LPS通过上调Toll样受体2和4表达增强IFN-α抗病毒效应的研究

Enhancement of LPS on the antiviral effect of IFN-α via increasing the expression of TLR2 and TLR4

目的探讨细菌脂多糖(LPS)对Toll样受体2和4(TLR2和TLR4)表达的影响及其在α-干扰素(IFN-α)抗病毒效应中可能发挥的作用及相关机制。方法以1 000 IU/ml IFN-α和5 mg/L LPS单独及联合作用于HepG2细胞24 h后,运用RT-PCR和Western blot法分析作用前后细胞表面TLR2、TLR4和IFN-αJAK-STAT途径分子STAT1、STAT2、IRF9表达情况,并进一步分析IFN-α诱导的MyD88、MxA、2'-5'寡腺苷合成酶(2'-5'OAS)、双链RNA依赖性蛋白激酶(PKR)抗病毒蛋白的表达。结果 RT-PCR结果显示,IFN-α作用于HepG2细胞24 h后,TLR2、TLR4和STAT1、STAT2、IRF9信号分子及MyD88、抗黏病毒A蛋白(MxA)、2'-5'寡腺苷合成酶(2'-5'OAS)、PKR抗病毒蛋白的表达上升,而单独LPS处理细胞24 h后,除TLR2、TLR4 mRNA显著升高外(P<0.05),其他分子的表达均无显著变化;联合处理组较IFN-α处理组除MyD88和MxA mRNA水平无显著变化,其他分子的表达均显著升高(P<0.05)。Western blot结果显示,与IFN-α处理组相比,联合处理组STAT1、STAT2蛋白表达显著增加(P<0.05),而MyD88蛋白表达无显著变化。结论LPS可能通过上调细胞表面TLR2、TLR4的表达而发挥增强IFN-α诱导的抗病毒蛋白的表达水平,从而参与IFN-α抗病毒效应。

Objective To analyse lipopolysaccharide （LPS） on the expression of Toll-like receptor 2 and 4 （TLR2, TLR4） and to explore its function and mechanism in the antiviral effects of interferon-α （IFN-α）. Methods HepG2 cells were treated with 1 000 IU/ml IFN-α, 5 mg/L LPS, and LPS combined with IFN-α for 24 hours, and the mRNA levels of TLRs, STAT1, STAT2, IRF9, MyD88 and MxA were assessed by RT-PCR. Meanwhile, the expression of MyD88 and STAT1, STAT2 was detected by Western blot. Results The mRNA levels of TLR2, TLR4, STAT1, MyD88 and MxA in the IFN-α group were significantly higher than those of the control group （P 〈 0. 05 ）. The expression of other moleculars in the LPS group was similar to the control group except TLR2 and TLR4. There were no significant difference of MyD88 and MxA between the combination group and the IFN-α group, however the mRNA levels of STAT1, STAT2, IRF9, 2＇ -5＇ OAS and ds-RNA-dependent protein kinase R （PKR） moleculars were increased significantly in the combination group （ P 〈 0. 05 ）. Western blot results indicated that, compared to the IFN-α group, STAT1, STAT2 protein was increased significantly （ P 〈 0.05 ） , while MyD88 protein was not increased in the combination group. Conclusion LPS can enhance the expression of TLR2 and TLR4 as well as the antiviral proteins induced by IFN-α, suggesting that LPS may participate in the antiviral effect of IFN-α via TLR2 and TLR4.