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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10 μg 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、MAP2K2、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10 μg HPS-LOS刺激细胞6、12和24 h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10 μg HPS-LOS刺激细胞6和12 h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、MAP2K2、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 investigate the role of rfaE gene in the transcriptional expression and MAPKs / NF-|ÊB signaling pathway of porcine alveolar macrophages (PAMs) signaling pathway stimulated by Haemophilus parasuis (HPS) lipopolysaccharide (LPS). PAMs were isolated from HPS SC096, rfaE gene-deficient (ΔrfaE) and complementary strains (cΔrfaE) and stimulated with 5 and 10 μg HPS-LOS, ΔrfaE-LOS and cΔrfaE-LOS respectively. Cells were harvested at different time points, RNA and protein extraction. The extracted RNA was reverse transcribed into cDNA, and the mRNA transcription levels of TLR4, MD2, NF-κB, MAP2K2, ERK, P38 and JNK were detected by RT-PCR. The concentration of extracted protein was determined. The expression of NF-κB p65 / phospho-NF-κB p65, IκBα, ERK1 / 2, JNK and p38 / phospho-p38 protein were detected by Western blot. The results showed that the mRNA transcription levels of TLR4, MD2, MAP2K2, ERK, P38 and JNK were significantly increased at 6, 12 and 24 h after stimulation with 5 and 10 μg HPS-LOS compared with those of the above transcription factors stimulated by ΔfafaE-LOS mRNA level (P <0.05), but there was no significant difference in NF-κB mRNA transcription level. In addition, IκBα protein expression was significantly lower than that of ΔfafaE-LOS-stimulated cells (P <0.05) after stimulation with 5 and 10 μg HPS-LOS for 6 and 12 h, NF-κB p65 and p38 Phosphorylation level and ERK1 / 2 and JNK protein expression were significantly higher than those of ΔfafaE-LOS phosphorylation of NF-κB p65 and p38 phosphorylation and ERK1 / 2 and JNK protein expression (P & lt; 0.05). At the same time, the mRNA transcription of TLR4, MD2, MAP2K2, ERK, P38 and JNK and the phosphorylation of NF-κB p65 and p38 and the levels of IκBα, ERK1 / 2 and JNK in PAMs stimulated by cΔrfaE-LOS were restored to HPS-LOS . The above test results confirmed that in the HPS-LOS-induced inflammatory response, the lack of rfaE gene by blocking the MAPKs / NF-κB signaling pathway to reduce the inflammatory response.