谢文宇; 张洪晨; 鲁传豪; 冯元; 张磊; 吕超; 时全星; 戴舒惠; 李侠

doi:10.3760/cma.j.cn115354-20220326-00171

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• 基础研究 •

脑缺血再灌注后STIM1通过内质网应激促进小胶质/巨噬细胞M1型活化的作用研究

中华神经医学杂志, 2022,21(8) : 762-769. DOI: 10.3760/cma.j.cn115354-20220326-00171

摘要

目的

探讨基质相互作用分子1(STIM1)对脑缺血再灌注损伤后小胶质/巨噬细胞M1型活化的影响及机制。

方法

(1)动物实验：采用随机数字表法将20只雄性C57BL/6J小鼠分为假手术组、大脑中动脉缺血再灌注(MCAO/R)组、MCAO/R+si-Ctrl组及MCAO/R+si-STIM1组，后3组小鼠建立MCAO/R模型，MCAO/R+si-Ctrl组及MCAO/R+si-STIM1组小鼠分别转染空载体对照病毒和STIM1基因敲除慢病毒。观察STIM1转染效率及各组小鼠中小胶质/巨噬细胞M1型活化标记物分化抗原86(CD86)的表达情况。(2)细胞实验：将原代小胶质细胞分为Ctrl组、氧糖剥夺/复氧(OGD/R)组、OGD/R+si-Ctrl组、OGD/R+si-STIM1组、OGD/R+溶媒组及OGD/R+4-苯基丁酸(4-PBA)组。后5组细胞构建OGD/R模型，OGD/R+si-Ctrl组、OGD/R+si-STIM1组细胞分别转染空载体对照病毒和STIM1基因敲除慢病毒；OGD/R+4-PBA组细胞在OGD/R造模前24 h使用1 mmol/L预处理1 h以抑制内质网应激(ERS)，OGD/R+溶媒组细胞同时间使用0.5%二甲基亚砜(DMSO)预处理1 h。采用Western blotting、ELISA、RT-qPCR等方法检测细胞模型中小胶质/巨噬细胞M1型活化标记物CD86、炎性因子肿瘤坏死因子-α(TNF-α) mRNA、IL-1β及ERS相关蛋白[转录因子C/EBP同源蛋白(CHOP)、活化转录因子4(ATF4)]的表达情况。

结果

(1)动物实验：MCAO/R+si-STIM1组STIM1表达水平较假手术组、MACO/R组及MCAO/R+si-Ctrl组均明显降低，差异均有统计学意义(P<0.05)。与MCAO/R组及MCAO/R+si-Ctrl组比较，MCAO/R+si-STIM1组小鼠缺血灶周围CD86与Iba-1共表达的小胶质/巨噬细胞数显著降低，差异有统计学意义(P<0.05)。(2)细胞实验：与OGD/R组及OGD/R+si-Ctrl组相比，OGD/R+si-STIM1组细胞STIM1、CD86、TNF-α mRNA表达水平及上清液中IL-1β含量均显著降低，差异均有统计学意义(P<0.05)。同样地，与OGD/R组及OGD/R+si-Ctrl组相比，OGD/R+si-STIM1组细胞ERS相关蛋白ATF4、CHOP表达水平亦显著降低，差异均有统计学意义(P<0.05)。此外，与OGD/R组及OGD/R+溶媒组相比，OGD/R+4-PBA组细胞中CD86表达水平、TNF-α mRNA表达水平及IL-1β含量均显著降低，差异均有统计学意义(P<0.05)。

结论

STIM1通过调控ERS水平影响脑缺血再灌注损伤后小胶质/巨噬细胞的M1型活化。

引用本文: 谢文宇, 张洪晨, 鲁传豪, 等. 脑缺血再灌注后STIM1通过内质网应激促进小胶质/巨噬细胞M1型活化的作用研究 [J] . 中华神经医学杂志, 2022, 21(8) : 762-769. DOI: 10.3760/cma.j.cn115354-20220326-00171.

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缺血性脑卒中(ischemic stroke，IS)是威胁人类健康的主要疾病之一，缺血/再灌注(ischemia/reperfusion，I/R)是其重要损伤过程。研究发现，小胶质/巨噬细胞M1型活化后致促炎因子大量释放，诱发一系列炎性级联反应并加重血脑屏障和周围神经细胞损伤可能是I/R后神经功能障碍的关键机理，但小胶质/巨噬细胞发生M1型活化的上游机制目前并不清楚。近年研究显示，内质网应激(endoplasmic reticulum stress，ERS)与小胶质/巨噬细胞活化以及中枢神经系统炎症反应密切相关，抑制ERS能减轻脂多糖诱导的BV2小胶质/巨噬细胞M1型活化，并抑制白细胞介素(IL)-1β、IL-18等促炎因子的释放^[1]。因此，探讨小胶质/巨噬细胞中ERS对于其炎症反应的作用有助于揭示I/R的具体调控机制。

贡献者信息

谢文宇

空军军医大学第一附属医院神经外科，西安　710032

张洪晨

空军军医大学第一附属医院神经外科，西安　710032

鲁传豪

空军军医大学第一附属医院神经外科，西安　710032

冯元

空军军医大学第一附属医院神经外科，西安　710032

张磊

空军军医大学第一附属医院神经外科，西安　710032

吕超

空军军医大学第一附属医院神经外科，西安　710032

时全星

战略支援部队特色医学中心神经外科，北京　100101

戴舒惠

空军军医大学第一附属医院神经外科，西安　710032

李侠

空军军医大学第一附属医院神经外科，西安　710032

通信作者

李侠

空军军医大学第一附属医院神经外科，西安　710032

Email：sjwklixia@sina.com

关键词

小胶质/巨噬细胞; 内质网应激; 缺血性脑卒中; 基质相互作用分子1;

基金项目

国家自然科学基金（81974188）

陕西省重点研发计划项目（2019SF-032）

陕西省创新能力支撑计划（2022TD-42）

利益冲突

所有作者均声明不存在利益冲突

历史

出版日期：2022-08-15

收稿日期：2022-03-26

本文编辑

张玲

Basic Research

Stromal interaction molecule 1 promotes microglia/macrophages M1 activation through endoplasmic reticulum stress after cerebral ischemia-reperfusion

Xie Wenyu, Zhang Hongchen, Lu Chuanhao, Feng Yuan, Zhang Lei, Lyu Chao, Shi Quanxing, Dai Shuhui, Li Xia

Published 2022-08-15

Cite as Chin J Neuromed, 2022, 21(8): 762-769. DOI: 10.3760/cma.j.cn115354-20220326-00171

Abstract

Objective

To investigate the influence and mechanism of stromal interaction molecule 1 (STIM1) in microglia/macrophages M1 activation after cerebral ischemia-reperfusion injury.

Methods

(1) Animal experiment: 20 male C57BL/6J mice were randomly divided into sham-operated (Sham) group, middle cerebral artery occlusion and reperfusion (MCAO/R) group, MCAO/R+si-Ctrl group, and MCAO/R+si-STIM1 group (n=5); MCAO/R models were established in mice of the latter 3 groups; empty vector control virus and STIM1 gene knockout lentivirus were transfected into mice in the MCAO/R+si-Ctrl group and MCAO/R+si-STIM1 group. The transfection efficiency of STIM1 and the expression of microglia/macrophages M1 activation marker cluster of differentiation 86 (CD86) in each group were observed. (2) Cell experiment: primary microglia were divided into Ctrl group, oxygen-glucose deprivation/re-oxygenation (OGD/R) group, OGD/R+si-Ctrl group, OGD/R+si-STIM1 group, OGD/R+solvent group, and OGD/R+4-phenylbutyric acid (4-PBA) group; OGD/R models were established in the later 5 groups; empty vector control virus and STIM1 gene knockout lentivirus were transfected into mice in the OGD/R+si-Ctrl group and OGD/R+si-STIM1 group; cells in the OGD/R+4-PBA group were pre-treated with 1 mmol/L 4-PBA for 1 h at 24 h before OGD/R modelling to inhibit endoplasmic reticulum stress (ERS), and cells in the OGD/R+solvent group were pre-treated with 0.5% dimethyl sulfoxide (DMSO) for 1 h at the same time. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), ELISA, Western blotting and other methods were used to detect the levels of CD86, tumour necrosis factor-α (TNF-α) mRNA, interleukin (IL)-1β, and ERS-related proteins (transcription factor C/EBP homologous protein [CHOP], activated transcription factor 4 [ATF4]) in these cells.

Results

(1) Animal experiment: the STIM1 expression in MCAO/R+si-STIM1 group was significantly lower than that in Sham group, MACO/R group and MCAO/R+si-Ctrl group (P<0.05); as compared with that in the MACO/R group and MCAO/R+si-Ctrl group, the number of microglia/macrophages co-expressing CD86 and Iba-1 around the ischemic foci of mice in the MCAO/R+si-STIM1 group was significantly decreased (P<0.05). (2) Cell experiment: as compared with those in the OGD/R group and OGD/R+si-Ctrl group, the expression levels of STIM1, CD86, and TNF-α mRNA, and supernatant IL-1β content in the OGD/R+si-STIM1 group were significantly decreased (P<0.05); as compared with those in the OGD/R group and OGD/R+si-CTRL group, the ATF4 and CHOP expression levels in OGD/R+si-STIM1 group were significantly decreased (P<0.05); as compared with those in the OGD/R group and OGD/R+solvent group, the CD86 level, TNF-α mRNA expression level and IL-1β content in the OGD/R+4-PBA group were significantly decreased (P<0.05).

Conclusion

STIM1 affects microglia/macrophages M1 activation after ischemia-reperfusion injury by regulating ERS level.

Key words:

Microglia/macrophage; Endoplasmic reticulum stress; Ischemic stroke; Stromal interaction molecule 1

Contributor Information

Xie Wenyu