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

发布时间：2018-06-10 05:36

本文选题：胆固醇调节原件结合蛋白裂解激活蛋白 + IL-β　；参考：《第三军医大学学报》2017年13期

【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(P0.01),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(P0.05)。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(P0.05),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(P0.01),同时显著促进细胞内pro-IL-1β剪切成熟并向细胞外分泌(P0.01),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(P0.05),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(P0.01),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 the dysfunction of cholesterol regulation original binding protein cleavage activator protein (regulatory element binding proteins cleavage-activating) to promote the activation of inflammatory corpuscles and the maturation of IL-1 尾 in THP-1 derived macrophages. Methods THP-1 derived macrophages were overexpressed by gene transfection and treated with P2X7 receptor P2X7 receptor P2X7R agonist (5 mmol / L) or inhibitor A438079 (100 渭 mol / L). The cells were divided into two groups: 1) the control group was divided into two groups: the control group was treated with A438079, and the control group was treated with A438079. The effect of overexpression of SCAP A438079 on the gene expression of P2X7Rpro-IL-1 尾 -NLRP3pro-caspase-1 in SCAP A438079 group was detected by RT PCR, and the expression of P2X7Rpro-IL-1 尾 -NLRP3pro-caspase-1 gene in SCAP A438079 group was detected by RT PCR. The expression of SCAP ATP A438079 was also detected by RT PCR. The effect of overexpression or inhibition of P2X7Rpro-IL-1 尾 -NLRP3pro-caspase-1 gene on the expression of P2X7Rpro-IL-1 尾 -NLRP3pro-caspase-1 gene was detected. Flow cytometry was used to detect the expression of P2X7R on the cell surface. Western blot was used to detect the protein level of IL-1 尾 in the culture supernatant. The same experiment was repeated 4 times at the cell level. Results 1) the overexpression of SCAP group was compared with that of control group. Its pro-IL-1 尾 -P2X7R gene expression level was significantly increased, and the expression of P2X7R was up-regulated on the surface of THP-1 macrophages. ATP and A438079 had no effect on the expression of SCAP and P2X7R mRNA in THP-1 derived macrophages. P0.05N. P2X7R agonist ATP could significantly promote THP-1 derived macrophages. The overexpression of p0.05R in pro-IL-1 尾 mRNA stimulated P2X7R on the basis of overexpression of SCAP. P2X7R could further activate pro-IL-1 尾 transcriptional gene P0.01a, and promote pro-IL-1 尾 shearing maturation and exocrine P0.01T significantly. Inhibition of P2X7R activity did not affect the up-regulation of pro-IL-1 尾 expression induced by overexpression of SCAP. However, the content of mature IL-1 尾 in the supernatant was decreased. Overexpression of NLRP3 and caspase-1 did not affect the expression of P0.05P, but the overexpression of P2X7R could significantly up-regulate the level of NLRP3 and caspase-1 gene and protein, which could be counteracted by P2X7R inhibitor A438079. Conclusion the dysfunction of cholesterol sensor SCAP can promote pro-IL-1 尾 expression in THP-1 derived macrophages, and activate NLRP3 inflammatory corpuscles by up-regulating P2X7R expression on the surface of THP-1 macrophages, thus promoting pro-IL-1 尾 maturation and exocrine secretion.
【作者单位】：重庆医科大学附属第一医院心血管内科;
【基金】：国家自然科学基金面上项目(81500341)~~
【分类号】：R363

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