Necrostatin-1在高糖环境下促巨噬细胞氧化应激反应中的作用及机制

doi:10.7518/hxkq.2021.06.008

华西口腔医学杂志 ›› 2021, Vol. 39 ›› Issue (6): 675-681.doi: 10.7518/hxkq.2021.06.008

Necrostatin-1在高糖环境下促巨噬细胞氧化应激反应中的作用及机制

周婷(), 周雪, 宋斌()

贵州省人民医院口腔科，贵阳 550002

收稿日期:2020-08-15 修回日期:2021-09-25 出版日期:2021-12-01 发布日期:2021-12-03
通讯作者: 宋斌 E-mail:zhouting061@163.com;songbin@vip.sohu.com
作者简介:周婷，副主任医师，硕士，E-mail：zhouting061@163.com
基金资助:
国家自然科学基金(81760198);贵州省科技厅联合基金(QKHLHZ[2016]7160);贵州省人民医院青年基金(GZSYQN[2016]06);贵州省人民医院国家自然科学基金培育基金(QKHPTRC[2018]5764-06)

Role and mechanism of necrostin-1 in promoting oxidative stress response of macrophages in high glucose condition

Zhou Ting(), Zhou Xue, Song Bin()

Dept. of Stomatology, Guizhou Provincial People’s Hospital, Guiyang 550002, China

Received:2020-08-15 Revised:2021-09-25 Online:2021-12-01 Published:2021-12-03
Contact: Song Bin E-mail:zhouting061@163.com;songbin@vip.sohu.com
Supported by:
The National Natural Science Foundation of China(81760198);The Natural Science Joint Foundation of Guizhou Province(QKHLHZ[2016]7160);The Foundation of Guizhou Provincial People’s Hospital for Young Scholars(GZSYQN[2016]06);The Training Fund of The National Natural Science Foundation of China for Guizhou Provincial People’s Hospital(QKHPTRC[2018]5764-06)

摘要/Abstract

摘要： 目的

探讨程序性细胞坏死特异性抑制剂Necrostatin-1（Nec-1）在高糖环境下，促巨噬细胞氧化应激反应中的作用及分子机制。

方法

将巨噬细胞在高糖组（25 mmol·L^-1葡萄糖）与对照组（5.5 mmol·L^-1葡萄糖）中培养72 h后，采用2’，7’-二氯荧光黄双乙酸盐（DCFA-DA）探针、丙二醛及超氧化物歧化酶（SOD）试剂盒分别检测其活性氧（ROS）含量、丙二醛及超氧化物歧化酶的活性；采用5 μmol·L^-1Nec-1对高糖组进行干预，作为高糖联合Nec-1处理组，再次检测氧化应激指标，并采用实时荧光定量聚合酶链反应（qRT-PCR）及蛋白质免疫印迹法（WB）检测受体相互作用蛋白激酶1（RIP1）的表达；沉默巨噬细胞中RIP1表达后，检测高糖环境对基因缺陷型细胞氧化应激反应的影响。

结果

在高糖刺激下，细胞ROS含量及丙二醛活性显著高于对照组（P<0.000 1），并且SOD活性显著低于对照组（P<0.000 1）；Nec-1干预后，高糖联合Nec-1处理组的ROS含量及丙二醛活性显著低于高糖组（P<0.000 1），并且高糖联合Nec-1处理组的SOD活性显著高于高糖组（P<0.01）。qRT-PCR及WB结果显示，高糖组中RIP1基因mRNA水平（P<0.001）及蛋白表达水平（P<0.000 1）均显著高于对照组，高糖联合Nec-1处理组的RIP1基因表达及蛋白表达显著低于高糖组（P<0.000 1）；同时RIP1得到有效沉默后（P<0.001），高糖+RIP1干扰组的ROS含量及丙二醛活性显著低于高糖+干扰对照组（si-NC）（P<0.001），SOD活性较高糖+干扰对照组显著上升（P<0.000 1）。

结论

高糖环境通过上调RIP1的表达诱发巨噬细胞的氧化应激反应。

关键词: 程序性细胞坏死抑制剂, 糖尿病, 牙周炎, 氧化应激, 受体相互作用蛋白-1

Abstract: Objective

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.

Methods

Macrophages were cultured in control (5.5 mmol·L^-1 glucose) or HG (25 mmol·L^-1 glucose) medium for 72 h. The HG+Nec-1 group was given HG and 5 μmol·L^-1 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.

Results

The HG group had increased ROS level and MDA activity (P<0.000 1) and decreased SOD activity (P<0.000 1) compared with the control group. The HG+Nec-1 group had higher ROS level and MDA activity (P<0.000 1) and lower SOD activity (P<0.01) than the HG group. The qRT-PCR and WB results showed that RIP1 mRNA level (P<0.001) and protein expression level (P<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 (P<0.000 1). After RIP1 was silenced effectively (P<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 (P<0.001), and SOD activity in the HG+si-RIP1 group increased than that in the HG+si-NC group (P<0.000 1).

Conclusion

HG promotes oxidative stress on macrophages by upregulating RIP1 expression.

Key words: necrostatin-1, diabetes, periodontitis, oxidative stress, receptor interacting protein 1

中图分类号:

R 781.4

周婷, 周雪, 宋斌. Necrostatin-1在高糖环境下促巨噬细胞氧化应激反应中的作用及机制[J]. 华西口腔医学杂志, 2021, 39(6): 675-681.

Zhou Ting, Zhou Xue, Song Bin. Role and mechanism of necrostin-1 in promoting oxidative stress response of macrophages in high glucose condition[J]. West China Journal of Stomatology, 2021, 39(6): 675-681.

图/表 6

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Necrostatin-1在高糖环境下促巨噬细胞氧化应激反应中的作用及机制

Role and mechanism of necrostin-1 in promoting oxidative stress response of macrophages in high glucose condition

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