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+10μM AG)组、高糖(30 mM)组、高糖+AG(30 mM葡萄糖+10μM 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 receptorγcoactivator-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pAMPK/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。
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 random-ized 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+10μM AG),high glucose(HG,30 mM)and HG+AG(30 mM glucose+10μM 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 receptorγcoactivator 1α(PGC-1α)and antiapoptotic protein B-cell lymphoma-2(Bcl-2)were determined by Western blot.The expression level of PGC-1αwas 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-1αprotein and Bcl-2 spiked in db/db+AG group(all P<0.01).As compared with control group,p-AMPK/AMPK ratio,PGC-1αprotein and Bcl-2 declined in HG group(P<0.001).As compared with HG group,p-AMPK/AMPK ratio,PGC-1αprotein and Bcl-2 jumped in HG+AG group(all P<0.01).As compared with db/db group,the expression of PGC-1αprotein was up-regulated in db/db+AG group(P<0.01).As compared with HG group,the expression of PGC-1αprotein rose in HG+AG group(P<0.01).In molecular docking,AG could stably conjugate with PGC-1αprotein.Conclusion AG improves mitochondrial function and alleviates kidney injury through AMPK/PGC-1αpathway,thereby delaying the progression of DKD.
作者
罗艺
汪梦晶
刘淑芬
袁爽
黄浩
Luo Yi;Wang Meng-jing;Liu Shu-fen;Yuan Shuang;Huang Hao(Affiliated Tianmen Hospital,Wuhan University of Science&Technology,Tianmen 431700,China;College of Medicine,Wuhan University of Science&Technology,Wuhan 430081,China)
出处
《临床肾脏病杂志》
2024年第11期930-937,共8页
Journal Of Clinical Nephrology
基金
湖北省自然科学基金面上类项目(2023AFB1105)。
