α-突触核蛋白激活NLRP3炎性小体介导神经细胞焦亡的发生

doi:10.12092/j.issn.1009-2501.2019.06.006

中国临床药理学与治疗学 ›› 2019, Vol. 24 ›› Issue (6): 637-643.doi: 10.12092/j.issn.1009-2501.2019.06.006

α-突触核蛋白激活NLRP3炎性小体介导神经细胞焦亡的发生

韩晨阳，张晓玲，杨毅，郭丽，官俏兵

嘉兴市第二医院，嘉兴 314001，浙江

收稿日期:2018-12-27 修回日期:2019-01-17 出版日期:2019-06-26 发布日期:2019-06-25
通讯作者: 官俏兵，女，本科，主任医师，研究方向：神经病学。 E-mail：qbguan@126.com
作者简介:韩晨阳，男，药师，硕士，研究方向：神经药理学。 E-mail：691513770@qq.com
基金资助:
浙江省自然科学基金项目（LYY19H280003）

α-synuclein activates NLRP3 inflammatory bodies to mediated nerve cell pyroptosis

HAN Chenyang, ZHANG Xiaoling, YANG Yi, GUO Li, GUAN Qiaobing

The Second Hospital of Jiaxing, Jiaxing 314001, Zhejiang, China

Received:2018-12-27 Revised:2019-01-17 Online:2019-06-26 Published:2019-06-25

摘要/Abstract

摘要：

目的:研究α-突触核蛋白（α-synuclein）对于神经细胞焦亡发生的影响和机制。方法:以小鼠海马神经元细胞HT22细胞株为对象，体外培养后用α-synuclein干预细胞，以CCK-8法检测细胞活力，确定IC50值。设置对照组（Con组）后，采用TUNEL染色法检测细胞焦亡水平，免疫荧光法观察Gasdermin-D-N（GSDMD-N）的表达，Western blot法检测细胞中NOD样受体热蛋白结构域相关蛋白3（NLRP3）、细胞凋亡相关斑点样蛋白（ASC）、Gasdermin-D（GSDMD）、GSDMD-N、Caspase-1的表达水平，酶联免疫吸附法检测培养基中IL-18、IL-1β的表达水平。采用Caspase-1抑制剂预处理HT22后，同样使用IC50值的α-synuclein干预细胞，同时设置α-synuclein组（单纯α-synuclein干预）和α-synuclein+ML132组，检测细胞焦亡的水平。结果:α-synuclein的IC50值为50 nmol/L。α-synuclein干预后细胞焦亡数目显著增高，TUNEL染色显示阳性细胞数目多于Con组，免疫荧光染色结果显示细胞中GSDMD-N的表达水平上调，细胞中NLRP3、ASC、GSDMD-N、Caspase-1的表达水平显著高于Con组，而GSDMD水平下调，同时细胞培养基中IL-18、IL-1β的水平上调。Caspase-1抑制剂处理后，50 nmol/L的α-synuclein干预后，细胞焦亡水平显著低于α-synuclein组，同时免疫荧光染色结果显示GSDMD-N的水平下调，细胞中NLRP3、ASC、GSDMD-N、Caspase-1的表达水平显著低于α-synuclein组，而GSDMD水平上调，培养基中IL-18和IL-1β的水平下调。结论:α-synuclein可以通过激活NLRP3炎性小体活化继发神经细胞焦亡的发生，在帕金森病的发生发展中具有一定的作用。

关键词: 细胞焦亡, α-突触核蛋白, 炎性小体, Gasdermin-D

Abstract:

AIM: To study the effect and mechanism of α-synuclein on nerve cell pyroptosis. METHODS: HT22 cell was cultured in vitro and interfered by α-synuclein. Cell viability was measured by CCK-8 method and IC50 value was determined. After setting up the control group (Con group), TUNEL staining was used to detect the pyroptosis level of cells, immunofluorescence was used to observe the expression of GSDMD-N, Western blot was used to detect the expression levels of NLRP3, ASC, GSDMD, GSDMD-N and Caspase-1 in cells, and enzyme-linked immunosorbent assay was used to detect the expression levels of IL-18 and IL-1β in culture medium. After HT22 was pretreated with Caspase-1 inhibitor, the cells were also treated with α-synuclein of IC50 value, and the α-synuclein group (only α-synuclein intervention) and α-synuclein+ML132 group were set up to detect the level of pyroptosis. RESULTS:The IC50 value of α-synuclein was 50 nmol/L. After the intervention of α-synuclein, the number of nerve cells increased significantly. TUNEL staining showed that the number of positive cells was more than that of Con group. Immunofluorescence staining showed that the expression level of GSDMD-N in cells was up-regulated. The expression levels of NLRP3, ASC, GSDMD-N and Caspase-1 in cells were significantly higher than those in Con group, while the level of GSDMD was down-regulated in cell culture medium. The levels of IL-18 and IL-1β were up-regulated. After treatment with Caspase-1 inhibitor, the pyroptotic level of 50 nmol/L α-synuclein was significantly lower than that of α-synuclein group. Immunofluorescence staining showed that the level of GSDMD-N was down-regulated. The expression levels of NLRP3, ASC, GSDMD-N and Caspase-1 in cells were significantly lower than those in α-synuclein group, while the level of GSDMD was up-regulated. The levels of IL-18 and IL-1β in culture medium were down regulation. CONCLUSION:α-synuclein may play a role in the development of Parkinson's disease by activating NLRP3 inflammatory bodies.

Key words: pyroptosis, α-synuclein, inflammatory bodies, GSDMD

中图分类号:

韩晨阳，张晓玲，杨毅，郭丽，官俏兵. α-突触核蛋白激活NLRP3炎性小体介导神经细胞焦亡的发生[J]. 中国临床药理学与治疗学, 2019, 24(6): 637-643.

HAN Chenyang, ZHANG Xiaoling, YANG Yi, GUO Li, GUAN Qiaobing. α-synuclein activates NLRP3 inflammatory bodies to mediated nerve cell pyroptosis[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2019, 24(6): 637-643.

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α-突触核蛋白激活NLRP3炎性小体介导神经细胞焦亡的发生

α-synuclein activates NLRP3 inflammatory bodies to mediated nerve cell pyroptosis

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