银杏叶提取物EGB761对糖尿病肾病小鼠肾小管损伤及内质网应激的影响

Effect of Ginkgo Leaves Extract EGB761 on Renal Tubular Injury and Endoplasmic Reticulum Stress in Mice with Diabetic Kidney Disease

目的探讨银杏叶提取物EGB761对糖尿病肾病(DKD)小鼠肾小管损伤及内质网应激的影响。方法将50只C57BL/6N小鼠随机分为正常组、DKD模型组、DKD+EGB761组(36 mg·kg-1)以及DKD+4-苯基丁酸(4-PBA)组(1 g·kg-1)。通过高脂饲料联合腹腔注射链脲佐菌素(STZ)制备糖尿病小鼠模型,糖尿病模型复制成功并继续高脂饲料喂养8周后检测各组尿蛋白,阳性者给药8周,给药期间观察各组小鼠一般情况。给药8周后检测血肌酐(Scr)、尿素氮(BUN)、24 h尿蛋白(24 h Pro)、空腹血糖(FBG)、肾脏肥大指数;HE染色、Masson染色、PAS染色观察各组小鼠肾脏病理改变;透射电镜观察肾小球及肾小管超微结构;ELLSA法检测各组小鼠肾小管损伤标志物尿β2-微球蛋白(β2-MG)、视黄醇结合蛋白4(RBP4)、中性粒细胞明胶酶相关脂质运载蛋白(NGAL)的表达;以免疫组化、蛋白免疫印迹法(Western blot)、实时荧光定量聚合酶链式反应(Realtime-PCR)检测肾组织内质网应激标志物GRP78、ATF6的表达。结果与正常组比,DKD模型组精神状态较差、反应迟钝、多饮多食多尿症状明显,Scr、BUN、24 h Pro、FBG、肾脏肥大指数均明显升高(P<0.01),肾脏病理损伤加重,尿β2-MG、RBP4、NGAL明显升高(P<0.01),肾组织GRP78、ATF6的表达明显升高(P<0.01)。与DKD模型组相比,DKD+EGB761组精神状态、反应情况及多饮多食多尿症状好转,Scr、BUN、24 h Pro、FBG水平均明显降低(P<0.01),肾脏肥大指数明显降低(P<0.05),肾脏病理损伤明显改善,尿β2-MG、RBP4、NGAL表达降低(P<0.05,P<0.01),肾组织GRP78、ATF6蛋白的表达降低(P<0.05,P<0.01)。结论银杏叶提取物EGB761可以改善DKD小鼠肾功能,减少蛋白尿,减轻肾小管损伤,其作用机制可能与抑制内质网应激相关。

Objective To investigate the effect of ginkgo leaves extract EGB761 on renal tubular injury and endoplasmic reticulum stress in mice with diabetic kidney disease(DKD).Methods Fifty adult male C57 BL/6 N mice were randomly divided into Normal group,DKD model group,DKD+EGB761 group(36 mg·kg-1),and DKD+4-PBA group(1 g·kg-1·d-1).High fat diet combined with intraperitoneal injection of streptozotocin was used to establish the model of diabetes.Eight weeks after successful establishment of diabetes model,urine protein levels of mice were tested.The corresponding drugs were given for 8 weeks.The general condition of mice in each group was observed during administration.After 8 weeks,serum creatinine(Scr),urea nitrogen(BUN),24 h urine protein(24 h Pro),fasting blood glucose(FBG),and renal hypertrophy index were detected.HE staining,Masson staining and PAS staining were used to observe the pathological changes of the kidneys of mice.Ultrastructure of glomeruli and renal tubules of mice were observed by transmission electron microscopy.Markers of renal tubular injury includingβ2-MG,RBP4,NGAL in urine of mice were detected by ELLSA.Expression of endoplasmic reticulum stress markers GRP78 and ATF6 were detected by immunohistochemical method,Western Blot and Real time-PCR.Results Compared with the Normal group,mice in the DKD model group had poor mental state,slower reaction,and obvious symptoms of polyphagia,polydipsia and polyuria;and increased Scr,BUN,24 h Pro,FBG,kidney hypertrophy index were observed(P<0.01).HE staining,Masson staining,PAS staining and transmission electron microscopy showed severe pathological damage.Urineβ2-MG,RBP4,NGAL were significantly increased,as well as the expression of GRP78 and ATF6 up-regulated(P<0.01).Compared with the DKD model group,Scr,BUN,24 h Pro,FBG of mice in DKD+EGB761 group were all decreased(P<0.01),kidney hypertrophy index were decreased(P<0.05),and the kidney pathological damage was improved significantly.Urineβ2-MG,RBP4,NGAL expression were all decreased(P<0.05,P<0.01).The expression of GRP78 and ATF6 were all decreased(P<0.05,P<0.01).Conclusion EGB761 can improve renal function of DKD mice,reduce proteinuria and renal tubule injury,and the mechanism may be related to inhibiting endoplasmic reticulum stress.