Dept. of Stomatology, Guizhou Provincial People's Hospital, Guiyang 550002, China.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2021 Dec 1;39(6):675-681. doi: 10.7518/hxkq.2021.06.008.
To investigate the role and molecular mechanism of necrostatin-1 (Nec-1), a specific programmed cell necrosis inhibitor, in promoting the oxidative stress response of macrophages under high glucose (HG) environment.
Macrophages were cultured in control (5.5 mmol·L glucose) or HG (25 mmol·L glucose) medium for 72 h. The HG+Nec-1 group was given HG and 5 μmol·L Nec-1. Reactive oxygen species (ROS) level, malondialdehyde (MDA) activity, and superoxide dismutase (SOD) activity were measured by 2'-7'dichlorofluorescin diacetate, MDA, and SOD enzyme linked immunosorbent assay kits, respectively. Moreover, receptor interacting protein 1 (RIP1) expression was assessed through real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot (WB). Finally, after the expression of RIP1 in macrophages was silenced, the effect of HG environment on oxidative stress response was evaluated in the gene-deficient cells.
The HG group had increased ROS level and MDA activity (<0.000 1) and decreased SOD activity (<0.000 1) compared with the control group. The HG+Nec-1 group had higher ROS level and MDA activity (<0.000 1) and lower SOD activity (<0.01) than the HG group. The qRT-PCR and WB results showed that RIP1 mRNA level (<0.001) and protein expression level (<0.000 1) in the HG group were significantly higher than those in the control group, and RIP1 mRNA and protein expression levels in the HG+Nec-1 group were significantly lower than those in the HG group (<0.000 1). After RIP1 was silenced effectively (<0.001) with si-RNA, the ROS level and MDA activity of the HG+si-RIP1 group decreased compared with those of the HG+si-negative control (si-NC) group (<0.001), and SOD activity in the HG+si-RIP1 group increased than that in the HG+si-NC group (<0.000 1).
HG promotes oxidative stress on macrophages by upregulating RIP1 expression.
研究特异性细胞程序性坏死抑制剂 necrostatin-1（Nec-1）在高糖（HG）环境下促进巨噬细胞氧化应激反应中的作用及其分子机制。
将巨噬细胞分别在对照（5.5 mmol·L 葡萄糖）或 HG（25 mmol·L 葡萄糖）培养基中培养 72 h。HG+Nec-1 组给予 HG 和 5 μmol·L Nec-1。通过 2'-7'-二氯荧光素二乙酸酯、丙二醛（MDA）和超氧化物歧化酶（SOD）酶联免疫吸附试验试剂盒分别测定活性氧（ROS）水平、MDA 活性和 SOD 活性。此外，通过实时定量聚合酶链反应（qRT-PCR）和 Western blot（WB）测定受体相互作用蛋白 1（RIP1）的表达。最后，沉默巨噬细胞中 RIP1 的表达后，在基因缺陷细胞中评估 HG 环境对氧化应激反应的影响。
与对照组相比，HG 组的 ROS 水平和 MDA 活性升高（<0.000 1），SOD 活性降低（<0.000 1）。与 HG 组相比，HG+Nec-1 组的 ROS 水平和 MDA 活性升高（<0.000 1），SOD 活性降低（<0.01）。qRT-PCR 和 WB 结果显示，HG 组的 RIP1 mRNA 水平（<0.001）和蛋白表达水平（<0.000 1）明显高于对照组，HG+Nec-1 组的 RIP1 mRNA 和蛋白表达水平明显低于 HG 组（<0.000 1）。用 si-RNA 有效沉默 RIP1 后（<0.001），与 HG+si-NC 组相比，HG+si-RIP1 组的 ROS 水平和 MDA 活性降低（<0.001），SOD 活性升高（<0.000 1）。
HG 通过上调 RIP1 表达促进巨噬细胞氧化应激。
