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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 cholesterol-regulated elemental binding protein cleavage-activating protein (SCAP) dysfunction in activation of THP-1-derived macrophage inflammatory corpus striatum and maturation of IL-1β. Methods SCAP was overexpressed in THP-1-derived macrophages by gene transfection and treated with P2X7 receptor (P2X7R) agonist ATP (5 mmol / L) or inhibitor A438079 (100 μmol / L) (1) control group, (2) ATP treatment group, (3) A438079 treatment group, (4) ATP + A438079 group, (5) Overexpression of SCAP group, (6) Overexpression of SCAP + ATP group, (7) Overexpression of SCAP + A438079 group, (8) Overexpression of SCAP + ATP + A438079 group. RTPCR was used to detect the effect of overexpression of SCAP on P2X7R, pro-IL-1β, NLRP3 and pro-caspase-1 gene expression by agonizing or inhibiting P2X7R. Flow cytometry was used to detect the expression of P2X7R on the cell surface. Western blot was used to detect the protein levels of SCAP, pro-IL-1β, NLRP3, caspase-1 (p20) and IL-1β in the culture supernatant. The same experiment was repeated 4 times at the cellular level. Results (1) Compared with the control group, the expression of pro-IL-1β and P2X7R in SCAP group was significantly increased (P <0.01), and the expression of P2X7R on the cell surface was significantly increased. (2) The P2X7R agonist ATP and inhibitor A438079 had no effect on the expression of SCAP and P2X7R m RNA in THP-1-derived macrophages (P> 0.05). P2X7R agonist ATP can significantly promote the pro-IL-1βmRNA expression of THP-1-derived macrophages (P <0.05), while P2X7R can activate pro-IL-1β gene transcription further on the basis of overexpression of SCAP ), And significantly enhanced the pro-IL-1β cleavage and extracellular secretion (P <0.01). Inhibition of P2X7R activity did not affect the up-regulation of pro-IL-1β expression induced by overexpression of SCAP, but decreased the expression of pro-IL- In mature IL-1β content. Overexpression of SCAP did not affect the expression of NLRP3 and caspase-1 (P> 0.05). However, full activation of P2X7R over-expression of SCAP significantly up-regulated NLRP3 and caspase-1 gene and protein levels (P <0.01) P2X7R inhibitor A438079 offsets. Conclusion Cholesterol sensor SCAP dysfunction can promote the expression of pro-IL-1β in THP-1-derived macrophages and activate the NLRP3 inflammasome by up-regulating P2X7R expression on the cell surface to promote pro-IL-1β maturation and its secretion to extracellular matrix .