“标本配穴”电针改善糖尿病肾病大鼠肾脏损伤机制的研究

Electroacupuncture of“Biaoben acupoints”relieves kidney injury by suppressing oxidative stress in diabetic nephropathy rats

目的:观察“标本配穴”电针对糖尿病肾病(DN)大鼠肾脏叉头状转录因子O1(FoxO1)和过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1α)的影响,探讨电针对DN大鼠肾脏的保护作用及可能机制。方法:雄性Wistar大鼠随机分为正常组10只、模型组12只、电针组11只、电针+抑制剂组11只和抑制剂组11只。采用高糖高脂饮食6周后联合链脲佐菌素腹腔注射建立DN大鼠模型。造模成功后,电针组、电针+抑制剂组予电针“足三里”“关元”“丰隆”“中脘”,15 min/次;电针+抑制剂组和抑制剂组予FoxO1靶向抑制剂AS1842856灌胃处理;均隔日治疗1次,共8周。检测并记录各组大鼠体质量、血糖、血肌酐(Scr)、尿素氮(BUN)、尿微量白蛋白(ALB)和肾脏超氧化物歧化酶(SOD)活性及丙二醛(MDA)、活性氧(ROS)含量;透射电镜观察肾脏亚细胞结构变化;Western blot法检测肾脏组织FoxO1、PGC-1α蛋白表达水平。结果:与正常组比较,模型组体质量、SOD活性和FoxO1、PGC-1α蛋白表达水平显著降低(P<0.01),血糖、Scr、BUN、ALB、MDA、ROS含量显著升高(P<0.01)。与模型组比较,电针组、电针+抑制剂组体质量、SOD活性和FoxO1、PGC-1α蛋白表达水平升高(P<0.01,P<0.05),血糖、Scr、BUN、ALB、MDA、ROS含量降低(P<0.01,P<0.05);抑制剂组体质量升高(P<0.05),血糖、Scr、BUN、MDA含量及FoxO1、PGC-1α蛋白表达水平均降低(P<0.01,P<0.05);其中,电针组上述指标升高或降低更加显著(P<0.05,P<0.01)。与电针+抑制剂组比较,抑制剂组BUN、ALB、ROS含量升高(P<0.05,P<0.01),SOD活性和FoxO1、PGC-1α蛋白表达水平降低(P<0.01)。电镜结果显示:与正常组比较,模型组肾脏结构不清晰,基底膜增厚,足突融合,细胞器损伤明显;电针组、电针+抑制剂组肾脏结构基本清晰,上述损伤不同程度减轻;抑制剂组较模型组无明显改善。结论:电针可能是通过上调FoxO1和PGC-1α在DN大鼠肾脏中的表达,从而减轻DN大鼠肾脏氧化应激反应,保护肾脏。

Objective To observe the effect of electroacupuncture(EA)of combined“Biao”-and“Ben”-acupoint(for treating symptoms and root causes of the disease,respectively)on the expression of kidney forkhead box O1(FoxO1)and peroxi-some proliferator-activated receptor-γcoactivator-1α(PGC-1α)in diabetic nephropathy(DN)rats,so as to explore its potential mechanisms underlying improvement of DN.Methods Wistar rats were randomly divided into normal control(n=10),DN model(n=12),EA(n=11),EA+inhibitor(AS1842856 targeting FoxO1,n=11)and inhibitor(n=11)groups.The DN model was established by high fat and high glucose diet for 6 weeks and intraperitoneal injection of streptozotocin(55 mg/kg).EA(2 Hz,1 mA)was applied to bilateral“Zusanli”(ST36),“Guanyuan”(CV4),“Fenglong”(ST40)and“Zhongwan”(CV12)for 15 min,once every other day for 8 weeks.The body mass was recorded,and blood glucose detected.The serum was sampled for detecting creatinine(Scr)content with Jaffe’s assay,urea nitrogen(BUN)content with urease method.Urine albumin(ALB)and renal reactive oxygen species(ROS)contents were detected with ELISA,renal superoxide dismutase(SOD)activity with xanthine oxidase method,and renal malondialdehyde(MDA)content with thiobarbituric acid method.The renal subcellular structure was observed under transmission electron microscopy,and the expression levels of PGC-1αand FoxO1 proteins in the kidney tissue were detected using Western blot.Results Compared with the normal control group,the levels of body mass,SOD activity,and FoxO1 and PGC-1αprotein expression were significantly reduced(P<0.01),while the contents of blood glucose,and serum Scr and BUN,urine ALB,renal MDA and ROS levels significantly increased in the model group(P<0.01).In comparison with the model group,the levels of body mass,SOD activity,and FoxO1 and PGC-1αexpression were significantly increased in the three treatment groups except SOD,expression of FoxO1 and PGC-1αin the inhibitor group(P<0.01,P<0.05),and the contents of blood glucose,Scr,BUN,ALB,MDA and ROS were obviously decreased in the three treatment groups except ALB and ROS in the inhibitor group(P<0.01,P<0.05).The therapeutic effect of EA was notably superior to that of EA+inhibitor and inhibitor in increasing body mass,SOD activity,and FoxO1 and PGC-1αexpression levels(P<0.05,P<0.01),and in down-regulating blood glucose,BUN,ALB and ROS levels(P<0.05,P<0.01),suggesting a reduction of the therapeutic effect of EA after administration of the inhibitor AS1842856 of FoxO1.Results of electron microscopy showed diffusely thickened and vague basement membrane,increased mesangial matrix,fused foot process,and reduced volume of endothelial cells with pykno-tic nucleus of the kidney tissue in the model group,which was obviously milder in both EA and EA+inhibitor groups particularly in the EA group.Conclusion EA increases the expression of FoxO1 and PGC-1αin the kidneys of DN rats,thereby reducing the oxidative stress response and protecting the kidneys.