Calcitonin Gene-related Peptide Inhibits The Function Of Macrophage Through Inhibiting The Activation Of Inflammasome

Background:Calcitonin gene related peptide is a polypeptide with many different functions by neural secretion,many experimental studies have shown that calcitonin gene-related peptide plays an important role in the inflammatory reaction,plays a very important function of [1] in innate immunity.Calcitonin gene related peptide can act directly on inflammatory cells such as macrophages and dendritic cells,inhibit the release of inflammatory factors,thereby blocking the presentation of antigen,and play an anti-inflammatory mechanism [2].In the fracture healing process,calcitonin gene related peptide in the regulation of [4] into cell proliferation and differentiation of [3],osteoclast proliferation and vascular permeability and inflammatory reaction in hematoma during bone healing,also play a role,but the specific mechanism is unknown.Inflammasome is a kind of receptor in the innate immunity of body,reduced coenzyme II oxidase and ROS-NOD like receptor protein 3(nicotinamide adenine dinucleotide phosphate oxidases-reactive oxygen species-NOD like receptor protein 3,NLRP3)is one of the main expression of [5],eosinophil neutrophil in macrophages and activated NLRP3.That will make the aspartic acid proteolytic zymogens containing cysteine-1(pro-cysteinyl aspartate specific proteinase,Procaspase-1)split into active cysteine containing aspartate protease-1(cysteinyl aspartate specific proteinase,Caspase-1),and further promote the secretion of inflammatory cytokines,inflammatory reaction,resistance to pathogens [6,7].Activated NLRP3 activates Caspase-1 and induces the processing and release of its downstream molecule,IL-1 beta.The activation mechanism of NLRP3 is very complex,so CGRP can regulate the activation of NLRP3 and then participate in the inflammatory reaction,which needs further verification.In this study,we investigated the effects and mechanisms of CGRP on the secretion of macrophage inflammatory factors IL-1 beta and TNF-alpha by using different concentrations of CGRP to stimulate RAW264.7 macrophages in resting state and LPS activated state.Method:1 in vitro mouse macrophage RAW264.7,allowing it to grow in DMEM medium(containing 10% FBS,1 x 105U/L 100mg/L penicillin,streptomycin),37 oC,5%CO2 culture conditions,3-4d was once passaged at 1:3.2.in vitro cultured mouse macrophages were pretreated with DMEM medium containing different concentrations of CGRP and / or LPS(LPS50ng/m L,CGRP,10ng/mL,CGPR,30ng/mL,CGRP,100ng/mL,CGRP,30ng/mL+LPS50ng/mL).3.macrophages,IL-1 beta,TNF-alpha and NLRP3 m RNA levels were detected in CGRP and / or LPS treated mice using qRT-PCR amplification apparatus4 the use of mouse interleukin-1 beta(Interleukin,IL),tumor necrosis factor(Tumor necrosis,factor,TNF)-a quantitative enzyme-linked immunosorbent assay(Enzyme-linked immuno sorbent assay,ELISA)kit on mouse macrophage CGRP and / or LPS after treatment for detection.5.protein immunoblot assay was used to detect the levels of NLRP3,Caspase-1 and Procaspase-1 proteins in RAW264.7 cells treated with different concentrations of CGRP and / or LPS.Result:1.cell cultures: under microscope,macrophages were round or oval,adherent to growth,and in good condition.2 in vitro mouse macrophage culture medium CGRP and / or LPS2 in mouse macrophages with different concentrations(LPS50ng/m L,CGRP 10ng/m L,CGPR 30ng/m L,CGRP 100ng/m L,CGRP 30ng/mL+LPS50ng/m L)medium culture conditions,3 days after the successful extraction of RNA and protein,preparation for the next step of the experiment.3 qRT-PCR mice were detected IL-1 beta,TNF-alpha,NLRP3 m RNA,LPS results indicated that CGRP activated macrophages,IL-1 beta,TNF-alpha m RNA levels than the control group,were significantly decreased(P < 0.05),and a concentration dependent.4 mice of IL-1 beta,TNF-alpha ELISA kit.The results suggest that CGRP(10ng/m L,30ng/mL,100ng/mL)after the treatment,IL-1 beta and TNF-alpha mRNA levels compared with the control group,decreased significantly(P < 0.05),and a concentration dependent.5.protein blot analysis of different concentrations of CGRP and / or LPS treatment of mouse RAW264.7 cells,NLRP3,Caspase-1 protein expression levels and mRNA consistent,and Procaspase-1 protein expression compared with the control group did not change significantly.Conclusions:1.,this study suggests that CGRP inhibits macrophage inflammatory activation and its mechanism may be related to the inhibition of NLRP3 expression and activity by CGRP;2.LPS can stimulate macrophages and increase the secretion of inflammatory factors;3.CGRP can inhibit the activity of NLRP3 and Caspase-1 in macrophage RAW264.7,and decrease the secretion of IL-1 beta and TNF-alpha,so as to regulate the inflammatory reaction.