摘要
探讨熊果酸通过改善高糖诱导的足细胞线粒体功能障碍,延缓足细胞损伤及凋亡的作用及作用机制。采用CCK-8法确定实验条件,实验分为对照组(CN组)、高糖组(200 mmol/L葡萄糖,HG组)、低剂量熊果酸组(200 mmol/L葡萄糖+0.703μmol/L熊果酸)、中剂量熊果酸组(200 mmol/L葡萄糖+1.406μmol/L熊果酸)、高剂量熊果酸组(200 mmol/L葡萄糖+2.813μmol/L熊果酸)。免疫荧光法观察线粒体形态,检测活性氧(reactive oxygen species,ROS)水平、线粒体通透性转换孔(mitochondrial permeability transition pore,mPTP)开放度、线粒体膜电位(mitochondrial membrane potential,MMP);流式细胞术检测ROS水平和足细胞凋亡率;Western Blot检测亲环蛋白D(cyclophilin D,Cyp D)、电压依赖性阴离子通道(voltage-dependent anion channel,VDAC)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、细胞色素c(cytochrome c,Cyt c)蛋白的表达。结果:CCK-8结果显示,与CN组比较,足细胞在葡萄糖200 mmol/L干预剂量下,细胞增殖率显著降低(P<0.01),故确定葡萄糖200 mmol/L为细胞造模剂量;与HG组比较,熊果酸在0.703、1.406、2.813μmol/L剂量下,显著提高足细胞增殖率(P<0.01);免疫荧光结果显示,与HG组比较,熊果酸不同剂量均能够显著恢复线粒体形态、抑制ROS过度产生、抑制mPTP异常开放、提高MMP;流式细胞术检测结果显示:与HG组比较,熊果酸不同剂量均能降低足细胞凋亡率;Western Blot结果表明,与HG组比较,不同剂量熊果酸均显著下调Cyp D、VDAC、Bax、Cyt c蛋白的表达,上调Bcl-2蛋白的表达。结论:熊果酸可通过抑制HG诱导的足细胞氧化应激所导致的线粒体功能障碍及细胞凋亡发挥其防治糖尿病肾病的作用。
Objective:This study aimed to investigate the protective effects of ursolic acid on mitochondrial dysfunction in podocytes induced by high glucose and to explore the underlying mechanism.Methods:The CCK-8 method was used to determine the experimental conditions and the experiments were divided into five groups:control(CN),high glucose(200 mmol/L,HG),low-dose ursolic acid(200 mmol/L glucose+0.703μmol/L ursolic acid),medium-dose ursolic acid(200 mmol/L glucose+1.406μmol/L ursolic acid)and high-dose ursolic acid(200 mmol/L glucose+2.813μmol/L ursolic acid).The immunofluorescence method was used to observe mitochondrial morphology and detect the reactive oxygen species(ROS)level,the degree of mitochondrial permeability transition pore(mPTP)opening and mitochondrial membrane potential(MMP).Flow cytometry was used to detect the ROS level and the rate of apoptosis of podocytes.Western Blot was used to detect the expression of cyclophilin D(Cyp D),voltage-dependent anion channel(VDAC),Bcl-2 associated X protein(Bax),B-cell lymphoma-2(Bcl-2)and cytochrome c(Cyt c)proteins.Results:Compared with the CN group,the proliferation rate of podocytes was significantly reduced by intervention of 200 mmol/L glucose(P<0.01),which was therefore used for modeling. Compared with the HG group, all doses of ursolic acid significantly increased the proliferationrate of podocytes (P < 0.01). Immunofluorescence assays indicated that they restored mitochondrial morphology, inhibitedROS overproduction, repressed the abnormal opening of mPTP and increased MMP. The results of flow cytometry showedthat ursolic acid at all three doses reduced the apoptosis rate of podocytes compared with the HG group (P < 0.01). TheWestern Blot results showed that compared with the HG group, they significantly down-regulated the expression of Cyp D,VDAC, Bax and Cyt c proteins but up-regulated the expression of Bcl-2 protein. Conclusion: Ursolic acid may exert its effectagainst diabetic nephropathy by inhibiting mitochondrial dysfunction and apoptosis caused by HG-induced oxidative stressin podocytes.
作者
顾惠贤
阿依夏木·麦提托合提
吴思宇
蒋祥龙
姚蓝
GU Huixian;AYIXIAMU·Maitituoheti;WU Siyu;JIANG Xianglong;YAO Lan(College of Traditional Chinese Medicine,Xinjiang Medical University,Ürümqi 830057,China;Xinjiang Key Laboratory of Famous Prescription and Science of Formulas,Ürümqi 830057,China)
出处
《食品科学》
北大核心
2025年第19期37-46,共10页
Food Science
基金
新疆维吾尔自治区天山英才计划第三期(2021241)。
关键词
熊果酸
糖尿病肾病
线粒体功能障碍
细胞凋亡
ursolic acid
diabetic nephropathy
mitochondrial dysfunction
apoptosis
作者简介
第一作者:顾惠贤(1998-)(ORCID:0009-0008-7808-8790),女,硕士研究生,研究方向为糖尿病及其并发症药理学。E-mail:1020030190@qq.com;通信作者:姚蓝(1982-)(ORCID:0009-0009-0274-1789),女,教授,博士,研究方向为糖尿病及其并发症药理学。E-mail:56174475@qq.com。
