黄芪苷通过氧化物酶体增殖物激活受体γ共激活因子1α通路改善线粒体功能减轻糖尿病肾脏疾病肾损伤

    Astragalin improved mitochondrial function through AMPK/PGC-1α pathway and alleviated renal injury in diabetic nephropathy

      摘要
    • 摘要:
      目的  探讨黄芪苷（astragalin, AG）减轻糖尿病肾脏疾病肾损伤的作用及机制。
      方法  16周龄 db/m、db/db小鼠按照随机数字表法分为db/m组、db/m+AG（5 mg/kg灌胃）组、db/db组、db/db+AG（5 mg/kg灌胃）组。体外培养原代人肾皮质近曲小管上皮细胞（human kidney-2，HK-2），并将其分为对照（5 mM葡萄糖）组、对照+AG（5 mM葡萄糖+10 μM AG）组、高糖（30 mM）组、高糖+AG（30 mM葡萄糖+10 μM AG）组。检测各组小鼠尿白蛋白肌酐比、血糖、体重水平；通过 PAS染色和Masson染色观察小鼠肾组织病理学改变；通过 蛋白质免疫印迹法测定腺苷酸活化蛋白激酶（adenosine monophosphate-activated protein kinase, AMPK）、磷酸化（p）-AMPK 、氧化物酶体增殖物激活受体γ共激活因子1α（peroxisome proliferator-activated receptor γ coactivator-1α，PGC-1α）及抗凋亡蛋白B淋巴细胞瘤-2基因（B-cell lymphoma-2，Bcl-2）表达情况；通过荧光染色检测PGC-1α蛋白的表达水平；通过分子对接检测AG与PGC-1α蛋白的结合力。
      结果  （1）与db/m组相比，db/m+AG组小鼠未出现体重、血糖变化及肾损伤（P＞0.05），提示口服AG具有一定的安全性；与db/db组相比，db/db+AG组小鼠体重下降，可降低血糖及尿白蛋白肌酐比水平，并缓解肾组织病变（P＜0.01）；（2）蛋白质免疫印迹法的结果显示与db/db组相比，db/db+AG组可增加p-AMPK/AMPK比值、PGC-1α蛋白的表达，并增加抗凋亡蛋白Bcl-2的表达（均P＜0.01）；与对照组相比，高糖组的p-AMPK/AMPK比值、PGC-1α蛋白表达下降，抗凋亡蛋白Bcl-2的表达也下降（均P＜0.001）；与高糖组相比，高糖+AG组可提高p-AMPK/AMPK比值、PGC-1α蛋白和抗凋亡蛋白Bcl-2的表达（均P＜0.01）；（3）荧光染色的结果显示与db/db组相比，db/db+AG组增加PGC-1α蛋白的表达增加（P＜0.01）；与高糖组相比，高糖+AG组PGC-1α蛋白的表达增加（P＜0.01）；（4）在分子对接中，黄芪苷AG可与PGC-1α蛋白稳定结合。
      结论  黄芪苷可通过AMPK/PGC-1α通路来改善线粒体功能，减轻肾损伤，从而延缓糖尿病肾脏疾病进展。

       

      Abstract:
      Objective  To explore the effect and mechanism of astragalin （AG） in alleviating renal injury in diabetic kidney disease(DKD).
      Methods  db/m and db/db mice aged 16 weeks were randomized into four groups of db/m, db/m+AG （5 mg/kg）, db/db and db/db+AG （5 mg/kg）. Primary Human renal cortex proximal convoluted tubule epithelial cells （HK-2） were cultured in vitro and assigned into four groups of control （5 mM glucose）, control +AG （5 mM glucose +10 μM AG）, high glucose （HG, 30 mM） and HG+AG （30 mM glucose +10 μM AG）. Urinary albumin-creatinine ratio, blood glucose （BG） and body weight were detected. The pathological changes of kidney were observed by （periodic acid-Schiff PAS） and MASSON stain. The expressions of adenosine monophosphate-activated protein kinase （AMPK）, phosphorylation （p）-AMPK, peroxisome proliferator-activated receptor γ coactivator 1α （PGC-1α） and antiapoptotic protein B-cell lymphoma-2 （Bcl-2） were determined by Western blot. The expression level of PGC-1α was detected by fluorescent stain. Molecular docking was performed for detecting the binding force of AG and PGC-1α.
      Results  As compared with db/m group, there were no changes in body weight, BG or kidney injury in db/m+AG group. It implied that oral AG had some safety. As compared with db/db group, body weight, BG and urinary albumin creatinine ratio decreased and renal tissue lesions lessened in db/db+AG group （P＜0.01）. As compared with db/db group, p-AMPK/AMPK ratio, PGC-1α protein and Bcl-2 spiked in db/db+AG group （all P＜0.01）. As compared with control group, p-AMPK/AMPK ratio, PGC-1α protein and Bcl-2 declined in HG group （P＜0.001）. As compared with HG group, p-AMPK/AMPK ratio, PGC-1α protein and Bcl-2 jumped in HG+AG group （all P＜0.01）. As compared with db/db group, the expression of PGC-1α protein was up-regulated in db/db+AG group （P＜0.01）. As compared with HG group, the expression of PGC-1α protein rose in HG +AG group （P＜0.01）. In molecular docking, AG could stably conjugate with PGC-1α protein.
      Conclusion  AG improves mitochondrial function and alleviates kidney injury through AMPK/PGC-1α pathway, thereby delaying the progression of DKD.

       

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