Department of Endocrinology, Second Clinical Medical College, Inner Mongolia University for Nationalities (Inner Mongolia Forestry General Hospital), Tongliao, Inner Mongolia, China (mainland).
Department of Endocrinology, The Centre Hospital of Wuhan, Wuhan, Hubei, China (mainland).
Med Sci Monit. 2020 Feb 3;26:e919399. doi: 10.12659/MSM.919399.
BACKGROUND The aim of this study was to explore the effects of NADPH oxidase 5 (NOX5) in high glucose-stimulated human glomerular mesangial cells (HMCs). MATERIAL AND METHODS Cells were cultured under normal glucose (NG) or high glucose (HG) conditions. Then, NOX5 siRNA was transfected into HG-treated HMCs. A Cell Counting Kit-8 assay, colony formation assay and 5-ethynyl-20-deoxyuridine (EDU) incorporation assay were applied to measure cell proliferative ability. In addition, the levels of oxidative stress factors including reactive oxygen species (ROS), malonaldehyde (MDA), NADPH, superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX), inflammatory cytokines including tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-1ß, and monocyte chemoattractant protein-1 (MCP-1) in HMCs were detected by kits. Moreover, the expression of TLR4/NF-kappaB signaling and extracellular matrix (ECM) associated genes were evaluated by western blotting. RESULTS The results revealed that the NOX5 was overexpressed in HG-treated HMCs. Silencing of NOX5 decreased proliferation of HMCs induced by HG. And NOX5 silencing alleviated the production of MDA and NADPH accompanied by an increase of SOD and GSH-PX levels. Additionally, the contents of TNF-alpha, IL-6, IL-1ß, and MCP-1 were reduced after transfection with NOX5 siRNA. Furthermore, silencing of NOX5 deceased the expression of collagen I, collagen IV, TGF-ß1, and fibronectin induced by HG stimulation. TLR4, MyD88, and phospho-NF-kappaB p65 expression were downregulated notably in NOX5 silencing group. CONCLUSIONS Taken together, these findings demonstrated that inhibition of NOX5 attenuated HG-induced HMCs oxidative stress, inflammation, and ECM accumulation, suggesting that NOX5 may serve as a potential therapeutic target for diabetic nephropathy (DN) treatment.
本研究旨在探讨烟酰胺腺嘌呤二核苷酸磷酸氧化酶 5（NOX5）在高糖刺激人肾小球系膜细胞（HMC）中的作用。
细胞在正常葡萄糖（NG）或高葡萄糖（HG）条件下培养。然后，将 NOX5 siRNA 转染到 HG 处理的 HMC 中。采用细胞计数试剂盒-8 测定法、集落形成测定法和 5-乙炔基-20-脱氧尿苷（EDU）掺入测定法测定细胞增殖能力。此外，通过试剂盒检测 HMC 中氧化应激因子包括活性氧（ROS）、丙二醛（MDA）、NADPH、超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-PX）、炎症细胞因子包括肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6、IL-1β和单核细胞趋化蛋白-1（MCP-1）的水平。此外，通过 Western 印迹评估 TLR4/NF-κB 信号和细胞外基质（ECM）相关基因的表达。
结果表明，NOX5 在 HG 处理的 HMC 中过度表达。沉默 NOX5 可降低 HG 诱导的 HMC 增殖。沉默 NOX5 可减轻 MDA 和 NADPH 的产生，同时增加 SOD 和 GSH-PX 水平。此外，转染 NOX5 siRNA 后 TNF-α、IL-6、IL-1β和 MCP-1 的含量减少。此外，沉默 NOX5 可降低 HG 刺激诱导的胶原 I、胶原 IV、TGF-β1 和纤维连接蛋白的表达。TLR4、MyD88 和磷酸化 NF-κB p65 的表达在 NOX5 沉默组中显著下调。
综上所述，这些发现表明抑制 NOX5 可减轻 HG 诱导的 HMC 氧化应激、炎症和 ECM 积聚，表明 NOX5 可能成为糖尿病肾病（DN）治疗的潜在治疗靶点。
