药学学报, 2017, 52(1): 80-85
引用本文:
崔鹤蓉, 李朋彦, 李雨萌, 王睿林, 何娟娟, 桑秀秀, 蔡光明, 牛明, 王伽伯, 柏兆方, 肖小河. 五味子甲素对NLRP3炎性小体活性的抑制作用及机制初步研究[J]. 药学学报, 2017, 52(1): 80-85.
CUI He-rong, LI Peng-yan, LI Yu-meng, WANG Rui-lin, HE Juan-juan, SANG Xiu-xiu, CAI Guang-ming, NIU Ming, WANG Jia-bo, BAI Zhao-fang, XIAO Xiao-he. nhibitory effect and mechanism of deoxyschizandrin on NLRP3 inflammasome[J]. Acta Pharmaceutica Sinica, 2017, 52(1): 80-85.

五味子甲素对NLRP3炎性小体活性的抑制作用及机制初步研究
崔鹤蓉1,2, 李朋彦1, 李雨萌3, 王睿林4, 何娟娟1,5, 桑秀秀2, 蔡光明1, 牛明1, 王伽伯1, 柏兆方1, 肖小河4
1. 解放军302医院全军中医药研究所, 北京 100039;
2. 承德医学院, 河北 承德 067000;
3. 秦皇岛市第一医院, 河北 秦皇岛 066000;
4. 解放军302医院中西医结合医学中心, 北京 100039;
5. 株洲千金药业股份有限公司, 湖南 株洲 412000
摘要:
探讨五味子甲素(deoxyschizandrin,schisandrin A)对小鼠骨髓来源巨噬细胞(BMDM)中NLRP3(NOD-like receptor family,pyrin domain containing 3)炎性小体活性的抑制作用及初步机制。CCK-8法检测细胞毒性,根据细胞存活率确定给药浓度。通过Western blot方法检测细胞培养上清中IL-1β和caspase-1的表达,分析不同浓度的五味子甲素(25、50、100和200 μmol·L-1)对尼日利亚菌素(nigericin,Nig)和三磷酸腺苷(adenosine triphosphate,ATP)诱导的NLRP3炎性小体活化的影响,检测细胞中pro-caspase-1、pro-IL-1β、ASC和NLRP3等蛋白的表达变化;免疫荧光技术分析NF-κB(p65)的入核情况。根据CCK-8法的结果,确定了五味子甲素的给药浓度范围为6.25~400 μmol·L-1。在25~200 μmol·L-1浓度内,五味子甲素能够抑制Nig和ATP引起的NLRP3炎症小体活化,进而抑制IL-1β的分泌。免疫荧光和Western blot实验结果显示,五味子甲素对NLRP3炎症小体活性的抑制作用不依赖于NF-κB通路活性及其诱导的NLRP3、ASC和pro-IL-1β等炎性小体蛋白的表达。综上,本实验首次证实,五味子甲素在一定浓度范围内通过阻断caspase-1活化,抑制caspase-1对pro-IL-1β的剪切活化,进而抑制NLRP3炎性小体的活性,减轻免疫炎症反应。本研究为五味子甲素防治NLRP3炎性小体相关的疾病提供了参考依据。
关键词:    五味子甲素      NLRP3炎性小体      炎症反应      caspase-1     
nhibitory effect and mechanism of deoxyschizandrin on NLRP3 inflammasome
CUI He-rong1,2, LI Peng-yan1, LI Yu-meng3, WANG Rui-lin4, HE Juan-juan1,5, SANG Xiu-xiu2, CAI Guang-ming1, NIU Ming1, WANG Jia-bo1, BAI Zhao-fang1, XIAO Xiao-he4
1. China Military Institute of Chinese Medicine, 302 Military Hospital, Beijing 100039, China;
2. Chengde Medical University, Chengde 067000, China;
3. First Hospital of Qinhuangdao, Qinhuangdao 066000, China;
4. Integrative Medicine Center, 302 Military Hospital, Beijing 100039, China;
5. Qianjin Pharmaceutical Co., LTD, Zhuzhou 412000, China
Abstract:
This study was conducted to investigate the inhibitory effect and the molecular mechanism of deoxyschizandrin on the activity of NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome. Bone marrow-derived macrophages were used to study the effects of deoxyschizandrin on inflammasome activation using inflammasome inducers (ATP and nigericin). Cytotoxic effect was evaluated with CCK-8. The expression of IL-1β, caspase-1 in the supernatant and the expression of pro-caspase-1, pro-IL-1 β, ASC, NLRP3 in cell was detected by Western blot for the inhibitory effect of deoxyschizandrin (25, 50, 100 and 200 μmol·L-1) on the activity of NLRP3 inflammasome. Immunofluorescence was applied to investigate NF-κB (p65) transportation to the nucleus. The results of CCK-8 showed that the optimum concentration of deoxyschizandrin was 6.25-400 μmol·L-1. Deoxyschizandrin (25, 50, 100, and 200 μmol·L-1) could inhibit the activation of NLRP3 inflammasome caused by nigericin and ATP, and inhibit the secretion of IL-1 β, which was associated with inhibiting the cleavage of pro-caspase-1. The results of immunofluorescence and Western blot also suggest that the inhibitory activity of deoxyschizandrin on NLRP3 inflammasome was not dependent on NF-κB pathway and protein expression of NLRP3, ASC, pro-caspase-1 and pro-IL-1 β mediated by NF-κB. Our results confirmed that deoxyschizandrin could suppress the cleavage of pro-caspase-1 and inhibit the activity of NLRP3 inflammasome at 25-200 μmol·L-1 to reduce the inflammation response.
Key words:    deoxyschizandrin    NLRP3 inflammasome    inflammatory response    caspase-1   
收稿日期: 2016-08-11
DOI: 10.16438/j.0513-4870.2016-0784
基金项目: 国家自然科学基金资助项目(81330090,81274026);国家自然科学基金面上项目(03772888).
通讯作者: 柏兆方,Tel/Fax:86-10-66933325,E-mail:baizf2008@126.com;肖小河,E-mail:pharmacy302@126.com
Email: baizf2008@126.com;pharmacy302@126.com
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