c-Jun氨基末端蛋白激酶在人角膜上皮细胞调控NLRP3炎症小体激活中的作用

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摘要目的：在高渗条件下，人角膜上皮细胞（HCECs）中c-Jun氨基末端蛋白激酶（JNK）对NLRP3炎症小体所介导的炎症因子白细胞介素1β（IL-1β）释放的作用。方法：设等渗、高渗、高渗+JNK抑制剂（SP600125）3组，其中等渗组和高渗组均加入DMSO使其浓度和高渗+JNK抑制剂组DMSO浓度相同。用RT-PCR检测各组JNK、炎症小体各聚合成分（NLRP3、ASC、caspase-1）和炎症因子（IL-1β、TNF-α、IL-8、IL-6）的mRNA水平；用caspase-1活性试剂盒检测细胞中caspase-1的活性，用AOEB染色法检测细胞凋亡情况；用免疫荧光检测NLRP3、IL-1β的蛋白表达变化；用Western blot检测JNK、p-JNK、NLRP3、caspase-1、ASC和IL-1β的蛋白表达情况。结果：RT-PCR检测结果显示JNK抑制剂预处理细胞能抑制高渗刺激引起的JNK1、JNK2、NLRP3、caspase-1、ASC、IL-1β、TNF-α、IL-8和IL-6的mRNA表达上调（P＜0.05）。caspase-1活性测定结果显示JNK抑制剂预处理细胞能抑制高渗刺激引起的caspase-1活性增强（P＜0.001）。AOEB染色检测显示JNK抑制剂预处理后，细胞凋亡减少。免疫荧光结果显示JNK抑制剂能抑制高渗引起的NLRP3、IL-1β蛋白表达上调。Western blot进一步提示该抑制剂能够抑制高渗刺激引起的p-JNK、炎症小体聚合成分NLRP3、caspase-1和炎症因子IL-1β的蛋白表达上调（P＜0.05）。结论：HCECs在高渗条件下，NLRP3炎症小体被激活，从而分泌更多活性IL-1β，而JNK在调控NLRP3炎症小体的激活以及下游的炎症因子释放中起到了重要作用。

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	谭秋凡
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关键词 ：高渗, c-Jun氨基末端蛋白激酶, 炎症小体, 白细胞介素1β, 干眼病

Abstract：Objective: To investigate the role of JNK (c-Jun N-terminal kinase) in hyperosmosis-induced NLRP3 (NLR family pyrin domain containing 3) up-regulation and IL-1β secretion in the human corneal epithelial cells. Methods: HCECs cultured in normal osmolar media (310 mOsm) were switched to 500 mOsm, with or without the JNK inhibitor SP600125 (20 μmol/L). The mRNA levels of JNK, NLRP3, ASC, caspase-1 and IL-1β were detected by RT-qPCR. Caspase-1 activity was tested by caspase-1 colorimetric assay kits. Cell apoptosis was tested by AOEB staining method. The protein expression of NLRP3 and IL-1β were detected by immunofluorescence staining. And the protein level of JNK, p-JNK, NLRP3, caspase-1, ASC and IL-1β were determined by Western blot. Results: Hyperosmosis-induced mRNA overexpressions of JNK1, JNK2, NLRP3, caspase-1, IL-1β, TNF-α, IL-8 and IL-6 were significantly inhibited by JNK inhibitor (P<0.05). Moreover, hyperosmosis-induced caspase-1 activity was decreased with JNK inhibitor treatment by caspase-1 colorimetric assay (P<0.001). JNK inhibitor was able to downregulate cell apoptosis by AOEB staining. The protein overexpressions of NLRP3 and IL-1β induced by hyperosmosis were reversed by JNK inhibitor with immunofluorescence staining assays. And the protein overexpressions of p-JNK, NLRP3, caspase-1 and IL-1β induced by hyperosmosis were also downregulated with the use of JNK inhibitor by Western blot (P<0.05). Conclusion: NLRP3 inflammasome activation and subsequent IL-1β secretion are key consecutive events in HCECs response to hyperosmotic stress, and JNK may play a pivotal role in the regulation of the inflammasomes activation and expression of inflammatory factors.

Key words： hyperosmotic c-Jun N-terminal kinase inflammasome interleukin-1beta xerophthalmia

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基金资助:浙江省自然科学基金资助项目（LY16H120008）

通讯作者: 陈蔚，主任医师，Email：chenweimd@hotmail.com

作者简介: 谭秋凡（1990-），女，湖北荆门人，住院医师，硕士

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2018/V48/I1/13

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