目的基于环磷酸腺苷-Ras相关蛋白1-哺乳动物雷帕霉素靶蛋白（cAMP-Rap1-mTOR）信号通路探究金匮肾气丸（JSP）对1-甲基-4-苯基吡啶鎓碘化物（MPP⁺）诱导人神经母细胞瘤细胞（SH-SY5Y）细胞稳态的调控机制。方法运用网络药理学预测JSP治疗帕金森病（PD）的潜在作用靶点及通路；采用MPP⁺诱导SH-SY5Y细胞建立PD体外模型，给予不同浓度JSP醇提物（25、50、100、200 μg·mL^-1）干预，细胞计数试剂盒-8（CCK-8）法检测细胞活力并得到最佳给药浓度；采用最佳给药浓度验证JSP的作用机制，ELISA法检测cAMP含量，流式细胞术检测细胞凋亡率，丹酰戊二胺（MDC）染色观察自噬体形成，Western blotting检测cAMP-Rap1-mTOR通路相关蛋白、凋亡相关蛋白及自噬相关蛋白的表达，并加入cAMP通路抑制剂ESI-09进行验证。结果网络药理学显示cAMP-Rap1-mTOR通路是JSP治疗PD的重要候选通路。体外实验表明，JSP可显著提高MPP⁺诱导的SH-SY5Y细胞活力（P＜0.001），升高cAMP含量（P＜0.001），上调cAMP激活的交换蛋白1（Epac1）、Rap1蛋白表达（P＜0.05、0.001），下调mTOR蛋白表达（P＜0.01）；降低细胞凋亡率（P＜0.001），下调Bcl-2相关蛋白（Bax）、剪切型半胱天冬酶-3（cleaved Caspase-3）蛋白表达（P＜0.01），上调B细胞淋巴瘤-2（Bcl-2）蛋白表达（P＜0.01）；增强自噬水平，上调贝克蛋白1（Beclin1）、微管相关蛋白3β/3α（LC3B/LC3A）蛋白表达（P＜0.05、0.01），下调泛素结合蛋白（P62）蛋白表达（P＜0.001）。加入ESI-09后可显著逆转JSP上述调控作用（P＜0.05、0.01、0.001）。结论 JSP可通过激活cAMP-Rap1-mTOR信号通路，抑制细胞凋亡、促进细胞自噬，恢复MPP⁺诱导的SH-SY5Y细胞稳态，发挥神经保护作用。

Objective To explore the regulatory mechanism of Jinkui Shenqi Pills (JSP) on cellular homeostasis of human neuroblastoma SH-SY5Y cells injured by 1-methyl-4-phenylpyridinium iodide (MPP⁺) via the cyclic adenosine monophosphate-Rasrelated protein 1-mammalian target of rapamycin (cAMP-Rap1-mTOR) signaling pathway. Methods Network pharmacology was used to predict the potential therapeutic targets and pathways of JSP against Parkinson’s disease (PD). An in vitro PD cell model was established by inducing SH-SY5Y cells with MPP⁺. The ethanol extract of JSP was prepared, and cells were intervened with JSP at different concentrations (25, 50, 100, 200 μg·mL^-1). Cell counting kit-8 (CCK-8) assay was applied to detect cell viability and screen the optimal administration concentration. The optimal concentration was further adopted to verify the mechanism of JSP. Enzymelinked immunosorbent assay (ELISA) was used to detect cAMP content; flow cytometry was performed to determine cell apoptosis rate; Monodansylcadaverine (MDC) staining was used to observe autophagosome formation; Western blotting assay was adopted to detect the expression levels of proteins related to the cAMP-Rap1-mTOR pathway, apoptosis-related proteins and autophagy-related proteins. In addition, the cAMP pathway inhibitor ESI-09 was added for mechanistic verification. Results Network pharmacology results indicated that the cAMP-Rap1-mTOR signaling pathway was a key candidate pathway for JSP in the treatment of PD. In vitro assays showed that JSP significantly enhanced the viability of MPP⁺-injured SH-SY5Y cells (P < 0.001), increased cAMP content (P < 0.001), upregulated the protein expression of exchange protein directly activated by cAMP 1 (Epac1) and Rap1 (P < 0.05, and 0.001), and downregulated mTOR protein expression (P < 0.01). JSP reduced the cell apoptosis rate (P < 0.001), downregulated the expression of Bcl-2-associated X protein (Bax) and cleaved Caspase-3 (P < 0.01), and upregulated B-cell lymphoma-2 (Bcl-2) protein expression (P < 0.01). Moreover, JSP elevated autophagy level, upregulated the protein expression of Beclin1 and microtubuleassociated protein light chain 3B/3A (LC3B/LC3A) (P < 0.05 and 0.01), and downregulated sequestosome-1 (P62) protein expression (P < 0.001). Intervention with ESI-09 markedly reversed the above regulatory effects of JSP (P < 0.05, 0.01, and 0.001). Conclusion JSP can restore the homeostasis of MPP⁺-injured SH-SY5Y cells and exert neuroprotective effects by activating the cAMP-Rap1-mTOR signaling pathway, inhibiting cell apoptosis and promoting cell autophagy.

R965

国家自然科学基金面上项目（ 85274609）；国家中医药管理局青年岐黄学者培养项目（国中医药人教函〔 2022〕 256号）；山西省青年拔尖人才支持计划项目（晋组办字〔 2019〕 35号文）；山西省中医药重点研究室建设专项（zyyyjs2024022）；山西省中医药创新团队建设专项（zyytd2024022）；山西省医学重点科研项目（2023XM018）