α-MSH对脂多糖诱导小鼠腹腔巨噬细胞CD14和TLR4 mRNA表达的影响

Effect of α-MSH on the expression of CD14 and TLR4 mRNA induced by LPS in mouse peritoneal macrophages

目的 :观察α -黑色素细胞刺激素 (α -MSH)对脂多糖 (LPS)诱导小鼠腹腔巨噬细胞CD14和TLR4mRNA表达的影响 ,探讨α-MSH拮抗LPS的作用机制。方法 :用半定量逆转录多聚酶链反应 (RT -PCR)的方法检测LPS诱导体外培养的小鼠腹腔巨噬细胞CD14和TLR4mRNA表达水平和给予α -MSH后对CD14和TLR4mRNA表达的影响。结果 :正常静息小鼠腹腔巨噬细胞只表达少量的CD14和TLR4mRNA ,给予LPS刺激后 6h ,两者表达明显强于正常对照 (P <0 . 0 1) ,并且其表达量随着LPS刺激时间的增加维持在高水平 ,2 4h达到峰值 ,在 4 8hCD14mRNA的表达降到正常水平 ,而TLR4mRNA的表达仍然维持在高水平。在LPS刺激的同时给予α -MSH ,CD14和TLR4mRNA的表达则明显低于LPS组 (P <0 .0 5 ) ,而且α-MSH这种效应与其使用浓度有关 ,0 .1nmol/Lα -MSH不影响LPS诱导的CD14和TLR4mRNA的表达 ,而当α -MSH的浓度达到 1、10、10 0nmol/L则能显著影响CD14和TLR4mRNA的表达(P <0 . 0 5 ) ,但各个浓度组之间的作用没有明显差别 (P >0 .0 5 )。结论 :α -MSH抗LPS的效应可能与其下调LPS信号转导通路关键受体CD14和TLR4mRNA的表达有关 ,从而干扰LPS跨膜信号转导 ,阻碍巨噬细胞活化。

AIM: To observe the effects of α-MSH on the expression of CD14 and TLR4 mRNA in mou se peritoneal macrophages induced by LPS and explore the mechanisms of α-MSH ag ainst LPS. METHODS: BALB/C mouse peritoneal macrophages were cultured in the presence of LPS or LPS plus α-MSH, and the expressions of CD14 and TLR4 mRNA were detected with the m ethod of reverse transcription polymerase chain reaction (RT-PCR). RESULTS: It was found that native murine macrophages o nly expressed a small amount of CD14 and TLR4 mRNA. Both CD14 and TLR4 mRNA expr ession to LPS in mouse macrophages increased significantly than those of contro l group at 6 h and maintained high level until 24 h when they reached to the pea k. Then the expression of CD14 mRNA backed to the normal gene expression baselin e, while TLR4 mRNA had excessive expression at 48 h. In presence of LPS and α- MSH, the expression of CD14 and TLR4 mRNA decreased remarkably than those of L PS group (P<0.05). Moreover, low concentration of α-MSH (0.1 nmol/L) had no inhibition on CD14 and TLR4 mRNA expression(P>0.05). Only when the dose s of α-MSH attained to 1, 10 or 100 nmol/L,α-MSH could affect LPS-stimulated e xpression of CD14 and TLR4 mRNA (P<0.05), however, the different effects of α-MSH among 1-100 nmol/L on the both gene expressions had not been observed ( P>0.05). CONCLUSION: These studies suggest that effects of α-MSH against LPS are associated with its suppressing the critical receptor (CD14 and TLR4) expression of the LPS signal transduction and inhibiting the activation of macrophages.