五味子合剂通过Wnt/β-catenin通路改善糖尿病肾病大鼠肾小管上皮-间质转分化的机制研究

Study on the mechanism of Schisandra chinensis Fruit Mixture improving EMT in diabetic nephropathy rats through Wnt/β-catenin pathway

目的:探究五味子合剂(SM)通过调控Wnt/β-catenin通路改善糖尿病肾病(DN)肾小管上皮-间质转分化(EMT)的作用及机制。方法:采取高糖高脂饲料联合腹腔小剂量注射链脲佐菌素(35mg/kg)构建DN大鼠模型。将造模成功的DN大鼠随机分为模型组,五味子合剂低、中、高剂量(1.5、3、6g·kg^(-1)·d^(-1))组和氯沙坦钾(20mg·kg^(-1)·d^(-1))组,另设对照组,每组6只。连续给药12周。12周后处死大鼠,记录体质量、右肾质量、血糖和24h尿蛋白定量(24hUTP),收取血清和肾组织标本。试剂盒测定大鼠Scr和BUN水平;HE、Masson染色观察肾组织病理变化;TEM观察肾组织超微结构变化;Western Blot检测大鼠肾组织Wnt4、β-catenin、E-cadherin、Vimentin、α-SMA蛋白表达;免疫组化检测大鼠肾组织β-catenin、E-cadherin、Vimentin、α-SMA的蛋白表达和定位。结果:与对照组比较,模型组大鼠肾质量/体质量比,血糖、24hUTP、Scr和BUN水平显著升高(P<0.01);肾脏病理表现为肾小球肥大、基底膜增厚、系膜增生、足突融合、足细胞丢失;肾组织中Wnt4、β-catenin、Vimentin、α-SMA表达增加,E-cadherin的表达下降(P<0.01)。与模型组比较,氯沙坦钾组和SM各剂量组大鼠肾质量/体质量比、24hUTP、Scr和BUN水平不同程度降低;肾组织中Wnt4、β-catenin、Vimentin、α-SMA蛋白表达不同程度下降,E-cadherin的蛋白表达不同程度升高(P<0.05,P<0.01)。结论:SM可以改善DN大鼠的肾功能和病理损伤,其机制可能与抑制Wnt/β-catenin信号通路,改善EMT有关。

Objective:To study the effect and mechanism of Schisandra chinensis Fruit Mixture(SM)in improving renal tubular epithelial-mesenchymal transition(EMT)in diabetic nephropathy(DN)by regulating the Wnt/β-catenin pathway.Methods:A high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin(35 mg/kg)in a single small dose was used to establish a DN rat model.The successfully modeled DN rats were randomly divided into model group,SM low(1.5 g kg^(-1)·d^(-1)),medium(3 g·kg^(-1)·d^(-1))and high(6 g kg"d")dose groups and losartan potassium(20 mg kgd")group,and a control group,with 6 rats in each group.The rats were administered continuously for 12 weeks.Twelve weeks later,body weight,right kidney weight,blood glucose and 24h urine protein quantification(UTP)were recorded,and serum kidney tissue specimens were collected.The levels of creatinine(Scr)and urea nitrogen(BUN)were measured by the kit,the renal histopathological changes were observed by hematoxylin-eosin(HE)and Masson staining,the ultrastructural changes were observed by transmission electron microscopy(TEM),and the Wnt/β-catenin signaling pathway proteins and EMT-related proteins E-cadherin,Vimentin,α-SMA were detected by Western Blot.Immunohistochemistry(IHC)to detect EMT-related proteins expression levels ofβ-catenin,E-cadherin,Vimentin andα-SMA in rat kidney tissues.Results:Compared with the control group,the renal weight/body weight ratio,blood glucose,24hUTP,Scr and BUN levels were significantly increased in the model group(P<0.01).Renal pathology showed glomerular hypertrophy,basement membrane thickening,thylakoid hyperplasia,pedicle fusion,and podocyte loss;the expression of Wnt4,β-catenin,Vimentin,α-SMA was increased,while the expression of E-cadherin was decreased in renal tissues(P<0.01).Compared with the model group,the renal weight/body weight ratio,24hUTP,Scr and BUN levels of the rats in the losartan potassium group and the Schisandra mixture groups were decreased to varying degrees;the protein expression of Wnt4,β-catenin,Vimentin,α-SMA and Collage in renal tissues were decreased to different degrees,and the protein expression of E-cadherin was increased to different degrees(P<0.05,P<0.01).Conclusion:SM combined can improve renal function and pathological injury in DN rats,and the mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway and improvement of EMT.