益智仁抑制PFKFB3介导的糖酵解对糖尿病肾病上皮间充质转化的影响  

Effect of inhibition of PFKFB3⁃mediated glycolysis on epithelial mesenchymal transition in diabetic kidney disease by Alpiniae Oxyphyllae Fructus

作　　者：李小燕 袁勇 王晶[3] 马天鹏 李丽丹 颜子杰 肖曼 谢毅强 LI Xiaoyan;YUAN Yong;WANG Jing;MA Tianpeng;LI Lidan;YAN Zijie;XIAO Man;XIE Yiqiang(Guizhou University of Traditional Chinese Medicine,Guiyang 550005,China;Sanya Hospital of Traditional Chinese Medicine,Sanya 572000,China;Chengdu University of Traditional Chinese Medicine,Chengdu 610075,China;College of Traditional Chinese Medicine,Hainan Medical University,Haikou 571199,China;Key Laboratory of Tropical Translational Medicine of Ministry of Education,School of Basic Medicine and Life Sciences,Hainan Medical University,Haikou 571199,China)

机构地区：[1]贵州中医药大学,贵州贵阳550005 [2]三亚市中医院,海南三亚572000 [3]成都中医药大学,四川成都610075 [4]海南医科大学中医学院,海南海口571199 [5]海南医科大学基础医学与生命科学学院,热带转化医学教育部重点实验室,海南海口571199

出　　处：《海南医科大学学报》2025年第7期481-489,共9页Journal of Hainan Medical University

基　　金：国家自然科学基金-面上项目(82174334);代谢性疾病中医药调控四川省重点实验2022年开放基金(SZKF202203)。

摘　　要：目的:利用体内和体外模型探究益智仁对糖尿病肾病上皮间充质转化的潜在治疗作用,并研究其作用机制。方法:体内实验:db/m小鼠作为空白组,将db/db小鼠分为模型组、阳性药物对照组(恩格列净组)、益智仁低、中、高剂量组。通过HE染色观察肾脏组织病理改变,采用RT-qPCR、Western blot检测肾脏组织中PFKFB3、E-cadherin、Col3α1和Vimentin的mRNA及蛋白表达水平。体外实验:将HK-2细胞分为空白组、模型组以及益智仁含药血清不同剂量组(低、中、高剂量组),同时设立恩格列净阳性对照组。经过24 h的药物干预后,首先通过划痕实验评估各组细胞的迁移能力,并据此确定最佳药物浓度。随后,采用RT-qPCR和Western blot技术,检测HK-2细胞中PFKFB3、E-cadherin、Col3α1和Vimentin的mRNA及蛋白表达水平;利用免疫荧光技术进一步验证EMT中关键蛋白Col3α1的表达情况。结果:体内实验:HE染色显示:与空白组相比,模型组肾小球体积增大、基底膜增厚,肾小管上皮细胞肿胀、管腔变窄,益智仁及恩格列净干预后各治疗组肾脏病理变化均有不同程度的减轻;在mRNA及蛋白表达水平中,与空白组相比,模型组E-cadherin表达水平显著下调(P<0.05),PFKFB3、Col3α1、Vimentin的表达水平则显著上调(P<0.05),与模型组比较,各给药组E-cadherin表达上调(P<0.05),PFKFB3、Col3α1、Vimentin的表达显著下调(P<0.05)。体外实验:与空白组相比,模型组细胞的迁移能力显著增强(P<0.05),与模型组比较,各含药血清组以及恩格列净组的HK-2细胞迁移能力均有所降低(P<0.05),其中以10%含药血清组降低最为明显;在mRNA及蛋白表达水平中,与空白组相比,模型组E-cadherin表达水平显著下调(P<0.05),PFKFB3、Col3α1、Vimentin的表达水平则显著上调(P<0.05),与模型组比较,各给药组E-cadherin的表达明显上调(P<0.05),PFKFB3、Col3α1、Vimentin的表达显著下调(P<0.05);与空白组比较,模型组Col3Objective:To investigate the potential therapeutic effect of Alpiniae Oxyphyllae Fructus on epithelial mesenchymal transition in diabetic kidney disease using in vivo and in vitro models and to study its mechanism of action.Methods:In vivo experiments:db/m mice were used as a blank group,and db/db mice were divided into the model group,the positive drug control group(the Empagliflozin group),and the low,medium,and high dose groups of Alpiniae Oxyphyllae Fructus.The pathological changes of renal tissues were observed by H&E staining,and the mRNA and protein expression levels of PFKFB3,E⁃cadherin,Col3α1 and Vimentin in renal tissues were detected by RT-qPCR and Western blot.In vitro experiments:HK-2 cells were divided into the blank group,the model group,the different dose groups(low,medium,and high dose groups)of Alpiniae Oxyphyllae Fructus-containing serum,and the positive control group of Empagliflozin.After 24 h of drug intervention,the migration ability of cells in each group was firstly assessed by scratch assay,and the optimal drug concentration was determined accordingly.Subsequently,the mRNA and protein expression levels of PFKFB3,E-cadherin,Col3α1 and Vimentin in HK-2 cells were detected by RT-qPCR and Western blot;the expression of the key protein Col3α1 in EMT was further verified by immunofluorescence.Re⁃sults:In vivo experiments:H&E staining revealed that,in comparison to the blank group,the model group exhibited an increase in glomerular volume,thickening of the basement membrane,swelling of tubular epithelial cells,and narrowing of the tubular lumen.After intervention with Alpiniae Oxyphyllae Fructus and Empagliflozin,renal pathological changes were observed to be reduced to varying degrees.The model group showed a significant downregulation of E-cadherin mRNA and protein expression(P<0.05)compared to the blank group,while the expression levels of PFKFB3,Col3α1,and Vimentin were significantly upregulated in each administered group(P<0.05).In contrast to the model group,the administered group

关 键 词：益智仁 糖尿病肾病 糖酵解 上皮间充质转化 

分 类 号：R285.5[医药卫生—中药学]