rfaE基因通过MAPKs/NF-κB信号通路对副猪嗜血杆菌LOS诱导猪肺泡巨噬细胞炎症反应中作用的研究

The Effect of rfaE Gene in Haemophilus parasuis Lipooligosaccharide Induced Inflammation via the MAPKs/NF-κB Signaling Pathways in Porcine Alveolar Macrophages

作者拟研究rfaE基因在副猪嗜血杆菌(Haemophilus parasuis,HPS)脂寡糖(LOS)刺激猪肺泡巨噬细胞(PAMs)信号通路分子的转录表达和MAPKs/NF-κB信号通路中的作用。提取HPS SC096株及其rfaE基因缺失株(ΔrfaE)和互补株(cΔrfaE)的LOS,分别用5和10μg HPS-LOS、ΔrfaE-LOS和cΔrfaE-LOS刺激PAMs,分别在不同时间点收集细胞,提取RNA和蛋白质。将提取的RNA反转录成cDNA,运用RT-PCR检测TLR4、MD2、NF-κB、MAP2 K2、ERK、P38和JNK的mRNA转录水平。测定提取蛋白质的浓度,利用Western blot方法检测NF-κB p65/phospho-NF-κB p65、IκBα、ERK1/2、JNK和p38/phospho-p38蛋白的表达量。结果表明用5和10μg HPS-LOS刺激细胞6、12和24h后,TLR4、MD2、MAP2K2、ERK、P38和JNK的mRNA转录水平均显著高于ΔrfaE-LOS刺激细胞后的以上转录因子的mRNA水平(P<0.05),但NF-κB的mRNA转录水平无显著差异。另外,用5和10μg HPS-LOS刺激细胞6和12h后,IκBα蛋白的表达量显著低于ΔrfaE-LOS刺激细胞后的IκBα蛋白的表达量(P<0.05),NF-κB p65和p38的磷酸化水平及ERK1/2和JNK蛋白的表达量显著高于ΔrfaE-LOS刺激细胞后NF-κB p65和p38的磷酸化水平及ERK1/2和JNK蛋白的表达量(P<0.05)。同时cΔrfaE-LOS刺激PAMs后TLR4、MD2、MAP2 K2、ERK、P38和JNK的mRNA转录水平以及NF-κB p65和p38的磷酸化水平和IκBα、ERK1/2和JNK蛋白水平能够恢复到HPS-LOS水平。以上试验结果证实在HPS-LOS诱导的炎症反应中,缺失rfaE基因后通过阻断MAPKs/NF-κB信号通路以减轻炎症反应。

The aim of this study was to study the role of rfaE gene in Haemophilus parasuis li- pooligosaccharide （LOS） which induced signal molecules mRNA transcription and MAPKs/ NF- kB signaling pathways in porcine alveolar macrophages （PAMs）. The LOS of H. parasuis SC096 strain, rfaE mutant and its complementation was extracted. The PAMs were stimulated with LOS （5 and 10 μg） from H. parasuis SC096, ArfaE mutant （ArfaE） and its complementation （cArfaE） for different time points. The RNA and protein was extracted from the collected cells. The extracted RNA was reversed into cDNA. The mRNA transcription of TLR4,MD2, NF-kB,MAP2K2 ,ERK,P38 and JNK were then detected by Real-time PCR. The protein of NF-nB p65/ Phospho-NF-~B p65,I^Ba,ERK,JNK and p38/ Phospho-p38 were detected by western blot. The results showed that the mRNA transcription of TLR4, MD2, MAP2K2, ERK, P38 and JNK in PAMs induced by 5 and 10 μg HPS-LOS for 6,12 and 24 h were up-regulated,and significantly higher than that in PAMs induced by 5 and 10/~g ArfaE-LOS （P〈0. 05）. The mRNA transcrip- tion of NF-kB in PAMs induced by 5 and 10 gg HPS-LOS for 6,12 and 24 h were higher than that in PAMs induced by 5 and 10μg ArfaE-LOS,but no significant difference was observed. The pro- tein of IkBa in PAMs induced by 5 and 10 /ag HPS-LOS for 6 and 12 h were significantly lower than that in PAMs induced by 5 and 10 μg ArfaE-LOS （P〈O. 05）. The p65 phosphorylation,p38 phosphorylation,ERK and JNK in PAMs induced by 5 and 10 μg HPS-LOS for 6 and 12 h were significantly higher than that in PAMs induced by 5 and 10 μg ArfaE-LOS （P〈O. 05）. At the same time, the signal molecules mRNA transcription level and the protein level in PAMs induced by cArfaE-LOS can restore to the level in PAMs induced by HPS-LOS. The date confirmed that the loss of rfaE gene could effectively reduced the inflammatory process in PAMs induced by HPS-LOS by blocking the MAPKs/ NF-kB signaling pathways.