活性维生素D3调控NF-κB信号通路对糖尿病肾病大鼠肾脏保护作用机制研究

A study on the mechanism of action of active vitamin D3 through regulating the NF-κB signaling pathway in renal protection of diabetic nephropathy rats

目的:探讨活性维生素D3调控NF-κB炎症信号通路对2型糖尿病肾病大鼠肾脏的保护作用及机制;寻找维生素D在治疗糖尿病肾病中的最佳浓度。方法:将SD大鼠70只随机分成正常对照组(10只)、DN模型组(60只),且DN模型组进一步分为花生油组、厄贝沙坦组、维生素D低剂量组、维生素D中剂量组、维生素D高剂量组(每组各10只)。正常组以普通饲料喂养,模型组分别以上述处理方式进行灌胃。6周后处死大鼠称取体质量,采血检测空腹血糖(fasting blood glucose,FBG),收集24 h尿液计算尿量、测24 h尿蛋白定量;留取肾脏组织,HE染色观察肾组织病理学变化;实时荧光定量PCR检测肾脏组织中NF-κB及其下游单核细胞趋化因子-1(monocyte chemotactic protein 1,MCP-1)的mRNA表达;Western blot检测炎症因子NF-κB及MCP-1蛋白表达。结果:①在血糖、24 h尿蛋白,维生素D低剂量组较花生油组明显下降[分别为18.20(16.25,21.25)、32.24(26.09,37.68)与23.55(22.75,24.60)、72.52(50.38,84.93),均P<0.05]。②正常组大鼠HE染色可见肾小球形态完整,系膜区无明显增生,毛细血管腔规则,毛细血管袢形态规则;花生油组肾小球体积缩小,系膜区明显增宽,肾小球系膜细胞增生,毛细血管壁增厚;厄贝沙坦组较花生油组增生减轻;维生素D高剂量组、中剂量组、低剂量组肾小球系膜区均有增宽,但维生素D低剂量组增宽程度最轻。③维生素D低剂量组的NF-κB及MCP-1的mRNA水平较正常组花生油组明显下降[分别为1.72±0.002、0.67±0.01;3.57±0.40、3.57±0.37,均P<0.05];但厄贝沙坦组与维生素D低剂量组比较无统计学差异。④NF-κB的蛋白表达水平在各组间均无统计学差异,维生素D低剂量组的MCP-1蛋白表达水平较花生油组、维生素D中剂量组下降最为明显[分别为0.47±0.17与0.91±0.30、1.22±0.18、0.93±0.43,均P<0.05]。结论:维生素D能通过下调NF-кB炎症信号通路,有效地缓解糖尿病肾病的肾脏损伤;维生素D低剂量[0.03μg/(kg·d)]在糖尿病肾病的保护作用中为最佳浓度。

Objective:①To investigate the mechanism of action of active vitamin D3 through regulating the NF-κB signaling pathway in renal protection of rats with type 2 diabetic nephropathy.②To determine the optimal concentration of vitamin D in the treatment of diabetic nephropathy.Methods:Seventy Sprague-Dawley rats were randomly divided into normal control group(10 rats)and DN model group(60 rats),and the DN model group was furhter divided into peanut oil group,irbesartan group,low-dose vitamin D group,medium-dose vitamin D group,and high-dose vitamin D group,with 10 rats in each group.The normal control group was fed with routine diet,while the rats in the model group were ad-ministered above-mentioned drugs by gavage.The rats were then sacrificed and weighed after 6 weeks.And fasting blood glucose(FBG)was measured after blood sampling,and 24-hour urine was also collected to calculate urine volume and quantify 24-hour urine protein.Renal tissue was retained and HE staining was used to observe the pathological changes of renal tissue.Meanwhile,real-time fluorescent quantitative PCR was used to determine the expression of nuclear factor-kappa B(NF-κB)mRNA and its downstream monocyte chemotactic protein-1(MCP-1)mRNA in renal tissue,and the protein expression of inflammatory factors,NF-κB and MCP-1,was measured by Western blot.Results:①The levels of FBG and 24-hour urine protein in the low-dose vitamin D group were significantly lower than those in the peanut oil group[18.20(16.25,21.25),32.24(26.09,37.68)vs.23.55(22.75,24.60),72.52(50.38,84.93),P<0.05].②In the normal control group,HE staining showed that the glomerular morphology was intact,the mesangial area had no obvious hyperplasia,and the capillary lumen and loops were both regularly shaped;in the peanut oil group,the glomerular volume was reduced,the mesangial area significantly broadened,the glomerular mesangial cells proliferated,and capillary wall became thickened as well.The irbesartan group showed less hyperplasia compared with the peanut oil group.The high-,medium-,and low-dose vitamin D groups all had a broadened glomerular mesangial area,but the low-dose group exhibited the lowest degree of broadening.③The mRNA levels of NF-κB and MCP-1 in the low-dose vitamin D group were significantly lower than those in the normal control group and the peanut oil group(1.72±0.002,0.67±0.01 vs.3.57±0.40,3.57±0.37,all P<0.05).However,there was no significant difference between the irbesartan group and the low-dose vitamin D group.④There were no significant differences between these groups regarding the expression of NF-κB protein.The low-dose vitamin D group had a significant reduction in the expression of MCP-1 protein compared with the peanut oil group and the medium-dose vitamin D group(0.47±0.17 vs.0.90±0.30,1.22±0.18,0.93±0.43,all P<0.05).Conclusion:①Vitamin D can effectively alleviate renal injury in diabetic nephropathy by down-regulation of the NF-κB inflammatory signaling pathway.②As indicated in the study,0.03μg/(kg·d)in the low-dose vitamin D group is the optimal concentration for renal protection in diabetic nephropathy.