基于Wnt/β-catenin通路探讨大黄糖络丸对糖尿病肾病db/db小鼠肾纤维化的作用机制

To investigate the mechanism of dabotangluo pills on renal fibrosis in db/db mice with diabetic nephropathy based on Wnt/β-catenin pathway

关键词:  大黄糖络丸  糖尿病肾病  肾纤维化  Wnt/β-catenin  GSK-3β  

Key words:  rhubarb sugar Luo pill  Diabetic nephropathy  Renal fibrosis  Wnt3a  β-catenin  Glycogen synthesis kinase-3β (GSK-3β)  Phosphorylated glycogen synthesis kinase-3 (p-GSK-3β)

摘要:

目的:观察大黄糖络丸对糖尿病肾病db/db小鼠肾功能及纤维化的影响,并基于Wnt/β-连环蛋白(β-catenin)通路探讨其可能的作用机制。方法:将40只db/db小鼠随机分为模型组、大黄糖络丸低、中、高剂量组(0.9,1.8,3.6 g·kg-1),阳性药物达格列净组(1.5mg·kg-1),每组8只。另取8只db/m小鼠为正常对照组。正常组和模型组给予等体积蒸馏水,6 组小鼠每日灌胃1次,连续10周。定期测量小鼠空腹血糖(FBG)、收集尿液并检测尿微量白蛋白、尿肌酐,尿微量白蛋白/肌酐比值(ACR)；眼球取血,用全自动生化分析仪检测小鼠胆固醇(CHO)、甘油三酯(TG)。Masson染色观察肾脏组织纤维化程度；实时荧光定量聚合酶链式反应(Real-time PCR)、蛋白免疫印迹法(Western blot)检测肾脏中 Wnt3a、β-catenin、糖原合成激酶-3β(GSK-3β)及磷酸化糖原合成激酶-3β(p-GSK-3β)中 mRNA以及蛋白定量表达水平。透射电子显微镜观察肾脏细胞超微结构的改变,并观测小鼠肾脏的病理损伤程度。采用SPSS统计软件进行数据分析。结果:与正常组相比,模型组小鼠肾脏出现明显损伤及纤维化,FBG、CHO、TG、ACR含量均显著升高(P<0.01),肾脏组织中Wnt3a、β-catenin、GSK-3β、p-GSK-3β等蛋白表达水平均明显提高(P<0.05)；与模型组相比,大黄糖络丸中、高剂量组和阳性药物组小鼠肾损伤明显改善,肾纤维化程度明显降低,FBG、CHO、TG、ACR水平明显降低(P<0.05),大黄糖络丸高、中剂量组和阳性药物组 Wnt3a、β-catenin、 GSK-3β及 p-GSK-3β蛋白表达水平均明显降低(P<0.05),大黄糖络丸低剂量组小鼠蛋白表达水平降低,但无统计学意义(P>0.05)。结论:大黄糖络丸可有效降低血糖、改善肾纤维化,其机制可能与抑制 Wnt/β-catenin通路有关。

Abstract:

Objective: To observe the effects of dabotangluo pills on renal function and fibrosis in db/db mice with diabetic nephropathy, and to explore the possible mechanism based on Wnt/ β-catenin pathway. Methods: 40 db/db mice were randomly divided into model group, low, medium and high dose groups (0.9, 1.8, 3.6g·kg-1) of rhubarb Tang Luo pill, and positive drug dagliprazin group (1.5mg·kg-1), with 8 mice in each group. Another 8 db/m mice were selected as normal control group. Normal group and model group were given equal volume normal saline (10mg·kg-1), and 6 groups were given intragastric administration once a day for 10 weeks. Fasting blood glucose (FBG) was measured regularly, urine was collected and microalbumin, urinary creatinine and microalbumin/creatinine ratio (ACR) were detected. Cholesterol (CHO) and triglyceride (TG) were detected by automatic biochemical analyzer. The degree of renal fibrosis was observed by Masson staining. Real-time fluorescent quantitative polymerase chain reaction (Real-Time PCR) and Western blot were used to detect Wnt3a, β-catenin, glycogen synthetic kinase-3β (GSK-3β) and phosphorylated glycogen synthetic kinase-3β (p-GSK-3β) in kidney Quantitative expression levels of mRNA and protein. The ultrastructure of kidney cells was observed by transmission electron microscope (TEM), and the pathological injury degree of mouse kidney was observed. SPSS statistical software was used for data analysis. Results: Compared with normal group, the kidney in model group showed obvious damage and fibrosis, the contents of FBG, CHO, TG and ACR were significantly increased (P<0.05), and the protein expression levels of Wnt3a, β-catenin, GSK-3β and P-GSK-3β in kidney tissues were significantly increased (P<0.05).Compared with model group, the renal injury of mice in high-dose group, medium-dose group and positive drug group was significantly improved, the degree of renal fibrosis was significantly decreased, and the levels of FBG, CHO, TG and ACR were significantly decreased (P<0.05).The protein expression levels of Wnt3a, β-catenin, GSK-3β and P-GSK-3β in high and medium dose groups and positive drug groups were significantly decreased (P<0.05, P<0.01), while the protein expression levels of mice in low dose group were not statistically significant (P>0.05).Conclusion: Rhubarb Tangluo pills can effectively reduce blood glucose and improve renal fibrosis, the mechanism of which may be related to the inhibition of Wnt/β-catenin pathway.