Mechanism and Effect of Inflammatory Cytokines on Activation of Astrocytes and Expression of sPLA2-IIA

doi:10.3969/j.issn.1673-5765.2018.10.003

Abstract

Abstract:

Objective To explore the effect of inflammatory cytokines on the activity of astrocytes and the expression of sPLA2-IIA secreted by astrocytes, and the possible mechanism. Methods On the basis of primary cell culture of rat cerebral cortical astrocytes in vitro, the fourth generation of cultured cells were used for the experiment, which were randomly divided into experimental group and control group. The experimental group included: 10 ng/ml IL-1β subgroup, 100 ng/ml IL-1β subgroup, 10 ng/ml TNF-α subgroup, 100 ng/ml TNF-α subgroup, 10 ng/ml IL- 1β plus 10 ng/ml TNF-α subgroup, 100 ng/ml IL-1β plus 100 ng/ml TNF-α subgroup. 24 hours later, cell viability was detected by CCK-8 assay. Then the 10 ng/ml IL-1β plus 10 ng/ml TNF-α subgroup was selected to continue the experiment. The expression of sPLA2-IIA was detected by qRT-PCR and ELISA, and the mRNA changes of IKKα, IKKβ and NF-κB were detected by qRTPCR. Results Through the separation and purification, the typical rat cortical astrocytes were obtained. The morphology of astrocytes was irregular, the neurites were longer and radial. Glial fibrillary acidic protein (GFAP) staining showed that the purity of astrocytes was over 95%. The activity of astrocytes in experimental group was higher than that in control group. Among them, the activity of astrocytes in 100 ng/ml IL-1β subgroup, 100 ng/ml TNF-α subgroup, 10 ng/ml IL-1β plus 10 ng/ml TNF-α subgroup and 100 ng/ml IL-1β plus 100 ng/ml TNF-α subgroup were significantly higher than that in control group (t =0.07, 0.12, 0.53, 0.39, P <0.05). Compared with the control group, the mRNA and protein expression of sPLA2-IIA significantly increased (t =9.615, 11.635, P <0.05), and the mRNA expression of IKKα, IKKβ and NF-κB in 10 ng/ml IL-1β and 10 ng/ml TNF-α subgroup significantly increased as well (t =4.015, 4.719, 6.509, P <0.05). Conclusions The combined application of inflammatory cytokines IL-1β and TNF-α can stimulate the activation of astrocytes, and promote the expression of sPLA2-IIA, which may be related to NF- κB pathway.

Key words: Astrocytes; Inflammatory cytokines; sPLA2-IIA

摘要：

目的探索在炎性因子作用下，星形胶质细胞的活性变化及其产生的分泌型磷脂酶A 2-ⅡA （secretory phospholipase A2 of group ⅡA，sPLA2-ⅡA）表达量的变化，进一步探讨其可能存在的机制。

方法在体外原代培养大鼠大脑皮质星形胶质细胞的基础上，取第4代培养的细胞用于实验。随机分为对照组和实验组，实验组分别为：10 ng/ml白介素-1β（interleukin-1 beta，IL-1β）组，100 ng/ml IL-1β组，10 ng/ml 肿瘤坏死因子-α（tumor necrosis factor，TNF-α）组，100 ng/ml TNF-α组，10 ng/ml IL-1β+10 ng/ml TNF-α组，100 ng/ml IL-1β+100 ng/ml TNF-α组。24 h后用细胞增殖-毒性检测试剂盒检测细胞活力。选取10 ng/ml IL-1β+10 ng/ml TNF-α组继续进行实验。通过实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction，qRT-PCR）和酶联免疫吸附测定，检测 sPLA2-ⅡA的表达量变化，并通过qRT-PCR检测核因子κB（nuclear factor-kappa B，NF-κB）、NF-κB 抑制蛋白激酶α（inhibitor of NF-κB kinase α，IKKα）、NF-κB抑制蛋白激酶β（inhibitor of NF-κB kinase β，IKKβ）的mRNA变化。

结果通过分离、纯化获得了形态典型的大鼠大脑皮质星形胶质细胞，细胞形态不规则，突起较多较长，呈放射状。胶质纤维酸性蛋白免疫染色显示纯度达95%以上。实验组细胞的活性均高于对照组。其中，100 ng/ml IL-1β组、100 ng/ml TNF-α组、10 ng/ml IL-1β+10 ng/ml TNF-α组和100 ng/ml IL-1β+100 ng/ml TNF-α组的细胞活性明显高于对照组（t =0.07、0.12、0.53、0.39，P＜0.05）。与对照组相比，10 ng/ml IL-1β+10 ng/ml TNF-α组sPLA2-ⅡA的mRNA和蛋白水平表达量显著升高（t =9.615、 11.635，P＜0.05），NF-κB、IKKα、IKKβ的mRNA均增高（t =4.015、4.719、6.509，P＜0.05）。

结论炎性因子IL-1β与TNF-α联合应用可刺激星形胶质细胞活化，并能够不同程度地刺激sPLA2- ⅡA的表达，该过程可能与NF-κB通路有关。

关键词: 星形胶质细胞; 炎性因子; 分泌型磷脂酶A2-ⅡA

LI Xue-Fen, CHEN Wen-Wu, LI Qing, GUO An-Chen, WANG Yong-Jun, WANG Qun. Mechanism and Effect of Inflammatory Cytokines on Activation of Astrocytes and Expression of sPLA2-IIA[J]. Chinese Journal of Stroke, 2018, 13(10): 1012-1018.

李雪粉, 陈文武, 李青, 郭安臣, 王拥军, 王群. 炎性因子对星形胶质细胞的活化及sPLA2-ⅡA表达的机制及影响[J]. 中国卒中杂志, 2018, 13(10): 1012-1018.

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