咖啡鞣酸对单纯疱疹病毒-1诱导单纯疱疹病毒感染过程中Toll样受体9信号通路的干预机制

The interference of chlorogenic acid on Toll-like receptor 9 signal pathway in herpes simplex virus-1 infected microglia

目的探讨咖啡鞣酸（CGA）对单纯疱疹病毒-1（（HSV-1）感染BV2小胶质细胞诱导单纯疱疹病毒性脑炎（HSE）细胞模型过程中Toll样受体9（TLR9）信号通路的干预机制。方法用HSV-1刺激的BV2细胞建立HSE的细胞模型，然后用不同浓度梯度（25、50、100 ng/ml）的CGA进行处理。通过噻唑蓝（MTT）法测定细胞存活率。分别通过反转录-聚合酶链反应（RT-PCR）和Western blot测定TLR9、骨髓分化因子88（Myd88） mRNA和蛋白的表达。通过酶联免疫吸附试验（ELISA）以及RT-PCR测定肿瘤坏死因子-α（TNF-α）的表达。结果CGA治疗后细胞存活率显著改善[模型组为：（43.0±8.7）%；CGA组分别为（57.0±8.4）%、（64.0±7.6）%、（84.0±4.2）%]，模型组TLR9、Myd88、TNF-α水平显著高于正常组（蛋白表达分别为：0.84±0.05比0.18±0.13，P＝0.000；0.89±0.03比0.76±0.11，P＝0.002；292.34±8.16比20.76±5.72，P＝0.000；mRNA相对表达量分别为：3.01±0.87，P＝0.000；5.79±0.72，P＝0.000；20.12±10.17，P＝0.000），CGA干预后，其水平显著低于模型组（P＝0.000）。结论在HSV-1感染BV2小胶质细胞诱导HSE细胞模型中，咖啡鞣酸可以通过抑制TLR9信号通路中TLR9、Myd88分子的表达，从而抑制促炎因子TNF-α的释放，减轻炎性反应。

Objective To investigate the interference of chlorogenic acid （CGA） on Toll-like receptor 9 （TLR9） signal pathway in herpes simplex virus （HSV）-1-induced responses in BV2 microglia.Methods The cellular model was established with BV2 cells stimulated by herpes simplex virus-1 （HSV-1） and then treated with CGA at different concentrations （25, 50, 100 ng/ml）. Cell viability was measured by the methyl thiazol tetrazolium （MTT） assay. The mRNA and protein expression of TLR9 and bone marrow differentiation factor 88 （Myd88） was detected by reverse transcriptase-polymerase chain reaction （RT-PCR）, and Western blotting respectively. The serum tumor necrosis factor-α （TNF-α） levels were determined by enzyme linked immunosorbent assay （ELISA） and RT-PCR.Results The cell survival rate was significantly improved after CGA treatment [model group： （43.0±8.7）%; CGA group： （57.0±8.4）%, （64.0±7.6）%, （84.0±4.2）%], and CGA inhibited the increases in TLR9, Myd88, TNF-α （comparison of protein expression： 0.84±0.05 vs. 0.18±0.13, P=0.000; 0.89±0.03 vs. 0.76±0.11, P=0.002; 292.34±8.16 vs. 20.76±5.72, P=0.000; relative expression of mRNA： 3.01±0.87, 5.79±0.72, 20.12±10.17, P=0.000） following HSV-1 challenge in BV2 cells.Conclusion In HSV-1 infected microglia cellular model, CGA inhibits the inflammatory reaction in herpes simplex virus encephalitis （HSE） via the suppression of TLR9, Myd88, TNF-α in TLR9 signal pathway.