Endothelial cells are a crucial component of the vessel-tissue wall and exert an important role in atherosclerosis (AS). To explore the role of Orientin in AS, human vascular endothelial cells (HUVECs) were induced by oxidized low-density lipoprotein (ox-LDL) to simulate the vascular endothelial injury during AS. Cell viability was detected by CCK-8 assay. Oxidative stress and inflammation related markers were measured using kits, RT-qPCR or western blot. Besides, cell apoptosis was assessed with TUNEL staining and cell autophagy was evaluated by LC3 immunofluorescent staining. Additionally, western blot was utilized to evaluate the expression of Sestrin 1 (SESN1) and proteins in AMPK/mTOR signaling. Afterwards, SESN1 was silenced to determine the expression of autophagy-related proteins. The further application of autophagy inhibitor 3-methyladenine (3-MA) was used to clarify the regulatory mechanism of Orientin on autophagy. Results showed that the decreased viability of HUVECs caused by ox-LDL induction was elevated by Orientin. Oxidative stress and inflammation were also attenuated after Orientin addition in HUVECs under ox-LDL condition. Moreover, Orientin suppressed apoptosis and induced autophagy of HUVECs stimulated by ox-LDL, accompanied by enhanced level of phospho (p)-AMPK and declined level of p-mTOR. Interestingly, SESN1 level was elevated by Orientin, and SESN1 depletion alleviated autophagy and reduced p-AMPK expression but enhanced p-mTOR expression. The further experiments indicated that SESN1 silencing or 3-MA addition reversed the inhibitory effects of Orientin on the oxidative stress, inflammation and apoptosis of HUVECs. Collectively, Orientin could induce autophagy by activating SESN1 expression, thereby regulating AMPK/mTOR signaling in ox-LDL-induced HUVECs.

内皮细胞是血管组织壁的重要组成部分，在动脉粥样硬化（AS）中发挥着重要作用。为了探讨荭草素在 AS 中的作用，通过氧化低密度脂蛋白 (ox-LDL) 诱导人血管内皮细胞 (HUVEC) 来模拟 AS 期间的血管内皮损伤。通过CCK-8测定检测细胞活力。使用试剂盒、RT-qPCR 或蛋白质印迹测量氧化应激和炎症相关标记物。此外，通过TUNEL染色评估细胞凋亡，通过LC3免疫荧光染色评估细胞自噬。此外，还利用蛋白质印迹评估了 AMPK/mTOR 信号传导中 Sestrin 1 (SESN1) 和蛋白质的表达。随后，沉默SESN1以确定自噬相关蛋白的表达。通过自噬抑制剂3-甲基腺嘌呤(3-MA)的进一步应用，阐明了荭草素对自噬的调控机制。结果表明，Orientin 可以提高由 ox-LDL 诱导引起的 HUVEC 活力下降。在 ox-LDL 条件下，在 HUVEC 中添加 Orientin 后，氧化应激和炎症也得到减轻。此外，Orientin 还可抑制 ox-LDL 刺激的 HUVEC 细胞凋亡并诱导自噬，并伴有磷酸 (p)-AMPK 水平升高和 p-mTOR 水平下降。有趣的是，Orientin 可以提高 SESN1 水平，并且 SESN1 缺失会减轻自噬并减少 p-AMPK 表达，但会增强 p-mTOR 表达。进一步的实验表明，SESN1沉默或添加3-MA可逆转Orientin对HUVECs氧化应激、炎症和凋亡的抑制作用。总的来说，Orientin 可以通过激活 SESN1 表达来诱导自噬，从而调节 ox-LDL 诱导的 HUVEC 中的 AMPK/mTOR 信号传导。