二甲双胍介导NF-κB信号通路对糖尿病肾病细胞损伤的保护作用

Protective Effects of Metformin Mediated NF-κB Signaling Pathway on Cell Damage in Diabetic Nephropathy

为探究二甲双胍介导核因子-κB(NF-κB)信号通路对糖尿病肾病细胞损伤的保护作用,体外培养人肾小球足细胞(HGPC),利用10、20、30和40 mmol/L D-葡萄糖进行干预,筛选构建HGPC高糖炎症损伤模型的D-葡萄糖最适浓度;再利用20、40、80、160 mmol/L二甲双胍进行干预,筛选最适二甲双胍浓度;随后将细胞分为对照组(Con组)、高糖组(HG组)、二甲双胍组(Met组,30 mmol/L D-葡萄糖+80 mmol/L二甲双胍)、抑制剂组(Y组,30 mmol/L D-葡萄糖+1μmol/L NF-κB通路抑制剂BAY 11-7082)、二甲双胍+抑制剂组(Met+Y组,30 mmol/L D-葡萄糖+80 mmol/L二甲双胍+1μmol/L BAY 11-7082)和二甲双胍+激动剂组(Met+A组,30 mmol/L D-葡萄糖+80 mmol/L二甲双胍+1μmol/L NF-κB通路激动剂Prostratin),干预24 h。利用细胞计数试剂盒-8(CCK-8)检测细胞活力;酶联免疫吸附试验(ELISA)法检测炎症因子水平;Transwell小室法测定细胞侵袭和迁移能力;蛋白免疫印迹(WB)法检测E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(vimentin)、纤连蛋白(FN)、NF-κB p65和p-NF-κB p65蛋白的表达水平。结果显示:选择30 mmol/L D-葡萄糖干预HGPC细胞24 h以构建HGPC高糖炎症模型,80 mmol/L为二甲双胍最适浓度;HG组细胞炎症因子肿瘤坏死因子(TNF-α)、白细胞介素-1β(IL-1β)水平、细胞侵袭、迁移能力以及N-cadherin、vimentin、FN和p-NF-κB p65蛋白水平高于Con组,E-cadherin蛋白表达水平低于Con组(P<0.05);与HG组相比,Met组和Y组细胞炎症因子TNF-α、IL-1β水平、细胞侵袭能力、迁移能力、N-cadherin、vimentin、FN和p-NF-κB p65蛋白表达水平显著降低,E-cadherin蛋白表达水平升高(P<0.05);与Met组相比,加入BAY 11-7082后,上述指标趋势更显著(P<0.05),Met+A组趋势与Met+Y组相反(P<0.05)。二甲双胍通过阻断NF-κB通路激活抑制D-葡萄糖诱导的HGPC炎症反应、侵袭、迁移和上皮间质转化进程,保护高糖诱导的足细胞损伤。

In order to explore the protective effect of metformin mediated nuclear factor-κB(NF-κB)signaling pathway on the damage of diabetic nephropathy cells,HGPC cells were cultured in vitro and treated with 10,20,30 and 40 mmol/L D-glucose to select the optimal concentration of D-glucose for the construction of HGPC hyperglycemic inflammatory injury model.Then 20,40,80 and 160 mmol/L metformin were used for intervention,and the optimal concentration of metformin was screened.The cells were then divided into control group(Con group),high glucose group(HG group)and metformin group(Met group,30 mmol/L D-glucose+80 mmol/L metformin),inhibitor group(Y group,30 mmol/L D-glucose+1μmol/L NF-κB pathway inhibitor BAY 11-7082),metformin+inhibitor group(Met+Y group,30 mmol/L D-glucose+80 mmol/L metformin+1μmol/L BAY 11-7082)and metformin+agonist groups(Met+A group,30 mmol/L D-glucose+80 mmol/L metformin+1μmol/L NF-κB pathway agonist Prostratin),intervention was conducted for 24 h.Cell count kit 8(CCK-8)was used to detect cell viability;the expression levels of inflammatory cytokines were detected by enzyme-linked immunosorbent assay(ELISA);cell invasion and migration were determined by Transwell assay.Western blotting(WB)was used to detect the expression levels of E-cadherin,N-cadherin,vimentin,Fibronectin(FN),NF-κB p65 and p-NF-κB p65.The results show that:HGPC cells were treated with 30 mmol/L D-glucose for 24 h to construct HGPC hyperglycemic inflammation model,and 80 mmol/L was the optimal concentration of metformin.Compared with Con group,the levels of inflammatory cytokines tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),invasion ability,migration ability,the expression levels of N-cadherin,vimentin,FN and p-NF-κB p65 protein in HG group significantly increased,while the expression level of E-cadherin protein decreased(P<0.05).Compared with HG group,the levels of inflammatory cytokines TNF-α,IL-1β,cell invasion ability,migration ability,the expression levels of N-cadherin,vimentin,FN and p-NF-κB p65 protein in Met group and Y group significantly decreased,and the expression level of E-cadherin protein increased(P<0.05).Compared with the Met group,the above indexes changed more significantly after the addition of BAY 11-7082(P<0.05),and the trend of Met+A group was opposite to that of Met+Y group(P<0.05).Metformin inhibits D-glucose-induced HGPC inflammatory response,invasion,migration and epithelial-mesenchymal transition process by blocking the activation of NF-κB pathway,and protects podocyte injury induced by high glucose.