摘要
目的探讨黄芪消渴汤调控血管内皮生长因子(VEGF)/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)信号通路对糖尿病肾病(DN)小鼠肾小管间质纤维化(TIF)的保护作用。方法采用高脂饮食联合腹腔注射链脲佐菌素构建小鼠DN模型,随机分为对照组、DN组、黄芪消渴汤低剂量组、黄芪消渴汤中剂量组和黄芪消渴汤高剂量组,各8只。造模成功后每日灌胃相应药物或溶剂1次,持续10周,给药结束后用试剂盒检测血尿素氮(BUN)、血肌酐(Scr)和24 h尿蛋白(UP)水平,自动分析仪测量总甘油三酯(TG)、总胆固醇(TC)和低密度脂蛋白(LDL-C),计算肾脏指数。采用苏木精和伊红、过碘酸希夫及马松三色染色,检测肾脏结构病理学改变、糖原沉积和TIF。透射电子显微镜检测肾小管超微结构。免疫组化检测肾皮质细胞外基质沉积。免疫荧光检测肾皮质VEGF表达和定位。蛋白质印迹检测肾皮质上皮细胞-间质细胞转化和VEGF/PI3K/AKT通路相关蛋白表达。结果与DN组比较,黄芪消渴汤低剂量组、黄芪消渴汤中剂量组和黄芪消渴汤高剂量组小鼠FBG和肾脏指数降低(P<0.05),呈浓度依赖性。与DN组比较,黄芪消渴汤低剂量组、黄芪消渴汤中剂量组和黄芪消渴汤高剂量组小鼠BUN、Scr、TC、LDL-C、TG和UP水平均降低,呈浓度依赖性(P<0.05)。与DN组比较,黄芪消渴汤低剂量组、黄芪消渴汤中剂量组和黄芪消渴汤高剂量组小鼠肾脏病理损伤程度逐渐减轻,肾小管损伤指数、纤维化面积占比、糖原沉积区域占比降低(P<0.05)。与DN组比较,黄芪消渴汤低剂量组、黄芪消渴汤中剂量组和黄芪消渴汤高剂量组小鼠肾组织中α-平滑肌肌动蛋白、波形蛋白和扭曲蛋白表达降低,E-钙黏蛋白表达升高,呈浓度依赖性(P<0.05)。与DN组比较,黄芪消渴汤低剂量组、黄芪消渴汤中剂量组和黄芪消渴汤高剂量组小鼠肾皮质中VEGF荧光强度和VEGF蛋白表达及磷酸化PI3K/PI3K和磷酸化AKT/AKT均升高,呈浓度依赖性,且VEGF荧光强度表达变化主要发生于肾小管上皮细胞。结论黄芪消渴汤可能部分通过激活VEGF/PI3K/AKT信号通路对DN小鼠TIF发挥保护作用。
Objective To explore the protective effect of Huangqi Xiaoke Decoction in regulating the vascular endothelial growth factor(VEGF)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway on tubulointerstitial fibrosis(TIF)in mice with diabetic nephropathy(DN).Methods A mouse DN model was established by a high-fat diet combined with intraperitoneal injection of streptozotocin.The mice were randomly divided into the control group,the DN group,the low-dose Huangqi Xiaoke Decoction group,the medium-dose Huangqi Xiaoke Decoction group,and the high-dose Huangqi Xiaoke Decoction group,with 8 mice in each group.After successful modeling,the corresponding drugs or solvents were administered by intragastric administration once a day for 10 weeks.After the administration ended,the levels of blood urea nitrogen(BUN),serum creatinine(Scr),and 24-hour urine protein(UP)were detected using a kit.Total triglycerides(TG),total cholesterol(TC),and low-density lipoprotein(LDL-C)were measured by an automatic analyzer,and the renal index was calculated.Hematoxylin and Eosin,periodic acid-Schiff,and masson staining were used to detect the structural pathological changes of the kidneys,glycogen deposition and TIF.The ultrastructure of renal tubules was detected by transmission electron microscopy.Immunohisto-chemical detection of extracellular matrix deposition in the renal cortex.Immunofluorescence was used to de-tect the expression and localization of VEGF in the renal cortex.Western blotting was used to detect the epi-thelial-mesenchymal transition of the renal cortex and the expression of proteins related to the VEGF/PI3K/AKT pathway.Results Compared with the DN group,the FBG and renal index of mice in the low-dose Huan-gqi Xiaoke Decoction group,medium-dose Huangqi Xiaoke Decoction group and high-dose Huangqi Xiaoke De-coction group decreased in a concentration-dependent manner(P<0.05).Compared with the DN group,the levels of BUN,Scr,TC,LDL-C,TG and UP in mice of the low-dose Huangqi Xiaoke Decoction group,medi-um-dose Huangqi Xiaoke Decoction group and high-dose Huangqi Xiaoke Decoction group were all decreased in a concentration-dependent manner(P<0.05).Compared with the DN group,the degree of renal pathologi-cal damage in the low-dose Huangqi Xiaoke Decoction group,medium-dose Huangqi Xiaoke Decoction group and high-dose Huangqi Xiaoke Decoction group gradually decreased,and the renal tubular injury index,the proportion of fibrotic area and the proportion of glycogen deposition area decreased(P<0.05).Compared with the DN group,the expressions ofα-smooth muscle actin,Vimentin and Twist in the renal tissues of mice in the low-dose,medium-dose and high-dose Huangqi Xiaoke Decoction groups decreased,while the expression of E-cadherin increased in a concentration-dependent manner(P<0.05).Compared with the DN group,the fluorescence intensity of VEGF,the expression of VEGF protein,and the phosphorylated PI3K/PI3K and phosphorylated AKT/AKT in the renal cortex of mice in the low-dose,medium-dose and high-dose groups of Huangqi Xiaoke Decoction were all increased in a concentration-dependent manner,and the changes in the expres-sion of VEGF fluorescence intensity mainly occurred in the renal tubular epithelial cells.Conclusion Huangqi Xi-aoke Decoction may exert a protective effect on TIF in DN mice partially by activating the VEGF/PI3K/AKT signaling pathway.
作者
谢鑫
崔燕
吴萍
XIE Xin;CUI Yan;WU Ping(Department of Endocrinology,Ankang Traditional Chinese Medicine Hospital,Ankang,Shaanxi 725000,China;Department of Pharmacy,Ankang Central Hospital,Ankang,Shaanxi 725000,China)
出处
《国际检验医学杂志》
2025年第24期2957-2964,共8页
International Journal of Laboratory Medicine
基金
全国中医学术流派第二轮建设项目(国中医药人教函[2019]62号)
陕西省中医药管理局科研项目(2019-ZZ-LC006)。
