| The neuropeptide alpha-melanocyte-stimulating hormone (a-MSH) is a 13-amino acid-long peptide derived from the special proteolytic cleavage of proopiomelanocortin. The concentration of a-MSH is high in the adenohypophysis, skin and location of inflammation. a-MSH has been recognized as having actions of pyretolysis, inhibiting food intake, protecting the skin from UV radiation-induced damage, anti-inflammation and immunomodulation. The significant protection of a-MSH on the animals with experimental autoimmune disease, transplant rejection, experimental lethal endotoxemia and acute respiratory distress syndrome has been proved by a great deal of the researches. All these effects of a-MSH are miediated via melanocortin receptors (MCRs). The distribution of MCRs is broad in many kinds of tissues and cells including the immune organs and immune cells, such as monocytes/macrophages, neutrophils, B cells and endothelial cells.T lymphocytes play a key role in mediating specific cellular immune response and assisting specific humoral immune response. In vitro and vivo experiments, a-MSH takes beneficial immunomodulatory action in T cell-mediated inflammation, some autoimmune diseases and graftrejection. Therefor, we presume that a-MSH may have a direct action on T cells. The previous experimental results in our lab showed that a-MSH binding sites were detecable on Jurkat cells, which are immortal human T lymphocytes. And a-MSH could inhibit the proliferation of PHA-induced Jurkat cells. Here, we investigate whether a-MSH can suppress the proliferation of PHA-induced human peripheral blood T lymphocytes and make elementary study on the mechanism of it if there are.A marked proliferation suppression was observed in the PHA-stimulated Tlymphocytes treated by a-MSH, with maximal inhibition at 1×10-7 M a-MSH. To examinethe mechanism of the growth inhibition effect of a-MSH, cell cycle analysis was performed by FACS. The result showed that PHA-induced T lymphocytes were greatly arrested in Gl phase of cell cycle. The effect of a-MSH on the T lymphocyte apoptosis induced by PHA was also investigated. PI staining followed by FACS analysis was used to detect the cell apoptosis. The suppression of PHA-stimulated T lymphocyte apoptosis by a-MSH was observed. The progression of the cell cycle is regulated by a complex network involving cyclins, cyclin-dependent kinases, and cyclin-dependent kinases inhibitors. The expression of several cell cycle-related proteins including cyclin E and p27 was analyzed by Western blotting. We found that in a-MSH+PHA treated T lymphocytes, the expression of p27 was significantly increased and the expression of cyclin E was significantly decreased. Taken together, our results suggested that a-MSH could inhibit the growth of PHA stimulated T lymphocytes, possibly via the up-regulation or down-regulation of cell cycle-related protein expression, resulting in the Gl-phase arrest of cell cycle. Two-dimensional electrophoresis (2-DE) was used to compare the global protein patterns between PHA-treated T lymphocytes and a-MSH+PHA-treated T lymphocytes. Two differential expression protein spots were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The data obtained from peptide mass fingerprinting were used in protein database search. Two protein spots that differently express in a-MSH+PHA-treated T lymphocytes were identified as tropomyosin isoform and zinc finger protein 585A. These results came to a conclusion that the upregulation of tropomyosin isoform and zinc finger protein 585A may be involved in the suppression of the PHA-induced T lymphocytes proliferation by a-MSH. The detection of cytokine level in the supernatant by ELISA showed that a-MSH suppressed the secretion of IL-2 and TNF-q, while increased the secreation of IFN-y.In summary, a-MSH suppressed PHA-induced T lymphocytes proliferation, causing Gl-phase arrest of cell cycle. a-MSH up-regulated the expression of cyclin dependent kinases inhibitor p27, tropomyosin isoform and zinc finger protein 585A, while down-regulated the expression of cyclin E in PHA-induced T lymphocytes, which maycontribute to the inhibitoty effect of a-MSH on T lymphocyte proliferation. Otherwise, a-MSH could suppress T lymphocyte apoptosis induced by PHA. It suggests that a-MSH is able to sustain the stability of immune system through regulating the "excitement" or activation of T lymphocytes. These data are subservient for elucidating the immunomodulatory mechanisms of a-MSH and provid a new proof for the application research of a-MSH in the prevention and treatment of inflammatory impairment mediated by T lymphocytes.
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