Effects Of Vasoactive Intestinal Peptide On IL-17 And IL-17R Expression In LPS-stimulated Lung And Its Mechanisms

Vasoactive intestinal peptide (VIP), a main transmitter secreted by non-cholinergic or non-adrenergic nerves in lung, which canregulate a variety of immune cell's proliferation, differentiation and biological functions. VIP can inhibit pulmonary alveolar macrophage and T cell's proliferation, decrease inflammatory mediators, promote injury epithelial cell's repair, scavenge oxygen free radicals and anti-apoptosis. Interleukin 17 (IL-17), a cytokine with a strong recruitment of polymorphonuclear cells, promoting the release of multiple inflammatory cytokines and inflammatory amplification, is a pro-inflammatory cytokine produced by Th17 Lymphocytes, a CD4+ T cell subset. IL-17 comprises six subtypes, IL-17A-F, among which IL-17A was firstly found and most important. IL-17 acts its biological effect mainly through combining with its receptor (IL-17R), which is identified with five subtypes (IL-17RA IL-17RD and SEF). Only IL-17RA and IL-17RC, while, can be combined with IL-17A. IL-17R was widely expressed in B lymphocytes, T lymphocytes, bone marrow mononuclear cells, fibroblasts and epithelial cells and other cells. tudies showed that the IL-17 in bronchoalveolar lavage fluid of patients with asthma and lung infection was significantly increased and there was abundant IL-17 in the airway of patients with cystic fibrosis; The inflammation and fibrosis significantly decreased in hypersensitivity pneumonitis mouse who without IL-17R. However, is the IL-17 mainly from Th17 or other cells in the lungs? Can pulmonary alveolar macrophages, an important cell in lung, secrete IL-17? How to change of the IL-17R in lung fibroblast? And the effect of VIP on expression regulation of the IL-17 and IL-17R in lung has not been reported.Objective:To observe the effect of VIP on the IL-17 and IL-17R expression in lung during LPS stress, and investigate the signal transduction pathway.Methods:1. Mice were intraperitoneal injected by LPS to establish ALI model. Tissue section of lung was stained with HE to observed the pathological changes; count and classify the white blood cell in BALF; ELISA assay was used to detect the IL-17 protein in BALF and lung tissue homogenate; RT-PCR was employed to determine the IL-17A mRNA and IL-17RA/C mRNA levels in lung tissue.2.The effect of VIP on IL-17 expression and secretion in LPS-induced macrophage:the IL-17 in culture supernatant of macrophage was detected by ELISA; use RT-PCR to detect the IL-17A mRNA expression in LPS-induced macrophage, and to observe the effect of PKC inhibitor H-7, PKA inhibitor H-89 on the regulatory role of VIP.3.The effect of VIP on IL-17R protein and IL-17RA/C mRNA expression in LPS-induced lung fibroblast:flow cytometry was used to determine the IL-17R level on the membrane of lung fibroblast; use RT-PCR to detect IL-17RA/C mRNA expression in fibroblast and observe the effect of H-7 and H-89 on the regulatory role of VIP.Results:1. The morphology and molecular changes in mouse lung tissue:(1) The lung tissue HE-staining showed that there, during ALI, were pulmonary alveoli interstice thickening, alveolar structure disorder, consolidation and a large number of inflammatory cells infiltration.(2) White blood cell count showed that the number of leukocytes in BALF of ALI group was significantly higher than that of control group, especially neutrophil was significantly increase (p<0.01).(3) ELISA results showed that IL-17 protein in BALF and lung homogenate of ALI group was significantly increased (P<0.05).(4) RT-PCR results showed that IL-17A mRNA expression in mice lung tissue of ALI group increased significantly (P<0.01); the IL-17RA mRNA expression did not changed, while IL-17RC mRNA was significantly increased (P<0.05).2. The effect of VIP on IL-17 expression and secretion of LPS-induced macrophage:(1) ELISA results showed that LPS at various time points (4,6,12,24 h) increased the IL-17 protein of macrophage, which reached the peak at 6 h (P<0.01); VIP partially reversed the up-regulation in the time-(4,6, 12,24 h) and dose-dependent (10-8mol/L-10-6 mol/L) (P<0.01) manners; and the effect of VIP (10-8 mol/L) on the secretion of IL-17 of macrophage could be partially blocked by H-7 and H-89 (P<0.01).(2)RT-PCR results showed that LPS at various time points (2,4,6,12, 24 h) increased the expression of IL-17A mRNA of macrophage, which reached the peak at 6 h (P<0.01); VIP could partially reverse the up-regulation in the time-(4,6,12,24 h) and dose-dependent (10-8 mol/L-10-6 mol/L) manners (P<0.01); and the effect of VIP (10-8 mol/L) on the expression of IL-17A mRNA of macrophage at 6 h point could be partially blocked by H-7 and H-89 (P<0.01).3. The effect of VIP on IL-17R and IL-17RA/C mRNA of LPS-induced lung fibroblast(1)Flow cytometry results showed that the expression of IL-17R was significantly increased of LPS-stimulated lung fibroblasts after 6 h, (P <0.05); VIP could reverse this effect (P<0.05) which could be partially blocked by H-7 and H-89 (P<0.05).(2) RT-PCR results showed that the expression of IL-17RA mRNA of lung fibroblast at all time points (2,4,6,12,24 h) after LPS-stimulating didn't significantly change; while the level of IL-17RC mRNA in LPS-stimulated lung fibroblasts after 6 h, was significantly increased (p P <0.05). VIP could partially reverse the up-regulation effect, which could be partially blocked by H-7 and H-89 (P<0.05).Conclusions:1. LPS increase dthe expression of IL-17, IL-17R albumen and IL-17RA/C mRNA in lung.2. Macrophage secreted IL-17 after LPS-stimulating, and VIP can down-regulate this effect, whose intracellular signal transduction pathway is relevant with PKC and PKA.3. LPS can up-regulate the expression of IL-17R and IL-17RC mRNA of lung fibroblasts, and VIP can reduce this effect, whose intracellular signal transduction pathway is relevant with PKC and PKA.