Effects Of Lung Regulatory Peptides On TNF-α And TGF-β₁ Expression Of LPS-induced Macrophages And Its Mechanisms

Background:Tumor necrosis factor-alpha(TNF-α) as a pro-inflammatory cytokines,could enhance the role of neutrophils, produced a strong stimulation of oxygen free radicals,which activated macrophages to release of inflammatory factors,thereby creating a vicious cycle.Transforming growth factor-beta(TGF-β₁) participated in pulmonary remodeling through inducing the proliferation and phenotypic alternation of fibroblast,encouraging the synthesis of collagen fibers and ECM.Alveolar macrophage-derived TNF-αand TGF-β₁ are closely related to the occurrence and development of ALI.Vasoactive intestinal peptides(VIP) and calcitonin gene-related peptide(CGRP) play a modulatory role in lung function.VIP has played a vital role in organism's physiological processes such as reducing the release of inflammation media,enhancing the wound repairing of airway epithelium, eliminating oxygen-derived free radicals,and CGRP can regulate the release of inflammatory mediators such as MMPs/TIMPs and IL-1.Therefore,we hypothesized that lung regulatory peptides(VIP, CGRP) may involve in the expression of TNF-αand TGF-β₁ in macrophage.Objective:To study aimed at determining whether lung regulatory peptides(VIP,CGRP) treatment affects TNF-αand TGF-β₁ secretion and gene expression in macrophage and to explore its possible signal transduction pathways.Methods:(1) The influence of VIP on the release of TNF-αand TGF-β₁ of LPS-induced macrophage:The secretory volume of TNF-αand TGF-β₁ of LPS-induced macrophage and the intracellular signaling pathway of VIP regulate were measured by immunocytochemistry and ELISA;the mRNA expression of TNF-αand TGF-β₁ were measured by RT-PCR.The effects of PKC inhibitor H-7,PKA inhibitor H-89,CaM antagonist W-7 and the MAPK inhibitor PD98059 on regulated-function of VIP by RT-PCR. (2) The influence of CGRP on the release of TNF-αand TGF-β₁ of LPS-induced macrophage:The secretory volume of TNF-αand TGF-β₁ of LPS-induced macrophage and the intracellular signaling pathway of CGRP regulate were measured by immunocytochemistry and the rnRNA expression of TNF-αand TGF-β₁ were measured by RT-PCR.Results:1.The influence of VIP on the release of TNF-αand TGF-β₁ of LPS-induced macrophage(1) The immunocytochemical results showed that,the brown intracytoplasmic TGF-β₁,and TNF-αparticles increase in LPS-stimulated macrophages and the brown granular cytoplasm of macrophages of VIP pretreatment group significantly less than LPS group.(2) ELISA test results showed that:the TGF-β₁,TNF-αof LPS -stimulated macrophages increased significantly(P＜0.01),VIP pre-tre atment may reverse the phenomenon(p＜0.05).The PKC and PKA inhibitor could block the function of VIP which regulate the TNF-αsecreted of LPS-stimulated macrophages;The PKA and CaM in hibitor could block the function of VIP which regulate the TGF-β₁, secreted of LPS-stimulated macrophages(P＜0.05).(3) RT-PCR test results show that:LPS stimulated TNF-αmRNA expression in a time-dependent(2h,4h,6h,12h,24h) manner an d VIP reverse the phenomenon in a concentration-dependent(10¹⁰mo 1/L-10^-6mol/L) manner(p＜0.01).The regulated effect of VIP with the TNF-αmRNA expression may be blocked by H-7,H-89(P＜0.05), indicating VIP could reduce the TNF-αmRNA expression of LPS-induced macrophage with PKA and PKC pathways.Compared with the control group,LPS stimulated TGF-β₁ mRNA expression in a time-dependent(2h,4h,6h,12h,24h) manner,and it was on the peak at the 6h and wasn't fall until 24h(P＜0.05 ),VIP in a co ncentration-dependent(10¹⁰mol/L-10^-6mol/L) manner(P＜0.01) increas e TGF-β₁ mRNA expression in 2h,4h(P＜0.05),while it can rev erse the effects in 6h,12h and 24h(P＜0.05).The effect may be blocked by H-89,W-7(P＜0.05)2.The influence of CGRP on the expression of TNF-αand T GF-β₁ of LPS-induced macrophage (1) The immunocytochemical results showed that,the brown g ranular cytoplasm of macrophages of CGRP pretreatment group sign ificantly more than LPS group.(2) RT-PCR test results show that:LPS stimulated TNF-αmR NA expression of macrophages,CGRP in a concentration-dependent(1 0^-10mol/L-10^-6mol/L) and time-dependent(2h,4h,6h,12h,24h) man ner enhance the effect(p＜0.05).The effect may be blocked by H-89,W-7 and PD98059(P＜0.05),it prompted that CGRP enhanc ed TNF-αmRNA expression of LPS-induced macrophage with PKA, CaM and MAPK pathways.Compared with the LPS group,CGRP could increase the TGF-β₁ mRNA expression of LPS-induced macr ophage in a concentration-dependent(10^-10mol/L-10^-6mol/L) and time -dependent(2h,4h,6h,12h,24h) manner(p＜0.05).The effect of CGRP may be diminished by H-7,H-89 and W-7(P＜0.05),indica ting that CGRP could amplify inflammatory response in the repairin g process of lung injury,and participate in the process of pulmon ary fibrosis through PKC,PKA and CaM pathways.Conclusions:1.After stimulated with LPS,TNF-αand TGF-β₁ secretion and expression of macrophage increased.2.VIP could inhibit the secretion and expression of TNF-αof LPS-induced macrophage through PKC and PKA pathways.3.VIP promote the expression of TGF-β₁ of LPS-induced macrophage at an early stage,inhibition of the expression of TGF-β₁ of LPS-induced macrophage in later period and its intracellular signal transduction pathways related to PKA,CaM.4.CGRP could up-regulate TNF-αexpression of LPS-induced macrophage through PKA,CaM and MAPK pathways;increase TGF-β₁ expression of LPS-induced macrophage with CaM,PKA and PKC pathways.