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目的探讨降钙素基因相关肽(CGRP)抑制巨噬细胞炎性反应的机制。方法使用不同浓度CGRP处理LPS活化/未活化的小鼠巨噬细胞(RAW264.7),实时荧光定量PCR(qRT-PCR)检测巨噬细胞内促炎细胞因子白介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)和还原型辅酶Ⅱ氧化酶-活性氧簇-NOD样受体蛋白3(NLRP3)mRNA表达水平;酶联免疫吸附法(ELISA)定量检测细胞上清分泌型IL-1β和TNF-α蛋白表达量;蛋白免疫印迹法(Western blot)检测细胞内NLRP3蛋白表达水平。结果 CGRP显著降低LPS活化/未活化RAW264.7细胞内炎性因子IL-1β、TNF-α、NLRP3 m RNA水平与蛋白水平,同时,NLRP3下游调节分子Caspase-1的蛋白水平被显著下调。在mRNA水平,NLRP3随CGRP剂量变化而变化的趋势与IL-1β、TNF-α非常相似。结论CGRP抑制巨噬细胞炎性激活,其机制可能与CGRP抑制NLRP3表达与活性有关。
Objective To investigate the mechanism of calcitonin gene-related peptide (CGRP) inhibiting macrophage inflammatory response. Methods Macrophages (RAW264.7) of LPS-activated / non-activated mice were treated with different concentrations of CGRP, and the levels of pro-inflammatory cytokines interleukin-1β (IL-1β) in macrophages were detected by qRT- (TNF-α) and reduced coenzyme Ⅱ oxidase-reactive oxygen species-NOD-like receptor protein 3 (NLRP3) mRNA expression levels were detected by enzyme-linked immunosorbent assay (ELISA) IL-1βand TNF-αprotein expression levels were detected by Western blot. Results CGRP significantly decreased the levels of IL-1β, TNF-α, NLRP3 mRNA and protein in LPS-activated / non-activated RAW264.7 cells. Meanwhile, the protein level of Caspase-1, a downstream regulator of NLRP3, was significantly down-regulated. At the mRNA level, the tendency of NLRP3 to change with the dose of CGRP is very similar to that of IL-1β and TNF-α. Conclusion CGRP inhibits the inflammatory activation of macrophages, which may be related to the inhibition of NLRP3 expression and activity by CGRP.