Alpha-MSH Inhibits Monocytes Adhesion To Vascular Endothelium

Atherosclerosis, one of the most common vascular diseases, involves a very complex pathological process with accumulation of modified lipids, inflamed endothelial cells and leukocytes in the arteries. Inflammation and its subsequent endothelial dysfunction have been reported to play a pivotal role in the initiation and progression of atherosclerosis. Increased circulating cytokines such as interleukin-1beta (IL-1B) and tumor necrosis factor alpha (TNF-a) are also associated with such cardiovascular events. TNF-a stimulates the expression of monocyte chemotactic protein-1(MCP-1), and adhesion molecules, including vascular adhesion molecule-1(VCAM-1), intercellular adhesion molecule-1(ICAM-1) and E-selectin. These chemokines and adhesion molecules play key roles in firm adhesion of monocytes to activated endothelial cells. Inhibiting the attachment of monocytes to endothelium is a potential therapeutic strategy for atherosclerosis treatment.The hormone a-melanocyte stimulating hormone (a-MSH) is an endogenous neuropeptide composed of thirteen amino acids which generated from a precursor hormone called proopiomelanocortin by post-translational processing. Multiple lines of evidence have shown that a-MSH has potent anti-inflammatory effects when administered systemically or locally. a-MSH and other melanocortins family members such as B-MSH, c-MSH and ACTH bind to melanocortin receptors (MC-Rs). MC-Rs belong to the superfamily of G-protein coupled receptors with seven transmembrane domains and bind the melanocortin peptides with differential affinity. Studies have shown that the anti-inflammatory effects of a-MSH in vitro are mediated mainly via engagement of melanocortin receptor1(MC-1R). Importantly, a-MSH has been reported to suppress the production of pro-inflammatory cytokines such as IL-1B, IL-6and TNF-a, as well as chemokines such as IL-8and interferon-c (IFN-c) upon treatment with a-MSH. However, whether a-MSH plays a role in regulating endothelial inflammation is still unknown. In this study, the effects of a-MSH on endothelial inflammation in HUVEC lines were investigated. It was found that a-MSH inhibits the expression of endothelial adhesion molecules and attenuates the adhesion of THP-1cells to the surface of endothelial cells.Objective:To study the a-MSH inhibits monocytes adhesion to vascular endothelium and endothelial inflammation which can provide new clues for mechanism of a-MSH treatment to atherosclerotic vascular disease.Methods:We used HUVECs and monocyte cell lines as the research objects following the cell culture medium in EBM-2and RPMI-1640. Using Qiazol technology to complete the extraction of RNA from cultured cells; and then select2ug RNA as a template for synthesis cDNA reverse transcription polymerase chain reaction (PCR); Real-time polymerase chain reaction with Step-One-Plus real-time fluorescent quantitative PCR system support using SYBR Green special quantitative PCR; Gene expression using standardized triphosphate glyceraldehyde△△ct method.HUVECs were randomly divided into two groups, adding a-MSH to experimental group, comparing with the control group, with TNF-a training induction, using0.2ml/L calcein red dot marked; Acquisition of images using video imaging system randomly for the statistical analysis of a section of the experimental field of vision. Each experiment field of adhesion molecules were standardized count compared with control group.HUVECs were fixed with4%paraformaldehyde at room temperature, the ice application through the effect of polyethylene-glycoloctyl-phenylether; which was standardized by goat serum blocking; using primary and secondary antibodies cultivation incubation at room temperature. Cells were fixed on the VECTASHIELD carrier medium containing DAPI, deconvolution microscopy techniques were applied, and three dimensional fluorescence signal images were recorded.HUVECs cell lysis buffer was used to cell lysis, and BCA protein assay was used for determination of protein concentration; Purification of protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electric transferred to Immobilon-P membrane; After blocking with TBS liquid for two hours at room temperature, the primary antibody and secondary antibody were incubated continuously. Molecularly imprinted by Immunocruz develop-ment finally.Results:First we study a-MSH for TNF-a mediated monocytes adhesion to vascular endothelial cells. HUVECs were used TNF-a stimulation after six hours, respectively, to add5μg/ml and10μg/ml a-MSH setting up two pretreatment group and control group without a-MSH. Then HUVECs by immune fluorescent tags monocytes were incubated with the surface. Comparing a-MSH pretreatment group with control group, the TNF-a mediated adhesion of monocytes to vascular endothelial cells decreased significantly; further comparing with two pretreatment groups,10ug/ml a-MSH pretreatment group weakened adhesion effect more apparent, showed that adhesion effect of attenuation and a-MSH dose correlation characteristic to some extent.Secondly, the mRNA level of vascular adhesion molecule-1(VCAM-1) and E-selectin were determined. Under the induction of TNF-a, VCAM-1secretion and E-selectin in mRNA level increased significantly; correspondingly, after a-MSH pretreatment group, VCAM-1and E-selectin in mRNA level decreased obviously. And, more importantly, protein imprinting method further confirmed in the protein level, VCAM-1and E-selectin expression is significantly reduced in a-MSH pretreatment group.Then through the NF-kB luciferase plasmid transfection for HUVECs, under the induction of TNF-a, NF-kB luciferase activity increased significantly. And the activity of this increase can be weakened by a-MSH. In addition, the immune dyeing results show that the TNF-a p65nuclear transfer induction can inhibit by a-MSH, which further confirmed the NF-kB inhibitory effect for a-MSH. When after transfection MC-1R. siRNA of endothelial cells, a-MSH inhibits monocytes adhesion to endothelium almost disappears. And more importantly, real-time PCR results show that when the MC-1R expression is inhibited, the a-MSH for VCAM-1and E-selectin inhibition also disappears. Luciferase report experiments further reveal the a-MSH will inhibit the activity of the NF-kB as MC-1R siRNA transfection.Conclusions:a-MSH inhibits TNF-a mediated monocytes adhesion of endothelial cells, VCAM-1and E-selectin in mRNA level significantly decreased, protein imprinting method shown in protein level, a-MSH can also inhibit VCAM-1and E-selectin expression. Immune dyeing results show that the induced by TNF-a p65nuclear transfer can be inhibited by a-MSH, which further confirmed by the NF-kB inhibitory effect for a-MSH. Inhibition of a-MSH on monocytes adhesion to endothelial cells is mainly through MC-1R.