Effect Of Calcitonin Gene-related Peptide On The Alternatively Activated Macrophages In Mouse Lung Tissue With Acute Lung Injury

Macrophages are the main effector cells of the immune system. Macrophages perform specific effects on the body by the micro-environment in which they lie. Activation of macrophages is divided into two types:the classically activated macrophages (M1) and the alternatively activated macrophages (M2). Alternative activation emerges in the late phase of inflammatory response; M2secretes a variety of anti-inflammatory and plerosis cytokines to facilitate the resolution of inflammation and tissue repair. In vitro, IL-4can induce alternatively activated macrophages; arginase-1and IL-10can be used as its molecular markers. Imbalance between different phenotypes of macrophages may exist in the formation and progression of many kinds of lung diseases. M2can effectively regulate the inflammatory response and accelerated the damage repair in the process of acute lung injury (ALI). Therefore, the research or development of endogenous and exogenous substances to promote macrophage alternatively activation, aiming to prevent the waterfall inflammatory response in ALI can be the effective strategies for its cure.Calcitonin gene-related peptide (CGRP) is the most abundant neuropeptide in the lung. Studies have shown that CGRP can regulate the immune function of macrophages mainly through regulation of its production of inflammation-mediated chemokines and cytokines. However, there has not been reported about the effect of CGRP on M2. Firstly, we observed the expression of CGRP in the lung tissue of ALI mice and LPS challenged mouse macrophages, then we explored the possible mechanisms of its effects on IL-4-induced mouse macrophage alternative activation.Object:To observe the changes of CGRP expression in the lung tissue of mice with ALI, to explore its possible mechanisms of CGRP on IL-4-induced mouse macrophage alternative activation.Methods:1. Intraperitoneal injection of LPS to copy ALI mouse models, RT-PCR was used to detect the relative expression of the different time points of CGRP mRNA in mouse lung.2. IL-4treated macrophages (RAW264.7(ATCC;TIB-7TM)) to induce M2and followed CGRP pretreatment, we used RT-PCR to detect the Arg-1and IL-10mRNA relative expression. Timeliness detection of Arg-1relative expression:Oh,8h,16h and24h after IL-4treatment, CGRP concentration was10-7M, pretreatment for1h. Dose-effect detection of Arg-1relative expression:IL-4treatment for24h, the concentration of CGRP was respectively used as10-8,5×10-8,10-7and 5×10-7M, pretreatment for1h.3. PKC signaling pathway inhibitor (H7), PKA signaling pathway inhibitor (H89) and calmodulin signaling pathway inhibitor (W7) pretreatment for0.5h, then the Arg-1mRNA relative expression was detected to find out its intracellular signal transduction mechanism.Results:1. CGRP mRNA expression in the mouse lung tissue with ALIRT-PCR analysis of CGRP mRNA relative expression in the lung show that:the expression of CGRP in mice lung tissue with ALI was significantly increased compared with the control group (P<0.05), which amounted to peak at6h while decreased at12h and24h.2. CGRP mRNA expression in mouse macrophages treated with LPSAfter LPS treatment for8h, RT-PCR was used to detect the CGRP mRNA expression of mouse macrophages. The results showed that, resting mouse macrophages had a basis expression of CGRP, LPS challenge could significantly increased its expression (P<0.05).3. Effect of CGRP on IL-4-induced mouse macrophage Arg-1and IL-10expression(1) Arg-1:RT-PCR analysis showed that:CGRP pretreatment had no effect on the expression of Arg-1mRNA in resting macrophages, but could promote the expression of Arg-1mRNA in IL-4-induced mouse macrophages in a time-dependent (0,8,16,24h) and dose-dependent manner (10-8,5×10-8,10-7,5×10-7M)(P<0.05).(2) IL-10:RT-PCR analysis showed that:resting mouse macrophages had a relatively low expression of IL-10mRNA while CGRP pretreatment could synergistically promote IL-4-induced IL-10mRNA expression (P <0.05).4. Intracellular signal transduction mechanism of CGRP on IL-4induced mouse macrophage Arg-1expressionRT-PCR results showed that,compared with normal group, CGRP could synergistically promote IL-4-induced Arg-1mRNA expression, but PKC signaling pathway inhibitor (H7), PKA signaling pathway inhibitor (H89) and calmodulin signaling pathway inhibitor (W7) pretreatment could inhibit the synergistic effect of CGRP (P<0.05). Hence, it was concluded that the synergistic effect of CGRP on IL-4-induced mouse macrophage Arg-1mRNA expression might be associated with PKC, PKA and calmodulin signaling pathway.Conclusion:1. CGRP mRNA expression increased in the mouse lung tissue with ALI.2. CGRP could promote IL-4-induced mouse macrophage alternative activation, while its intracellular signal transduction might be associated with PKC, PKA and calmodulin signaling pathway.