电针对糖尿病肾病大鼠肾功能及肾脏自噬表达的影响

Effect of electroacupuncture on renal function and expression of autophagy-related proteins in kidney of rats with diabetic nephropathy

目的:观察电针对糖尿病肾病(DN)大鼠肾功能及自噬表达的影响,探讨电针治疗DN的可能机制。方法:将Wistar大鼠随机分为正常组、模型组和电针组,每组8只。采用高脂饮食+链脲佐菌素腹腔注射(35 mg/kg)法制备DN大鼠模型。电针组电针双侧"肾俞""足三里",每次20 min,每周5次,连续6周。分别检测治疗前后24 h尿量(24 h-UV)、24 h尿蛋白定量(24 h-UP)、空腹血糖(FBG)及治疗后血尿素氮(BUN)、血清肌酐(Scr)水平;PAS染色法观察大鼠肾组织病理改变,透射电镜观察大鼠肾小球超微结构及足细胞自噬,免疫荧光法检测大鼠足细胞标志蛋白Nephrin定位表达,Western blot法检测大鼠肾组织中自噬相关蛋白LC3、Belcin-1、p62及足细胞蛋白Nephrin的表达。结果:治疗前,与正常组比较,模型组、电针组24 h-UV、24 h-UP、FBG显著升高(P<0.01)。治疗后,与正常组比较,模型组24 h-UV、24 h-UP、FBG、BUN、Scr水平及p62蛋白表达显著升高(P<0.01),LC3Ⅱ、LC3Ⅱ/Ⅰ、Beclin-1、Nephrin蛋白表达显著降低(P<0.01);与模型组比较,电针组24 h-UV、24 h-UP、FBG、BUN水平及p62蛋白表达显著降低(P<0.01,P<0.05),LC3Ⅱ、LC3Ⅱ/Ⅰ、Beclin-1、Nephrin蛋白表达显著升高(P<0.01,P<0.05)。病理结果显示,模型组肾小球系膜细胞、系膜基质及肾小管上皮细胞大量增生,基底膜增厚,足突融合或脱落增加,部分可见基底膜裸露,足细胞内自噬体或自噬泡数目减少,Nephrin蛋白荧光表达强度减弱;与模型组比较,电针组系膜细胞和基质增生减少,足突融合或脱落减轻,Nephrin荧光表达强度及足细胞内自噬体、自噬泡数目增加。结论:电针治疗可有效减轻DN大鼠肾损害,其机制可能与电针激活肾脏自噬表达有关。

Objective To observe the effect of electroacupuncture(EA)on renal function and autophagy-related protein expression in diabetic nephropathy(DN)rats,so as to explore its mechanisms underlying improvement of DN.Methods Male Wistar rats were randomly divided into normal control,DN model and EA groups(n=8 rats in each group).The DN model was established by high-fat diet(for 4 weeks)+streptozotocin(STZ,35 mg/kg,i.p.).EA(2 Hz/15 Hz,1 mA)was applied to bilateral“Shenshu”(BL23)and“Zusanli”(ST36)for 20 min,5 times a week for 6 consecutive weeks.The urine volume in 24 h(24 h-UV)was recorded,the levels of urine protein in 24 h(24 h-UP),blood urea nitrogen(BUN)and serum creatinine(Scr)were detected by using an automatic biochemical analyzer,and the fasting blood glucose(FBG)was detected using rapid blood glucose paper test.The histopathological changes of glomerulus of the left kidney were observed after periodic acid-Schiff stain(PAS)and the ultra-microstructural changes,the numbers of autophagosomes and autophagic vacuoles of the right kidney were observed by transmission electronic microscope(TEM).The expression and localization of podocyte marker protein Nephrin was detected by using immunofluorescence assay,and the expression levels of autophagy-related proteins microtubule associated protein 1(LC3),Beclin-1,p62 and Nephrin in the kidney tissue measured by Western blot.Results Before treatment,compared with the normal control group,the levels of 24 h-UV,24 h-UP and FBG were significantly increased(P<0.01)in the model and EA groups.After treatment,compared with the normal group,the levels of 24 h-UV,24 h-UP,FBG,BUN,Scr and p62 protein expression were signi-ficantly increased(P<0.01),and the expression levels of LC3Ⅱ,Beclin-1 and Nephrin proteins and ratio of LC3Ⅱ/Ⅰwere significantly decreased(P<0.01)in the model group.Compared with the model group,the levels of 24 h-UV,24 h-UP,FBG,BUN and p62 were apparently decreased(P<0.01,P<0.05),while the expression levels of LC3Ⅱ,Beclin-1 and Nephrin proteins and ratio of LC3Ⅱ/Ⅰwere obviously up-regulated(P<0.01,P<0.05)in the EA group.No significant changes were found in the level of Scr after EA(P>0.05)and in the expression levels of LC3Ⅰafter modeling and EA(P>0.05).Outcomes of PAS showed blurred structure of the glomerulus with obvious proliferation of glomerular mesangial cells,increase of cell matrix,and thickening of basement membrane of the glomerulus and renal tubule,atrophy of epithelia cells of renal tubules and infiltration of inflammatory cells in the renal interstitum;and TEM revealed fusion and exfoliation of some foot processes,bareness or thickening of some basement membrane,and reduction of number of autophagosomes or autophagobubbles in podocytes in the model group,which was relatively milder in the EA group.Conclusion EA can effectively alleviate kidney damage in DN rats,which may be related to its function in facilitating autophagy in the kidney.