肾消通络方对糖尿病db/db小鼠肾脏TLR4/NF-κB信号通路的影响

Effects of Shenxiao Tongluo Formula on TLR4/NF-κB signaling pathway in kidney of diabetic db/db mice

目的:观察肾消通络方对自发性2型糖尿病动物模型db/db小鼠肾脏TLR4/NF-κB信号通路的影响,探讨肾消通络方对db/db小鼠肾脏的保护作用和机制。方法:将24只SPF级8周龄的雄性db/db小鼠随机分为中药组、西药组、模型组,每组8只;另取8只同遗传背景野生型正常db/m小鼠为正常组,连续治疗8周。于治疗前与治疗后每隔2周检测空腹血糖,每隔4周收集24h尿液,用于检测24h尿微量白蛋白水平。给药8周后处死小鼠,摘眼球取血,用于检测血清肌酐(Scr)、血尿素氮(BUN)水平;分离肾脏,放入4%多聚甲醛中固定,用于后续病理检测。HE和PAS染色观察肾组织病理改变;免疫组织化学法检测肾组织中Toll样受体4(TLR4)、核转录因子-κBp65(NF-κB p65)、单核细胞趋化蛋白-1(MCP-1)蛋白的表达。结果:与正常组比较,模型组小鼠空腹血糖、Scr、BUN、24h尿微量白蛋白显著升高(P<0.01);肾组织结构排列紊乱,肾小球肥大,基底膜增厚,系膜细胞增生、毛细血管管腔变窄;肾组织TLR4、NF-κB p65、MCP-1蛋白表达显著升高(P<0.01)。与模型组比较,中药组和西药组血清Scr、BUN、24h尿微量白蛋白均显著降低(P<0.01);肾脏病理改变减轻;TLR4、NF-κB p65、MCP-1蛋白表达显著减弱(P<0.01)。结论:肾消通络方能够抑制db/db小鼠肾脏炎症因子的表达从而减轻肾脏损害,其机制可能与下调TLR4/NF-κB信号通路相关蛋白表达有关。

Objective:To observe the effects of Shenxiao Tongluo Formula on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway of db/db mice model of spontaneous type 2 diabetes,and to investigate the renal protective effect and mechanism of Shenxiao Tongluo Formula on db/db mice.Methods:Twenty-four female db/db mouse aged 8 weeks old were randomly divided into three groups including model group,Chinese medicine group and Western medicine group with 8 mice in each group.Another 8 female db/db mice aged 8 weeks old were used as a normal control group.Continuous treatment for 8 weeks.The fasting blood glucose(FBG)was detected every 2 weeks and the urine of 24 h was collected every 4 weeks.The collected urine was used for the detection of microalbumin of different groups before and after treatment.The mice were sacrificed after 8 weeks of administration.The blood was collected for Scr and BUN.The kidneys were separated to observe the morphological changes with hematoxylin-eosin and periodic acid Schiff staining,and detect the expressions of TLR4,NF-κB p65 and MCP-1 with immunohistochemistry.Results:Compared with normal group,FBG,Scr,BUN and 24 h UMALB in model group increased(P<0.01).Disturbance of renal tissue structure,the glomerular hypertrophy,basement membrane thickening,the mesangial cell proliferation and the stenosis of capillary lumen.Protein expression levels of TLR4,NF-κB p65 and MCP-1 in model group were up-regulated(P<0.01).Compared with model group,Chinese medicine group and Western medicine group could reduce the levels of Scr,BUN and 24 h UMALB(P<0.01).The pathological damage of the kidney was inhibited.The protein expression of TLR4,NF-κB p65 and MCP-1 were decreased(P<0.01).Conclusion:Shenxiao Tongluo Formula can inhibit the expression of inflammatory factors in the kidney of db/db mice and reduce the renal damage.The mechanism may be related to the down-regulation of TLR4/NF-κB signal pathway related protein expression.