电针预处理通过沉默信息调节因子3/锰超氧化物歧化酶通路对2型糖尿病大鼠肾脏损伤及氧化应激的影响

Electroacupuncture preconditioning alleviates renal injury and oxidative stress through Sirt3/MnSOD pathway in type 2 diabetic rats

目的:观察电针预处理对2型糖尿病大鼠肾脏的保护效应和对沉默信息调节因子3(Sirt3)、锰超氧化物歧化酶(MnSOD)表达的影响,探讨其作用机制。方法:Wistar大鼠随机分为对照组、模型组、电针组、电针+抑制剂组、抑制剂组,每组10只。除对照组外,各组大鼠均予高糖高脂饮食联合链脲佐菌素复制2型糖尿病模型。造模前,电针组、电针+抑制剂组给予电针“足三里”“关元”“丰隆”“中脘”,15 min/次,隔日1次,共8周;电针+抑制剂组、抑制剂组给予腹腔注射Sirt3抑制剂3-TYP(50 mg/kg),隔日1次,共3次。测量大鼠双肾质量及体质量,计算肾脏指数;ELISA法检测尿微量白蛋白(ALB)、24 h尿液中8-羟基脱氧鸟苷(8-OHdG)和肾脏组织活性氧(ROS)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性;紫外分光光度法检测肾脏组织线粒体呼吸链酶复合物(RCCⅠ—RCCⅣ)活性;HE染色法、Masson染色法和透射电镜观察肾脏病理变化;Western blot法和荧光定量PCR法检测肾脏Sirt3、MnSOD蛋白和mRNA表达水平。结果:与对照组比较,模型组尿ALB含量及肾脏指数、肾脏组织ROS活性、24 h尿液中8-OHdG含量和肾脏胶原容积分数(CVF)升高(P<0.01),肾脏组织SOD、CAT、GSH-Px活性和RCCⅠ—RCCⅣ活性、Sirt3和MnSOD蛋白和mRNA表达水平降低(P<0.01)。与模型组比较,电针组尿ALB含量及肾脏指数、肾脏组织ROS活性、24 h尿8-OHdG含量和肾脏CVF降低(P<0.01,P<0.05),肾脏组织SOD、CAT、GSH-Px活性和RCCⅠ—RCCⅣ活性、Sirt3和MnSOD蛋白和mRNA表达水平升高(P<0.01,P<0.05);电针+抑制剂组RCCⅡ活性、肾脏组织MnSOD mRNA表达水平均升高(P<0.05);抑制剂组尿ALB、24 h尿液中8-OHdG含量及CVF升高(P<0.05),肾脏组织SOD活性、Sirt3和MnSOD蛋白及mRNA表达水平降低(P<0.05)。与电针组比较,电针+抑制剂组和抑制剂组尿ALB、肾脏指数、肾脏组织ROS、24 h尿液中8-OHdG及CVF升高(P<0.05,P<0.01),肾脏组织SOD、CAT、GSH-Px活性和RCCⅠ、RCCⅡ活性及Sirt3、MnSOD蛋白和mRNA表达水平降低(P<0.05,P<0.01),抑制剂组RCCⅢ、RCCⅣ活性降低(P<0.05)。与电针+抑制剂组比较,抑制剂组尿ALB、24 h尿液中8-OHdG含量及肾脏CVF升高(P<0.01),肾脏组织SOD活性、RCCⅡ活性和Sirt3、MnSOD蛋白及mRNA表达水平降低(P<0.01,P<0.05)。模型组和抑制剂组肾小球肥大,肾脏纤维化严重,足突大量消失,电针组和电针+抑制剂组肾脏损伤不同程度减轻。结论:电针预处理能减轻糖尿病所导致的肾脏损伤,其机制可能与促进Sirt3、MnSOD的表达,改善肾脏氧化应激有关。

Objective To explore the protective effect and molecular mechanism of electroacupuncture(EA) preconditioning on renal injury in type 2 diabetic rats. Methods Fifty male Wistar rats were randomly divided into control, model, EA, EA+inhibitor, and inhibitor groups, with 10 rats in each group. Diabetes model was established by high fat and high glucose diet and intraperitoneal injection of streptozotocin(40 mg/kg). EA(2 Hz, 1 mA) preconditioning was applied to “Guanyuan”(CV4), “Zhongwan”(CV12), bilateral “Zusanli”(ST36) and “Fenglong”(ST40) for 15 min, once every other day for 8 weeks. Rats of the inhibitor and EA+inhibitor groups were given intraperitoneal injection of 3-TYP(50 mg/kg) once every other day for a total of 3 times. The body weight, kidney mass, and renal index were recorded. The contents of urine microalbumin(ALB), 24 h urine 8-hydroxydeoxyguanosine(8-OHdG) and activities of reactive oxygen species(ROS), superoxide dismutase(SOD), catalase(CAT), glutathione glycine peroxidase(GSH-Px) in kidney were detected by ELISA. The activities of mitochondrial respiratory chain enzyme complex(RCCⅠ—RCCⅣ) in kidney were detected using spectrophotometric method. HE staining, Masson staining and transmission electron microscopy were used to observe the changes of renal structure. The protein and mRNA expressions of silent information regulator 3(Sirt3), manganese superoxide dismutase(MnSOD) in kidney were detected by Western blot and quantitative real-time PCR, respectively. Results After modeling and compared with the control group, the contents of ALB, the renal index, activity of ROS and content of 8-OHdG, and the renal collagen volume fraction(CVF) were increased(P<0.01), while the activities of SOD, CAT and GSH-Px, RCCⅠ—RCCⅣ, and the mRNA and protein expressions of Sirt3 and MnSOD were decreased(P<0.01). After the treatment and compared with the model group, the contents of ALB, the renal index, ROS, 8-OHdG, and the CVF were decreased in the EA group(P<0.01, P<0.05), while the activities of SOD, CAT, GSH-Px, RCCⅠ—RCCⅣ, and Sirt3 and MnSOD expression levels were increased(P<0.01, P<0.05);the RCCⅡ activity and the expression level of MnSOD mRNA were increased(P<0.05) in the EA+inhibitor group;the ALB and 8-OHdG contents and the CVF in the inhibitor group were increased(P<0.05), while the activity of SOD, and Sirt3 and MnSOD expression levels were decreased(P<0.05). In comparison with the EA group, the contents of ALB, the renal index, activities of ROS and 8-OHdG contents, and the CVF were increased(P<0.05, P<0.01), activities of SOD, CAT and GSH-Px and RCCⅠ and RCCⅡ, and the mRNA and protein expressions of Sirt3 and MnSOD were decreased(P<0.05, P<0.01) in both EA+inhibitor group and inhibitor group, whereas the activities of RCCⅢ and RCCⅣ were decreased in the inhibitor group(P<0.05). The therapeutic effect of inhibitor was notably inferior to that of EA+inhibitor in decreasing ALB and 8-OHdG contents, and CVF(P<0.01), and in up-regulating SOD and RCCⅡ activities, Sirt3 and MnSOD expression levels(P<0.01, P<0.05). Conclusion EA preconditioning can increase the expressions of renal Sirt3 and MnSOD in type 2 diabetic rats, thereby reducing the oxidative stress response and protecting the kidneys.