Rapamycin is known to be effective in suppressing senescence and the senescence-associated secretory phenotype (SASP). Therefore, it is highly expected to represent an anti-aging drug. Its anti-aging effect has been demonstrated at the mouse individual level. However, there are not many clinical findings with respect to its activity in humans. Here, we aimed to clarify the effect of rapamycin on human endothelial cells (ECs) as an in vitro model of human blood vessels. Over the course of oxidative stress-induced senescence using hydrogen peroxide, we examined the effect of rapamycin on human coronary artery ECs (HCAECs). Senescence was evaluated by detecting senescence-associated β-galactosidase (SA-β-Gal) activity and the real-time PCR analysis of p16INK4a. Furthermore, expression levels of SASP factors were examined by real-time PCR and the expression of senescence-related antigens, such as intercellular adhesion molecule-1 (ICAM-1) and ganglioside GM1, were examined by fluorescence-activated cell sorting analysis and immunostaining. The inhibitory effect of rapamycin on mTOR signaling was examined by immunoblotting. The adhesion of leukocytes to HCAECs was evaluated by adhesion assays. Endothelial–mesenchymal transition (EndMT) induced by rapamycin treatment was evaluated by real-time PCR analysis and immunostaining for EndMT markers. Finally, we checked the activation of autophagy by immunoblotting and examined its contribution to EndMT by using a specific inhibitor. Furthermore, we examined how the activation of autophagy influences TGF-β signaling by immunoblotting for Smad2/3 and Smad7. A decrease in SA-β-Gal activity and the suppression of SASP factors were observed in HCAECs undergoing stress-induced premature senescence (SIPS) after rapamycin treatment. In contrast, ICAM-1 and ganglioside GM1 were upregulated by rapamycin treatment. In addition, leukocyte adhesion to HCAECs was promoted by this treatment. In rapamycin-treated HCAECs, morphological changes and the promotion of EndMT were also observed. Furthermore, we found that autophagy activation induced by rapamycin treatment, which led to activation of the TGF-β pathway, contributed to EndMT induction. We revealed that although rapamycin functions to inhibit senescence and suppress SASP in HCAECs undergoing SIPS, EndMT is induced due to the activation of autophagy.

中文翻译：

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雷帕霉素通过自噬激活促进应激诱导的早衰过程中的内皮-间质转化。

已知雷帕霉素可有效抑制衰老和与衰老相关的分泌表型（SASP）。因此，非常期望代表一种抗衰老药物。已经在小鼠个体水平上证明了其抗衰老作用。然而，关于其在人类中的活性，没有多少临床发现。在这里，我们旨在阐明雷帕霉素作为人血管的体外模型对人内皮细胞（EC）的作用。在使用过氧化氢的氧化应激诱导的衰老过程中，我们检查了雷帕霉素对人冠状动脉EC（HCAEC）的影响。通过检测与衰老相关的β-半乳糖苷酶（SA-β-Gal）活性和p16INK4a的实时PCR分析来评估衰老。此外，通过实时PCR检测SASP因子的表达水平，并通过荧光激活细胞分选分析和免疫染色检测衰老相关抗原的表达，例如细胞间粘附分子-1（ICAM-1）和神经节苷脂GM1。通过免疫印迹检查雷帕霉素对mTOR信号转导的抑制作用。通过粘附测定法评估白细胞对HCAEC的粘附。雷帕霉素治疗诱导的内皮-间质转化（EndMT）通过实时PCR分析和EndMT标记物的免疫染色进行评估。最后，我们通过免疫印迹检查了自噬的激活，并通过使用特异性抑制剂检查了其对EndMT的贡献。此外，我们研究了自噬激活如何通过免疫印迹Smad2 / 3和Smad7影响TGF-β信号传导。在雷帕霉素治疗后经历应激诱导的早衰（SIPS）的HCAEC中观察到SA-β-Gal活性降低和SASP因子抑制。相反，雷帕霉素治疗上调了ICAM-1和神经节苷脂GM1的表达。另外，这种处理促进了白细胞对HCAEC的粘附。在雷帕霉素处理的HCAEC中，还观察到形态变化和EndMT的促进。此外，我们发现雷帕霉素处理诱导的自噬激活导致TGF-β途径的激活，促成EndMT的诱导。我们发现，尽管雷帕霉素在经历SIPS的HCAEC中起抑制衰老和抑制SASP的作用，但由于自噬的激活而诱导EndMT。另外，这种处理促进了白细胞对HCAEC的粘附。在雷帕霉素处理的HCAEC中，还观察到形态变化和EndMT的促进。此外，我们发现雷帕霉素处理诱导的自噬激活导致TGF-β途径的激活，促成EndMT的诱导。我们发现，尽管雷帕霉素在经历SIPS的HCAEC中起抑制衰老和抑制SASP的作用，但由于自噬的激活而诱导EndMT。另外，这种处理促进了白细胞对HCAEC的粘附。在雷帕霉素处理的HCAEC中，还观察到形态变化和EndMT的促进。此外，我们发现雷帕霉素处理诱导的自噬激活导致TGF-β途径的激活，促成EndMT的诱导。我们发现，尽管雷帕霉素在经历SIPS的HCAEC中起着抑制衰老和抑制SASP的作用，但由于自噬的激活而诱导了EndMT。