降钙素相关基因肽在小鼠巨噬细胞炎症调控中的作用研究

Role of CGRP in regulating inflammatory response of murine macrophages

摘要:

目的 探讨降钙素相关基因肽（CGRP）在炎症调控中的作用，验证CGRP可通过活性氧簇-核苷酸结合低聚体结构域样受体3（ROS-NLRP3）信号通路抑制小鼠巨噬细胞炎症因子的分泌。方法 实验分为对照组、脂多糖（LPS）100?ng/ml组（LPS组）、CGRP 10?ng/ml组（CGRP 10组）、CGPR 30?ng/ml组（CGRP 30组）、CGRP 100?ng/ml组（CGRP 100组）及CGRP 30?ng/ml+LPS 100?ng/ml组（CGRP 30+LPS组）。作用12?h后，分别收集各组细胞的培养上清，酶联免疫吸附试验（ELISA）检测上清液中白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）的蛋白表达。同时收集各组细胞，实时荧光定量聚合酶链反应（qRT-PCR）检测炎症因子IL-1β、TNF-α和炎症复合体NLRP3 mRNA的表达水平。流式细胞术检测各组细胞内活性氧簇（ROS）水平，Western blotting检测ROS-NLRP3信号通路相关蛋白NLRP3、Caspase-1、IL-1β及TNF-α的蛋白表达。结果 与对照组比较，LPS组细胞中IL-1β、TNF-α及NLRP3 mRNA表达水平升高，培养上清液中IL-1β和TNF-α的蛋白表达水平升高，细胞内ROS水平升高，且细胞内NLRP3、Caspase-1、IL-1β及TNF-α的蛋白表达水平升高（P?<0.05）；与LPS组比较，CGRP 10组、CGRP 30组、CGRP 100组、CGRP 30+LPS组细胞中IL-1β、TNF-α及NLRP3 mRNA表达水平降低，培养上清液中IL-1β和TNF-α蛋白表达水平降低，细胞内ROS水平降低，且细胞内NLRP3、caspase-1、IL-1β及TNF-α蛋白表达水平也降低，所有指标中CGRP 30组降低最明显（P?<0.05）。结论 CGRP可降低巨噬细胞内ROS和NLRP3的表达，使炎症因子IL-1β和TNF-α的释放减少，CGRP可能在减弱局部炎症反应中发挥重要的调控作用。

Abstract:

Objective To investigate the role of calcitonin gene related peptide (CGRP) in inflammation regulation in mouse macrophages. Methods Mice were divided into control group, lipopolysaccharide (LPS) (100 ng/ml group, LPS group), CGRP (10?ng/ml group, CGRP 10 group), CGPR (30?ng/ml group, CGPR 30 group), CGRP (100?ng/ml, CGRP 100 group) and CGRP 30?ng/ml + LPS 100?ng/ml group (CGRP 30 + LPS group). After 12?h, cells and supernatants were collected. Expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay (ELISA). Expression levels of IL-1β, TNF-α and NLRP3 mRNA were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The levels of ROS in each group were detected by flow cytometry. The expression of ROS-NLRP3 signaling pathway associated protein NLRP3, Caspase-1, IL-1β and TNF-α were detected by Western blotting. Results Compared with the control group, LPS group experienced increased levels of IL-1β, TNF-α, NLRP3 mRNA, and ROS in cells, upregulated levels of IL-1β and TNF-α protein in the cell supernatant, and enhanced levels of NLRP3, caspase-1, IL-1β and TNF-α protein in cells, which were reversed with treatment of CGRP. Mice in CGRP30 group exerted the least levels of the mentioned markers. Conclusions CGRP inhibits the expressions of ROS and NLRP3 in macrophages, reducing the release of inflammatory cytokines such as IL-1β and TNF-α.