[1]伍莎莎,欧阳南,何泉,等.胆固醇敏感器SCAP功能失调通过上调P2X7受体表达促进THP-1源性巨噬细胞炎症小体活化[J].第三军医大学学报,2017,39(13):1327-1332.
 WU Shasha,OUYANG Nan,HE Quan,et al.Dysfunction of cholesterol sensor SCAP promotes inflammasome activation by up-regulating P2X7R in THP-1 macrophages[J].J Third Mil Med Univ,2017,39(13):1327-1332.
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胆固醇敏感器SCAP功能失调通过上调P2X7受体表达促进THP-1源性巨噬细胞炎症小体活化(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
39卷
期数:
2017年第13期
页码:
1327-1332
栏目:
基础医学
出版日期:
2017-07-15

文章信息/Info

Title:
Dysfunction of cholesterol sensor SCAP promotes inflammasome activation by up-regulating P2X7R in THP-1 macrophages
作者:
伍莎莎欧阳南何泉周超
重庆医科大学附属第一医院:心血管内科,肾脏内科
Author(s):
WU Shasha OUYANG Nan HE Quan ZHOU Chao

Department of Cardiology, Department of Nephrology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China

关键词:
胆固醇调节原件结合蛋白裂解激活蛋白IL-1&beta炎症P2X7RNLRP3炎症小体
Keywords:
sterol regulatory element binding proteins cleavage-activating protein IL-1&beta inflammation P2X7R NLRP3 inflammasome
分类号:
R392.12; R392.32; R394.3
文献标志码:
A
摘要:

目的         探讨胆固醇调节原件结合蛋白裂解激活蛋白(sterol regulatory element binding proteins cleavage-activating protein,SCAP)功能失调促进THP-1源性巨噬细胞炎症小体活化及IL-1β成熟的分子机制。方法       采用基因转染方法在THP-1源性巨噬细胞中过表达SCAP,结合使用P2X7受体(P2X7 receptor, P2X7R)激动剂ATP(5 mmol/L)或抑制剂A438079 (100 μmol/L)处理THP-1源性巨噬细胞,将细胞分为:①对照组、②ATP处理组、③A438079处理组、④ATP+A438079组、⑤过表达SCAP组、⑥过表达SCAP+ATP组、⑦过表达SCAP+A438079组、⑧过表达SCAP+ATP+A438079组。RTPCR检测过表达SCAP以及激动或抑制P2X7R对P2X7R、pro-IL-1β、NLRP3、pro-caspase-1基因表达水平的影响。流式细胞术检测细胞表面P2X7R的表达。Western blot检测SCAP、pro-IL-1β、NLRP3、caspase-1(p20)以及培养上清中IL-1β的蛋白水平。同一实验在细胞水平重复4次。结果        ①过表达SCAP组与对照组比较,其pro-IL-1β、P2X7R基因表达水平显著升高(P<0.01),细胞表面P2X7R表达明显上调。②P2X7R激动剂ATP与抑制剂A438079对THP-1源性巨噬细胞SCAP及P2X7R mRNA表达无影响(P>0.05)。P2X7R激动剂ATP能够显著促进THP-1源性巨噬细胞pro-IL-1β mRNA表达(P<0.05),在过表达SCAP基础上激动P2X7R能够进一步激活pro-IL-1β基因转录(P<0.01),同时显著促进细胞内proIL-1β剪切成熟并向细胞外分泌(P<0.01),抑制P2X7R活性并不影响过表达SCAP致pro-IL-1β表达上调的作用,但将减少上清中成熟IL-1β的含量。   过表达SCAP并不影响NLRP3及caspase-1表达(P>0.05),但在过表达SCAP基础上充分激动P2X7R将显著上调NLRP3及caspase-1基因及蛋白水平(P<0.01),上述变化能够被P2X7R抑制剂A438079抵消。结论        胆固醇敏感器SCAP功能失调能够促进THP-1源性巨噬细胞内pro-IL-1β表达,同时通过上调细胞表面P2X7R表达激活NLRP3炎症小体,促进pro-IL-1β成熟及其向细胞外分泌。

Abstract:

Objective        To investigate the molecular mechanism of inflammasome activation and IL-1β maturation promoted by sterol regulatory element binding proteins cleavage-activating protein (SCAP) dysfunction in THP-1 macrophages. Methods        Full length SCAP over-expression plasmid pCMV-scap was transfected into THP-1 macrophages. THP-1 macrophages were treated with P2X7 receptor (P2X7R) agonist ATP (5 mmol/L) or antagonist A438079 (100μmol/L). Thus, the THP-1 macrophages were divided into control group, ATP group, A438079 group, SCAP overexpression group, SCAP over-expression plus ATP group, SCAP over-expression plus A438079 group, and SCAP over-expression plus both ATP and A438079 group. The mRNA expression levels of P2X7R, pro-IL-1β, NLRP3 and pro-caspase-1 were investigated by real time-PCR. The expression of P2X7R on the cell surface was detected by flow cytometry. The protein expression and the mature IL-1β level in the supernatants were estimated by Western blotting. The experiments were repeated for 4 times at the cellular level. Results        ① Over-expression of SCAP significantly increased the expression of pro-IL-1β and P2X7R at mRNA level (P<0.01),and enhanced the P2X7R protein expression on the cell surface. ② The agonist ATP or the antagonist A438079 of P2X7R did not affect the mRNA expression levels of SCAP and P2X7R in THP1 macrophages (P>0.05). The agonist ATP could promote the mRNA level of pro-IL-1β (P<0.05), which would be further enhanced when combined with overexpression of SCAP (P<0.05), and the combination also improved the cleavage, maturation and secretion of intracellular pro-IL-1β (P<0.01), while suppressing the activity of P2X7R by A438079 had no effect on the up-regulation of pro-IL-1β induced by SCAP over-expression, but decreased the production of mature IL-1β in the supernatant. ③ Though over-expression of SCAP showed no effect on the expression of NLRP3 and caspase-1 at mRNA and protein levels (P>0.05), it combined with P2X7R agonist ATP up-regulated the expression of the 2 molecules dramatically (P<0.01). The up-regulating effects of SCAP over-expression on NLRP3 and caspase-1 above were reconciled by the treatment with P2X7R antagonist A438079. Conclusion         The dysfunction of cholesterol sensor SCAP increases pro-IL-1β production, promotes NLRP3 inflammasome activation via up-regulating P2X7R, and then facilitates the maturation and secretion of pro-IL-1β in THP-1 macrophages.
 

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更新日期/Last Update: 2017-07-11