The Study Of Calcitonin Gene Related Peptide On The Activation Of Human Vascular Endothelial Cells And Regulation Of The Secretion Of Chemokines

Objective:Psoriasis is a chronic inflammatory and autoimmune skin disease with a high incidence and the pathogenesis of the disease is not quite clear. As an important predisposing factor, neuropsychiatric factor plays a crucial role in the occurrence and development of psoriasis. The present studies show that the calcitonin gene related peptide(CGRP) is a neuropeptide which is closely related to the skin immune cells in the neurotransmitter associated with skin. By studying the role of CGRP on the activation of endothelial cells and the secretion of chemokines and the regulation of signaling pathways, we further reveal the role of the abnormal immune regulation in the pathogenesis of psoriasis.Materials and methods1?Take fresh umbilical cord and human umbilical vein endothelial cells(HUVEC)were cultured by enzyme digestion method in vitro, using M200 to culture it and passage 3-4 generations. Add different concentrations of CGRP(10-7mol/L ?10-8mol/L?10-9mo L) to stimulate the endothelial cells. Stain on activated endothelial cells with PE labeled CD62 E monoclonal antibody respectively after the stimulation of 6h, 12 h and 24 h. Untreated group as negative control group while cells stimulated by TNF-? as positive control group. Respectively test the groups with flow cytometry on the plane and choose the optimal concentration and time(10-7mol/L, 12h).2?Culture HUVEC in vitro, stimulate endothelial cells with 10-7mol/L CGRP as stimulation group, stimulate the cells with MEK1/2 antagonist for 1h before the stimulation of 10-7mol/L CGRP for 12 h as the intervention group and the non stimulated endothelial cells as the control group. Apply the method of real-time fluorescence quantitative PCR(RQ-PCR) to detect the m RNA expression level of chemokine CCL17?CCL22 and CX3CL1 secreted by endothelial cells.3?Culture HUVEC in vitro, stimulate endothelial cells with 10-7mol/L CGRP for24 h as stimulation group, stimulate the cells with MEK1/2 antagonist for 1h before the stimulation of 10-7mol/L CGRP for 24 h as the intervention group and the non stimulated endothelial cells as the control group, apply the method of enzyme linked immunosorbent assay(ELISA) to detect the protein expression level of chemokine CCL17 and CCL22 in supernatant secreted by endothelial cells.4?Culture HUVEC in vitro and stimulate endothelial cells with 10-7mol/L CGRP, the normal cells as the control group, extract total protein of adherent cells from two groups and apply the method of Western-blot to detect the phosphorylation level of ERK1/2 protein.Result1?Detect the cell activation with the method of flow cytometry, compared with the negative control group, the activation percentage and fluorescence intensity of endothelial cell of the CGRP-stimulate-group were significantly increased and the difference was statistically significant. The optimum activation concentration and time is 10-7mol/L, 12 h.2?Detect the m RNA expression level of cytokine with the method of RT-PCR,compared with the blank control group, the m RNA expression levels of CCL17 and CCL22 of the CGRP-stimulate-group are significantly increased and the difference is statistically significant(P < 0.05),while the elevated m RNA levels of CCL17 and CCL22 in the-intervention-group are lower than the stimulated-group and the difference is statistically significant(P < 0.05); the m RNA expression level of CX3CL1 were increased compared with the control group, but the difference was not statistically significant(P > 0.05).3?Detect the secretion level in the supernatant protein with the method of ELISA,compared with the blank control group, the protein expression level of CCL17 and CCL22 of the CGRP-stimulate-group are significantly increased and the difference is statistically significant(P < 0.05),while the elevated levels of CCL17 and CCL22 in the-intervention-group are lower than the stimulated-group and the difference is statistically significant(P < 0.05).4?Detect the phosphorylation level of ERK1/2 protein with the method of Western-Blot, the level of the experimental group was higher than the control group and the difference was statistically significant(P<0.05).Conclusion1?CGRP can effectively stimulate endothelial cell to activate, and the optimal concentration and activation time point is 10-7mol/L, 12 h.2?Through the level of m RNA and protein, CGRP can effectively stimulate endothelial cell to activate and secrete the chemokine CCL17 and CCL22, and this effect can weaken by inhibiting the MEK1/2 signaling pathway.3?Stimulate endothelial cell with CGRP, the phosphorylation level of ERK1/2protein was increased in the activated endothelial cells.