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目的:探讨当归补血汤通过单磷酸腺苷激活的蛋白激酶/哺乳动物雷帕霉素靶蛋白(AMPK/mTOR)通路调控人肾足细胞自噬的机制。方法:人肾足细胞分为空白对照组、模型对照组、高糖+正常对照血清组、高糖+10%当归补血汤含药血清组。UPLC-PDA分析当归补血汤的主要成分；CCK-8法检测各组细胞的增殖情况；透射电镜法观察细胞的自噬改变；qRT-PCR法检测自噬相关因子mRNA的表达；采用Wertern blot法检测AMPK/mTOR通路及自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ的表达。结果:咖啡酸、阿魏酸、芒柄花黄素、毛蕊异黄酮和藁本内酯这五种当归补血汤主要成分同时被检出；当归补血汤含药血清能有效改善细胞活力，减少高糖条件下的人肾足细胞自噬小体、自噬溶酶体和隔离膜的数量；与正常对照血清组相比，当归补血汤含药血清组能够促进高糖条件下自噬相关因子Beclin1、Ulk 1、Lc3-Ⅱ/Lc3-Ⅰ、Ampk的mRNA表达，下调P62的mRNA表达(P<0.05),能够上调p-AMPK/AMPK和LC3-Ⅱ/LC3-Ⅰ蛋白表达上调，下调p-mTOR/mTOR蛋白的表达(P<0.05)。结论:当归补血汤对高糖条件下人肾足细胞具有保护作用，机制是通过AMPK/mTOR信号通路激活下游细胞自噬而实现。
Abstract：Objective: To investigate the mechanism of Danggui Buxue Decoction(当归补血汤) in regulating human renal podocyte autophagy through the adenosine 5′-monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) pathway. Methods: Human renal podocytes were divided into a blank control group, a model control group, a group of high glucose + blank serum, and a group of high glucose + 10% Danggui Buxue Decoction-containing serum. The main components of Danggui Buxue Decoction were analyzed by ultra-performance liquid chromatography with photodiode array(UPLC-PDA), and the proliferation of cells in each group was detected by the cell counting kit-8(CCK-8) method. Autophagic changes in cells were observed by transmission electron microscopy, and the expression of autophagy-related factor mRNA was detected by quantitative real-time fluorescent polymerase chain reaction(qRT-qPCR).The expressions of the AMPK/mTOR pathway and autophagy-related microtubule-associated protein light chain 3(LC3)-Ⅱ/LC3-Ⅰ were detected by Western blotting. Results: Five main components of Danggui Buxue Decoction were detected, including caffeic acid, ferulic acid, formononetin, calycosin, and ligustilide. Danggui Buxue Decoction-containing serum effectively improved cell viability and reduced the number of autophagosomes, autolysosomes, and isolation membranes in human renal podocytes under high glucose conditions. Compared with the blank serum group, the group of Danggui Buxue Decoction-containing serum exhibited the promoted expressions of autophagy-related factors, including Beclin1, Unc-51 like autophagy activating kinase 1(Ulk1), Lc3II/Lc3I, and Ampk mRNAs and the reduced expression of P62 mRNA under high glucose conditions(P<0.05). Meanwhile, through Danggui Buxue Decoction-containing serum, the expressions of p-AMPK/AMPK protein and autophagy-related LC3-II/LC3-Ⅰ protein were up-regulated, and the expression of phosphorylated mammalian target of rapamycin(p-mTOR)/mTOR protein was down-regulated(P<0.05). Conclusion: Danggui Buxue Decoction exerted a protective effect on human renal podocytes under high glucose conditions by activating downstream cellular autophagy via the AMPK/mTOR signaling pathway.
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基本信息:
DOI：10.13412/j.cnki.zyyl.20250425.002
中图分类号:R285.5
引用信息:
[1]王楠,张紫薇,周静,等.当归补血汤通过AMPK/mTOR通路改善人肾足细胞自噬的机制研究[J].中药药理与临床,2025,41(04):15-21.DOI:10.13412/j.cnki.zyyl.20250425.002.
基金信息:
2024年江苏省研究生科研创新计划项目(编号：KYCX24＿2330); 国家自然科学基金青年科学基金项目(编号：81904085); 国家中医药管理局高水平中医药重点学科建设项目(编号：GSPZDXK-FJXYB003)