Objective: To study the protective effect of electroacupuncture (EA) at "Biao"-acupoints, "Fenglong"(ST40) and "Zhongwan"(CV12), used for treating symptoms of the disease, and "Ben"-acupoints, "Zusanli"(ST36) and "Guanyuan"(CV4), for treating the root cause of the disease on oxidative stress injury of renal mitochondria through SIRT1/PGC-1α signal pathway in rats with diabetic nephropathy (DN).
Methods: A total of 33 male Wistar rats were randomized into normal (n=10), model (n=12) and EA (n=11) groups.The DN model was established by feeding the rats with high-sugar and high-fat diet for 6 weeks combined with streptozotocin (STZ, 35 mg/kg, i.p.). EA (4 Hz/60 Hz, 1 mA)was applied to ST36-ST40 and CV4-CV12 for 15 min, once every other day for 8 weeks. The rats' body weight was recorded, urine in 24 hours (24-h UP) was collected to measure the urine protein level, and the fasting blood glucose (FBG) level detected by using a glucometer. The levels of serum glycosylated hemoglobin (HbA1c), creatinine (Scr) and urea nitrogen (BUN) were assayed using immunoturbidi-metry, picric acid method and urease method, respectively, and those of serum triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) detected using an automatic biochemical analyzer. The kidney tissue was collected for assaying the activity of superoxide dismutase (SOD) with xanthine oxidase method, glutathione (GSH) activity with dithio-dinitrobenzoic acid method, catalase （CAT） activity with ammonium molybdate spectrometric method, and malondialdehyde (MDA) content with thiobarbituric acid method. Histopathological changes of the kidney tissue were observed by microscope after hematoxylin-eosin staining (HE), periodate Schiff staining (PAS) and Masson staining, separately, and its subcellular structure was observed under transmission electron microscopy. The expression levels of renal SIRT1 and PGC-1α mRNAs and proteins were detected by quantitative real-time PCR and Western blot, and the immunoactivity of renal α-smooth muscle actin (α-SMA), and immunofluorescence density of renal collagen Ⅰ (Col Ⅰ), collagen Ⅳ(Col Ⅳ) and fibronec-tin (FN) detected by immunohistochemistry and immunofluorescence assay, respectively.
Results: Compared with the normal group, the levels of FBG, HbA1c, BUN, Scr, 24-h UP, TG, TC, LDL-C, MDA, α-SMA, Col Ⅰ, Col Ⅳ and FN proteins were significantly increased (P<0.01), and the levels of body weight, HDL-C, SOD, GSH, CAT, SIRT1 and PGC-1α mRNAs and proteins were decreased in the model group (P<0.01, P<0.05). In comparison with the model group, the levels of FBG, HbA1c, BUN, Scr, 24-h UP, TG, TC, LDL-C and MDA, and the expressions of α-SMA, Col Ⅰ, Col Ⅳ and FN proteins were markedly down-regulated (P<0.05, P<0.01), and those of body weight, HDL-C, SOD, GSH, CAT, and the expressions of SIRT1 and PGC-1α mRNAs and proteins significantly up-regulated (P<0.01, P<0.05) in the EA group. HE staining showed mesangial dilatation, glomerular hypertrophy and mesangial matrix accumulation; PAS staining showed an increase of the glomerular extracellular matrix deposition; and Masson staining displayed an enhancement of glomerular fibrosis and interstitial space expansion; and electron microscope revealed foot process fusion, basement membrane thickening and organelle injury in the rat's kidney of the model group, which were relatively milder in the EA group.
Conclusion: EA of ST36-ST40 and CV4-CV12, "Biao-Ben" acupoints combination, can alleviate oxidative stress and mitochondrial dysfunction in DN rats, which may be associated with its functions in up-regulating SIRT1/PGC-1α signaling, and decreasing renal fibrosis.
目的：观察“标本配穴”电针通过调控沉默信息调节因子1(SIRT1)/过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1α)信号通路对糖尿病肾病(DN)大鼠肾脏线粒体氧化应激损伤的保护作用。方法：Wistar大鼠随机分为正常组10只、模型组12只和电针组11只。采用高糖高脂饮食喂养6周联合链脲佐菌素注射法建立DN大鼠模型。电针组予“足三里”“关元”“丰隆”“中脘”电针治疗, 15 min/次, 隔日1次, 连续8周。检测并记录各组大鼠体质量、空腹血糖(FBG)、24小时尿蛋白定量(24-h UP)；免疫比浊法检测血清糖化血红蛋白(HbA1c)水平；苦味酸法和脲酶法检测血清肌酐(Scr)、尿素氮 (BUN)含量；全自动生化分析仪检测血清甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)含量；黄嘌呤氧化法、二硫代二硝苯甲酸法、钼酸铵法检测肾脏组织超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)、过氧化氢酶(CAT)活性；硫代巴比妥钠法检测肾脏组织丙二醛(MDA)含量；苏木精-伊红(HE)染色、过碘酸希夫(PAS)染色、马松(Masson)染色观察肾脏组织病理学改变；透射电镜观察肾组织超微结构；荧光定量PCR法、Western blot法检测大鼠肾脏组织中SIRT1、PGC-1α mRNA和蛋白表达；免疫组织化学法检测肾脏组织中α-平滑肌肌动蛋白(α-SMA)的表达；免疫荧光染色法检测肾脏组织中胶原蛋白Ⅰ(Col Ⅰ)、胶原蛋白Ⅳ(Col Ⅳ)、纤维连接蛋白(FN)的表达。结果：与正常组比较, 模型组大鼠FBG、24-h UP显著升高(P<0.01), 体质量显著降低(P<0.01)；血清中HbA1c、BUN、Scr、TG、TC、LDL-C含量显著升高(P<0.01), HDL-C含量显著降低(P<0.01)；肾脏组织中SOD、GSH、CAT活性显著降低(P<0.01, P<0.05), MDA含量显著升高(P<0.01)；肾脏组织中SIRT1、PGC-1α mRNA和蛋白表达水平显著降低 (P<0.01), α-SMA、ColⅠ、Col Ⅳ、FN蛋白表达显著升高(P<0.01)。与模型组比较, 电针组FBG、24-h UP显著降低(P<0.05, P<0.01),体质量显著升高(P<0.05)；血清中 HbA1c、BUN、Scr、TG、TC、LDL-C含量显著降低(P<0.05, P<0.01), HDL-C含量显著升高 (P<0.05)；肾脏组织中SOD、GSH、CAT活性显著升高 (P<0.05), MDA含量显著降低(P<0.01)；肾脏组织中SIRT1、PGC-1α mRNA和蛋白表达显著升高(P<0.05, P<0.01), α-SMA、Col Ⅰ、Col Ⅳ、FN蛋白表达显著降低(P<0.01)。模型组HE染色显示肾组织系膜扩张、肾小球肥大、系膜基质聚集, PAS染色显示肾小球细胞外基质沉积, Masson染色显示肾小球纤维化增强、间质间隙扩大, 电镜显示肾组织出现足突融合、基底膜增厚、细胞器损伤；电针组上述损伤均有不同程度减轻。结论：“标本配穴”电针可能通过激活SIRT1/PGC-1α信号通路减轻肾脏氧化应激和线粒体功能障碍, 改善DN大鼠肾组织纤维化。.
Keywords: Diabetic nephropathy; Electroacupuncture; Oxidative stress; SIRT1/PGC-1α signaling; “Biao-Ben” acupoints combination.