（保定市第二中心医院检验科，保定 072750）
摘　要：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+1 μmol/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+1 μmol/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+1 μmol/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。
关键词：糖尿病肾病；二甲双胍；核因子-κB信号通路；D-葡萄糖诱导；人肾小球足细胞炎症
中图分类号： R587.1                           文献标志码：ADOI：10.3969/j.issn.1007-7146.2024.03.011

(Department of Laboratory Medicine, the Second Central Hospital of Baoding, Baoding 072750, China)
Abstract: 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+1 μmol/L NF-κB pathway inhibitor BAY 11-7082), metformin+inhibitor group (Met+Y group, 30 mmol/L D-glucose+80 mmol/L metformin+1 μmol/L BAY 11-7082) and metformin+agonist groups (Met+A group, 30 mmol/L D-glucose+80 mmol/L metformin+1 μmol/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.
Key words: diabetic nephropathy; metformin; nuclear factor-κB signaling pathway; D-glucose induction; inflammation of human glomerular podocytes
（Acta Laser Biology Sinica, 2024, 33(3): 275-283）