海南医科大学学报

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Col3α1荧光表达水平升高，给予药物处理后Col3α1荧光表达水平下降。结论：益智仁通过抑制PFKFB3介导的异常糖酵解改善糖尿病肾病上皮间充质转化。

Objective: 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. Results: 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 groups demonstrated a significant upregulation of E-cadherin expression(P<0.05) and a significant downregulation of PFKFB3, Col3α1, and Vimentin expression(P<0.05).In vitro experiments: The model group's cell migration ability was significantly enhanced(P<0.05) compared to the blank group. The migration ability of HK-2 cells was reduced in all serum-containing groups and the Empagliflozin group(P<0.05), with the 10% serum-containing group showing the most pronounced reduction.The model group displayed a significant downregulation of E-cadherin mRNA and protein expression(P<0.05) and an upregulation of PFKFB3, Col3α1, and Vimentin mRNA and protein expression(P<0.05) compared to the blank group. In the drug-administered groups, E-cadherin expression was significantly upregulated(P < 0.05), while the expression of PFKFB3, Col3α1, and Vimentin was significantly downregulated(P<0.05) when compared to the model group. Additionally, the fluorescence expression level of Col3α1 was elevated in the model group compared to the blank group, and this level decreased following drug treatment. Conclusion: Alpiniae Oxyphyllae Fructus improves epithelial mesenchymal transition in diabetic nephropathy by inhibiting PFKFB3-mediated abnormal glycolysis.