The Effects Of α-MSH On The Expression Of CD14, TLR4 And SOCS-3 MRNA Induced By LPS In Mouse Peritoneal Macrophages

AIM: CD14 and Toll-like receptor 4 (TLR4) play important roles in the LPS signal transductions. It is reported that SOCS is a negative regulator of the LPS signal cascades, a -melanocyte-stimulating hornone( a -MSH) reprents a feature of anti-LPS, however, the mechanisms of a -MSH against LPS are incompletely characterized. Therefore, our studies focus attention on the effects of a -MSH on the expressions of CD14,TLR4 and SOCS-3 mRNA and the production of NO in mouse peritoneal macrophages induced by LPS.METHODS: BALB/c mouse peritoneal macrophages were cultured in the presence of LPS or LPS plus a -MSH , and the expressions of CD 14, TLR4 and SOCS-3 mRNA were detected with the method of reverse transcription polymerase chain reaction (RT-PCR). At the same time, the concentration of NO in the supernatant of cultured cells was measured by Griess reagent.RESULTS: It was found that the unstimulated murine macrophages only produced a small quantity of NO. LPS could strongly stimulate macrophages to release NO (P<0.01), and a -MSH could abolish NO production in response to LPS in mouse macrophages(P<0.01). On the other hand, native murine macrophages only expressed a small amount of CD14 and TLR4 mRNA. Both CD14 and TLR4 mRNA over-expression to LPS in mouse macrophages were shown at 6h and maintained high level until 24h when they reached to the peak. Then the expression of CD 14 mRNA backed to the normal gene expression baseline, while TLR4 mRNA had excessive expression at 48h. In presence of LPS, a -MSH could remarkably decrease the expressions of CD 14 and TLR4 mRNA (P<0.05). Moreover, too low concentration of a -MSH (0.1nmol/L) had no inhibition on CD14 and TLR4 mRNA expressions(P>0.05). Only when the dosage of a -MSH attained to 1, 10 or 100nmol/L, a -MSH could inhibit LPS-stimulated expressions of CD14 and TLR4 mRNA(P<0.05), however, the different effects of a -MSH among 1-100nmol/Lon the both gene expressions had not been observed (P>0.05). Meanwhile, trace of SOCS-3 mRNA could be sensed in macrophages without any stimulation. Culturedmacrophages with LPS for 3h, SOCS-3 mRNA expression was significantly elevated (P<0.01). When macrophages were cultured with a -MSH and LPS, the expression of SOCS-3 mRNA was markedly reduced (P<0.01 ) . a -MSH alone was unable to affect the realese of NO or CD14, TLR4 and SOCS-3 gene expressions in macrophages (P>0.05).CONCLUSIONS: These studies suggested that effects of a -MSH against LPS were associated with its function that suppressed the critical receptor expressions of the LPS signal trusductions. During the inflammation produced by LPS, SOCS-3 might be induced through TLR4 and involved in the negative regulation of LPS responses, a -MSH could dramatically depress the expressions of CD14 and TLR4 mRNA, which down-regulated the SOCS-3 mRNA expression and implied that SOCS might not be involved in the anti-LPS mechanism of a -MSH. It proposed that other pathways might be concerned with the mechanisms of a -MSH against LPS. All of our results help to further investigate the immunoloregulation of a -MSH.