Hypercholesterolemia increases the risk of tendon pain and tendon rupture. Tendon-derived stem cells (TDSCs) play a vital role in the development of tendinopathy. Our previous research found that high cholesterol inhibits tendon-related gene expression in TDSCs. Whether high cholesterol has other biological effects on TDSCs remains unknown. TDSCs isolated from female SD rats were exposed to 10 mg/dL cholesterol for 24 h. Then, cell apoptosis was assessed using flow cytometry and fluorescence microscope. RFP-GFP-LC3 adenovirus transfection was used for measuring autophagy. Signaling transduction was measured by immunofluorescence and immunoblotting. In addition, Achilles tendons from ApoE −/− mice fed with a high-fat diet were histologically assessed using HE staining and immunohistochemistry. In this work, we verified that 10 mg/dL cholesterol suppressed cell proliferation and migration and induced G0/G1 phase arrest. Additionally, cholesterol induced apoptosis and autophagy simultaneously in TDSCs. Apoptosis induction was related to increased expression of cleaved caspase-3 and BAX and decreased expression of Bcl-xL. The occurrence of autophagic flux and accumulation of LC3-II demonstrated the induction of autophagy by cholesterol. Compared with the effects of cholesterol treatment alone, the autophagy inhibitor 3-methyladenine (3-MA) enhanced apoptosis, while the apoptosis inhibitor Z-VAD-FMK diminished cholesterol-induced autophagy. Moreover, cholesterol triggered reactive oxygen species (ROS) generation and activated the AKT/FOXO1 pathway, while the ROS scavenger NAC blocked cholesterol-induced activation of the AKT/FOXO1 pathway. NAC and the FOXO1 inhibitor AS1842856 rescued the apoptosis and autophagy induced by cholesterol. Finally, high cholesterol elevated the expression of cleaved caspase-3, Bax, LC3-II, and FOXO1 in vivo. The present study indicated that high cholesterol induced apoptosis and autophagy through ROS-activated AKT/FOXO1 signaling in TDSCs, providing new insights into the mechanism of hypercholesterolemia-induced tendinopathy. High cholesterol induces apoptosis and autophagy through the ROS-activated AKT/FOXO1 pathway in tendon-derived stem cells.

中文翻译：

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高胆固醇通过源自肌腱的干细胞中的ROS激活的AKT / FOXO1途径诱导凋亡和自噬。

高胆固醇血症会增加肌腱疼痛和肌腱断裂的风险。肌腱来源的干细胞（TDSC）在肌腱病的发展中起着至关重要的作用。我们先前的研究发现，高胆固醇会抑制TDSC中与肌腱相关的基因表达。高胆固醇是否对TDSC具有其他生物学影响尚不清楚。从雌性SD大鼠分离的TDSC暴露于10 mg / dL胆固醇24小时。然后，使用流式细胞仪和荧光显微镜评估细胞凋亡。RFP-GFP-LC3腺病毒转染用于测量自噬。通过免疫荧光和免疫印迹测量信号转导。另外，使用HE染色和免疫组织化学从组织学上评估高脂饮食喂养的ApoE-/-小鼠的跟腱。在这项工作中 我们证实10 mg / dL胆固醇抑制细胞增殖和迁移并诱导G0 / G1期停滞。此外，胆固醇在TDSC中同时诱导凋亡和自噬。凋亡诱导与裂解的caspase-3和BAX的表达增加以及Bcl-xL的表达减少有关。自噬通量的发生和LC3-II的积累表明胆固醇诱导自噬。与单独的胆固醇治疗相比，自噬抑制剂3-甲基腺嘌呤（3-MA）增强了细胞凋亡，而凋亡抑制剂Z-VAD-FMK减少了胆固醇诱导的自噬。此外，胆固醇触发了活性氧（ROS）的产生并激活了AKT / FOXO1途径，而ROS清除剂NAC阻断了胆固醇诱导的AKT / FOXO1途径的激活。NAC和FOXO1抑制剂AS1842856挽救了胆固醇诱导的细胞凋亡和自噬。最后，高胆固醇升高体内裂解的caspase-3，Bax，LC3-II和FOXO1的表达。本研究表明，高胆固醇通过ROS激活的AKT / FOXO1信号传导诱导TDSC中的细胞凋亡和自噬，为高胆固醇血症引起的肌腱病机理提供了新见识。高胆固醇通过源自肌腱的干细胞中的ROS激活的AKT / FOXO1途径诱导凋亡和自噬。本研究表明，高胆固醇通过ROS激活的AKT / FOXO1信号传导诱导TDSC中的细胞凋亡和自噬，为高胆固醇血症引起的肌腱病机理提供了新见识。高胆固醇通过源自肌腱的干细胞中的ROS激活的AKT / FOXO1途径诱导凋亡和自噬。本研究表明，高胆固醇通过ROS激活的AKT / FOXO1信号传导诱导TDSC中的细胞凋亡和自噬，为高胆固醇血症引起的肌腱病机理提供了新见识。高胆固醇通过源自肌腱的干细胞中的ROS激活的AKT / FOXO1途径诱导凋亡和自噬。