Objectives: To observe the effect of electroacupuncture (EA) preconditioning on podocytes and phosphatase and tension homologous protein (PTEN)/phosphoinositide-3-kinase (PI3K) signaling pathway in rats with type 2 diabetic kidney injury, so as to explore its potential mechanisms.
Methods: Fifty male Wistar rats were randomly divided into control, model, EA, inhibitor, and sham EA groups, with 10 rats in each group. Diabetes model was established by high-fat and high-sugar diet and intraperitoneal injection of streptozotocin (40 mg/kg). For rats in the EA group, EA (2 Hz, 1 mA) preconditioning was applied to "Guanyuan" (CV4), "Zhongwan" (CV12), and bilateral "Zusanli" (ST36), "Fenglong" (ST40) for 15 min, once every other day for 8 weeks. And rats in the sham EA group were given acupuncture at the subcutaneous areas of the same acupoints without EA stimulation. Rats of the inhibitor group were given intraperitoneal injection of BPV (HOpic, 0.6 mg/kg, 0.5 mg/mL) combined with the same EA stimulation as EA group once a week for 8 weeks. The blood glucose level of rats was recorded. Urinary albumin (ALB) content in rats was detected by ELISA. The contents of serum urea (Urea), creatinine (Crea), total cholesterol (TC) and triglyceride (TG) were detected by automatic biochemical analyzer. HE staining, PAS staining and Masson staining were used to observe the pathological changes of renal tissues. The ultrastructural changes of podocytes were observed by transmission electron microscopy. The relative expression levels of Synaptopodin, microtubule-associated protein light chain 3 Ⅱ(LC3-Ⅱ), PTEN and PI3K proteins in kidney were detected by Western blot.
Results: Compared with the control group, the blood glucose, content of serum ALB, Urea, Crea, TC, TG and renal PI3K protein expression in the model group were significantly increased (P<0.01), while the expression levels of renal Synaptopodin, LC3-Ⅱ and PTEN proteins were decreased (P<0.01). Compared with the model group, the blood glucose, content of serum ALB, Urea, Crea, TC, TG and renal PI3K protein expression in the EA group decreased (P<0.01, P<0.05), while the expression levels of renal Synaptopodin, LC3-Ⅱ and PTEN proteins increased (P<0.01). In comparison with the EA group, the blood glucose, ALB, Urea, Crea, TC, TG content and PI3K protein expression level were increased (P<0.05, P<0.01), while the expression levels of Synaptopodin, LC3-Ⅱ and PTEN proteins were decreased (P<0.01) in the inhibitor group；whereas, the blood glucose, ALB, Urea, TC, TG content and PI3K protein expression level were increased (P<0.05, P<0.01), while the expression levels of Synaptopodin, LC3-Ⅱ and PTEN proteins were decreased (P<0.01) in the sham EA group. HE staining and PAS staining showed glomerular hypertrophy and glomerular glycogen deposition；and Masson staining displayed an enhancement of glomerular fibrosis；and electron microscope revealed foot process fusion, basement membrane thickening and autophagosomes reduction in the rat' s kidney of the model, inhibitor and sham EA groups, which were relatively milder in the EA group.
Conclusions: EA promotes the expression of PTEN gene-encoded protein in the kidneys of type 2 diabetic rats, thereby inhibiting the activation of PI3K, increasing the autophagy level and protecting the podocytes.
目的: 观察电针预处理对2型糖尿病肾脏损伤大鼠足细胞和磷酸酶与张力蛋白同源物（PTEN）/磷脂酰肌醇-3-激酶（PI3K）信号通路的影响，探讨其减轻2型糖尿病的可能机制。方法: Wistar大鼠随机分为空白组、模型组、电针组、抑制剂组、假电针组，10只/组。空白组正常饮食，不予处理；余组予高脂高糖饮食并联合链脲佐菌素腹腔注射造模。电针组造模前电针“中脘”“关元”及双侧“足三里”“丰隆”，假电针组取穴同电针组，针至皮下不通电，隔日1次，每次15 min，抑制剂组在电针组的基础上腹腔注射BPV（HOpic，0.6 mg/kg，0.5 mg/mL），所有治疗均持续8周。检测大鼠随机血糖，ELISA法检测尿微量白蛋白（ALB）含量，全自动生化分析仪检测血清尿素（Urea）、肌酐（Crea）、总胆固醇（TC）、甘油三酯（TG）含量，HE染色、PAS染色、Masson染色观察大鼠肾脏组织病理学变化，透射电镜观察肾脏足细胞超微结构改变，Western blot法检测肾脏Synaptopodin、微管相关蛋白轻链3-Ⅱ（LC3-Ⅱ）、PTEN和PI3K蛋白表达水平。结果: 与空白组相比，模型组大鼠血糖、血清ALB、Urea、Crea、TC、TG含量及肾脏PI3K蛋白表达水平显著上升（P<0.01），Synaptopodin、LC3-Ⅱ、PTEN蛋白表达水平降低（P<0.01），肾小球肥大，肾小球内糖原沉积，胶原纤维增多，基底膜增厚，足突融合或消失，自噬体明显减少；与模型组相比，电针组大鼠血糖、血清ALB、Urea、Crea、TC、TG含量及肾脏PI3K蛋白表达水平下降（P<0.01，P<0.05），Synaptopodin、LC3-Ⅱ、PTEN蛋白表达水平上升（P<0.01），肾小球肥大和基底膜增厚减轻，糖原沉积明显减少，足突基本正常，并见自噬体；与电针组相比，抑制剂组大鼠血糖、ALB、Urea、Crea、TC、TG含量及肾脏PI3K蛋白表达水平不同程度上升（P<0.05，P<0.01），Synaptopodin、LC3-Ⅱ、PTEN蛋白表达水平下降（P<0.01），假电针组血糖、ALB、Urea、TC、TG含量及肾脏PI3K蛋白表达水平上升（P<0.05，P<0.01），Synaptopodin、LC3-Ⅱ、PTEN蛋白表达水平下降（P<0.01），两组肾小球肥大和基底膜增厚较明显，可见糖原沉积，足突不同程度融合，自噬体少见。结论: 电针预处理对大鼠肾脏足细胞的保护作用可能是通过促进PTEN基因编码蛋白的表达从而抑制PI3K的激活，提高足细胞自噬水平而实现的。.
Keywords: Autophagy; Electroacupuncture; Kidney injury; Phosphatase and tension homologous protein /phosphoinositide-3-kinase pathway; Podocyte; Type 2 diabetes.