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  

Dysfunction of cholesterol sensor SCAP promotes inflammasome activation by upregulating P2X7R in THP-1 macrophages

作　　者：伍莎莎[1] 欧阳南[1] 何泉[1] 周超[1] 

机构地区：[1]重庆医科大学附属第一医院心血管内科,重庆400016

出　　处：《第三军医大学学报》2017年第13期1327-1332,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81500341)~~

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(100μmol/L)处理THP-1源性巨噬细胞,将细胞分为:(1)对照组、(2)ATP处理组、(3)A438079处理组、(4)ATP+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。RTPCR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。(2)P2X7R激动剂ATP与抑制剂A438079对THP-1源性巨噬细胞SCAP及P2X7R m RNA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-1βm RNA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。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 wi

关 键 词：胆固醇调节原件结合蛋白裂解激活蛋白 IL-1Β 炎症 P2X7R NLRP3炎症小体 

分 类 号：R392.1[医药卫生—免疫学] R392.32[医药卫生—基础医学]