抑制效应。综上，本研究数据表明，AsIV可通过抑制CAPN1介导的mTOR活化通路，减少细胞凋亡与自噬，从而缓解缺氧诱导的心肌肥厚。