基于TLR4/MyD88/AMPK信号通路探索“从风论治”治法治疗糖尿病肾病的作用机制

Based on TLR4/MyD88/AMPK signaling pathway,explore the mechanism of“treating diabetic kidney disease through wind”therapy

目的基于TLR4/MyD88/AMPK信号通路探索“从风论治”治法治疗糖尿病肾病(diabetic kidney disease,DKD)的作用机制。方法db/db小鼠分为对照组、糖尿病肾病组、干预组,干预时间为8周。留取血清、尿液,检测24小时尿蛋白定量、白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的表达水平。留取肾组织,光镜下观察肾组织病理改变,同时采用Western blot法及免疫组织化学法检测肾组织Toll样受体4(Toll-like receptor 4,TLR4)、髓样分化因子(myeloid differentiation factor 88,MyD88)、AMP活化蛋白激酶(AMP-activated protein kinase,AMPK)、过氧化物酶体增殖物激活受体γ-辅激活因子1-α(peroxisome proliferator-activated receptorγcoactivator 1-alpha,PGC-1α)蛋白的相对表达情况。结果干预组24小时尿蛋白在药物干预后明显减少,且干预组与糖尿病肾病组对比差异有统计学意义。Elisa结果显示:干预组与糖尿病肾病组相比,db/db小鼠血清中IL-6水平与TNF-α水平均有降低,差异具有统计学意义(P<0.05)。对照组肾组织的肾小球结构完整,糖尿病肾病组系膜细胞及系膜基质均增生,间质少量炎细胞浸润,干预组较糖尿病肾病组有减轻。Western blot及免疫组化结果显示:干预组TLR4、MyD88蛋白表达程度明显低于糖尿病肾病组(P<0.01);干预组p-AMPK、PGC-1α蛋白的表达程度显著高于糖尿病肾病组(P<0.01),而干预组AMPK蛋白的表达水平与糖尿病肾病组差异无统计学意义(P>0.05)。结论“从风论治”治法通过TLR4/MyD88/AMPK信号通路能够减轻DKD炎性因子的表达延缓DKD进展。

Objective Based on TLR4/MyD88/AMPK signaling pathway,explore the mechanism of the treatment of diabetic kidney disease(DKD)with“wind based treatment”.Methods db/db mice were divided into control group,diabetic kidney disease group and intervention group,intervention time is 8 weeks.Collect serum and urine,detect the 24-hour urine protein quantification and the level of expression of interleukin-6(IL-6),and tumor necrosis factor detection-α(TNF-α).Retain renal tissue and observe pathological changes under light microscopy.At the same time,Western blot and immunohistochemical methods were used to detect the level of relative expression of Myeloid differentiation factor 88(MyD88),Toll-like receptor 4(TLR4),AMP-activated protein kinase(AMPK),and peroxisome proliferator-activated receptorγcoactivator 1-alpha(PGC-1α)proteins in renal tissue.Results The 24-hour urinary protein in the intervention group was significantly decreased after the drug intervention,and there was a statistically significant difference between the intervention group and the diabetic kidney disease group.Elisa results showed that compared with the diabetic kidney disease group,the level of IL-6 and TNF-αin the serum of db/db mice in the intervention group were all decreased,and the difference was statistically significant(P<0.05).The glomerular structure of renal tissue in the control group was intact,mesangial cells and mesangial matrix were proliferating in the diabetic kidney disease group,and a small amount of inflammatory cells were infiltrating into the interstitium.The intervention group was less than the diabetic kidney disease group.The results of Western blot and immunohistochemical showed that the expression of TLR4 and MyD88 proteins in the intervention group was significantly lower than that in the diabetic kidney disease group(P<0.01);the expression of p-AMPK,PGC-1αin the intervention group was significantly higher than that in the diabetic kidney disease group(P<0.01).There was no significant difference in the expression of AMPK protein between the intervention group and the diabetes nephropathy group(P>0.05).Conclusion The treatment method of“treating DKD based on wind”can down-regulate the expression of inflammatory factors in DKD and delay the progression of DKD through the TLR4/MyD88/AMPK signaling pathway.