基于AMPK/Rheb/mTOR通路探讨补阳还五汤加味含药血清对高糖下HRCECs自噬的抑制作用

Inhibitory Effect of Buyang Huanwu Decoction Modified Serum on Autophagy in HRCECs under High Glucose via AMPK/Rheb/mTOR Pathway

摘要: 目的：明确补阳还五汤加味含药血清对高糖环境下人视网膜微血管内皮细胞(HRCECs)自噬的影响，并从AMPK/Rheb/mTOR通路探讨其机制。方法：制备含药血清与空白血清。将HRCECs分为空白对照组、高糖模型组、空白血清组、含药血清组及多种干预组(如AMPK激动剂/抑制剂组、Rheb沉默/过表达组)。Western Blot检测p-AMPK、p-mTOR、Beclin-1等蛋白表达，RT-PCR检测相关mRNA水平。结果：高糖模型组AMPK磷酸化水平及Beclin-1表达显著升高，mTOR磷酸化水平显著降低。与模型组相比，含药血清组能显著抑制AMPK过度磷酸化，恢复mTOR活性，并下调Beclin-1表达。AMPK激动剂可逆转含药血清的上述作用；干预Rheb表达能显著影响药物对mTOR和自噬的调控效果。结论：补阳还五汤加味含药血清可能通过抑制AMPK过度激活、恢复Rheb/mTOR信号活性，从而抑制高糖诱导的HRCECs过度自噬。

Abstract: To investigate the effect of Buyang Huanwu Decoction Modified (BYHWD-M) serum on autophagy in human retinal microvascular endothelial cells (HRCECs) under high glucose conditions and explore its mechanism via the AMPK/Rheb/mTOR pathway. Methods: Drug-containing serum and blank serum were prepared. HRCECs were divided into control, high glucose model, blank serum, drug-containing serum, and various intervention groups. Western Blot was used to detect p-AMPK, p-mTOR, Beclin-1, and other proteins; RT-PCR was used to detect mRNA levels. Results: The high glucose model group showed increased AMPK phosphorylation and Beclin-1 expression, while mTOR phosphorylation decreased. Compared with the model group, BYHWD-M serum significantly inhibited AMPK overactivation, restored mTOR activity, and downregulated Beclin-1. AMPK agonist reversed these effects, and Rheb modulation influenced drug efficacy on mTOR and autophagy. Conclusion: BYHWD-M serum may inhibit high glucose-induced excessive autophagy in HRCECs by suppressing AMPK overactivation and restoring Rheb/mTOR signaling.