Regulation Effect Of Alph α-Melanocyte Stimulating Hormone And Its Analogue On The Expression Of Tissue Factor And Tissue Factor Pathway Inhibitor

Objection: After separated and cultured, the mouse brain microvascular endothelial cells were used in vitro as the target cell of the research. The established mouse model of endotoxaemia were used to research and evaluate the immunomodulation effect ofα-melanocyte stimulating hormone(α-MSH) and its analogue on the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in mouse endotoxaemia and brain microvascular edotheial cells.Methods: Cells obtained by isolation and primary culture the mouse brain microvascular endothelial cells, which were used in this research in vitro. To compare and evaluate the effect ofα-MSH, NDP-MSH, peptide 39 (10-7mol/L) on the brain microvascular edotheial cells stimulated with LPS for different time, the supernant of culture and the cell were collected at the time of 6h and 8h, then the expression of TF and TFPI were analyzed with ELISA and RT-PCR. The model mouse of endotoxaemia was established by injection intraperitoneally with D-Gal (20mg) and LPS (1μg) in PBS. After administration withα-MSH, NDP-MSH, peptide 39 (2.5mg/kg) at different time, then different group of the model mouse were injected with LPS. After 6, 8 hours, the blood sample were taken from the orbit, and the tissue of lung, spleen, and kidney were collected after cervical dislocation. The expression of TF and TFPI in serum was assayed with ELISA, and the mRNA from different tissues was analyzed with RT-PCR.Results: In vitro, the results of research shows that allα-MSH, NDP-MSH and peptide 39 (10-7mol/L) can reduce inhibit the TF production in the mouse brain microvascular endothelial cells in primary culture(p<0.01), and peptide 39 shows higher degree of inhibition effect than the others. All the three reagents show no effects of up-regulation in the production of TFPI. The results in vivo shows that all the three peptides can significantly down-regulate the expression of TF (p﹤0.01), and no significance difference were found between group 6h and 8h after statistic analysis. All the three peptides show effect of up-regulation in TFPI expression(p<0.05). Especially the group 6h (blood taken after treatment for 6 hours) , group 2h (after 2 hours of stimulation with LPS ) and peptide 39 show higher up-regulation effect than other groups and all the three peptides ( p﹤0.05). While the blood collected after 8 hours of treatment, administration with LPS at the same time or after 1h show higher up-regulation effect on TFPI expression than others (p﹤0.05), and peptide 39 superior to other peptides. Conclusion: The results of our research showsα-MSH, NDP-MSH and peptide 39 can regulate the expression of TF and TFPI, which was not reported previously. The results provide strong evidence for further illuminate the mechanism of anti-inflammatory effects ofα-MSH, NDP-MSH and peptide 39, and its clinical application in the future.