H9N2禽流感病毒PB2蛋白627位点突变对小鼠肺脏RIP3、Slit2和Robo4表达和分布的影响

Effects of E627K Mutation in the PB2 Protein of H9N2 Avian Influenza Virus on the Expression and Distribution of RIP3,Slit2 and Robo4 in the Lungs of Mice

PB2蛋白627位点被证实是决定甲型流感病毒宿主范围和致病性的关键因子,为深入探究H9N2禽流感病毒(avian influenza virus,AIV)及其PB2蛋白627位点突变株对BALB/C小鼠肺脏RIP3、Slit2和Robo4基因表达和组织分布的影响,该研究使用H9N2 AIV(V_(K627))和该毒株PB2蛋白627位点突变株(rV_(K627E))感染BALB/C小鼠,分别在攻毒后第1、3、5、6 d采集肺组织,通过qRT⁃PCR和免疫组织化学技术对RIP3、Slit2和Robo4基因在小鼠肺组织中的特异性表达和分布进行研究。结果显示,攻毒组小鼠肺脏RIP3的表达量与对照组相比显著上升,同时VK627组RIP3的表达量显著高于rV_(K627E)组;Slit2和Robo4的表达量在攻毒后第1 d明显低于对照组,之后rVK627E组的表达量与对照组和VK627E组相比显著上升。免疫组织化学检测结果显示,RIP3蛋白主要分布在小鼠肺泡壁上皮细胞以及肺动脉血管内皮细胞,呈弱阳性表达,而攻毒组RIP3蛋白在气管粘膜下层细胞也有中至强阳性表达;Slit2蛋白在对照组和rVK627E组小鼠的肺泡上皮细胞以及气管粘膜上皮细胞中呈现中强阳性表达,而在VK627组中呈现弱阳性表达。Robo4蛋白的分布和表达情况与Slit2蛋白相似。综上所述,H9N2 AIVs感染小鼠后,会显著影响RIP3和Slit2/Robo4的表达和分布,且其表达和分布与H9N2 AIVs对小鼠的致病性密切相关;PB2蛋白627位点的突变在H9N2 AIVs影响RIP3、Slit2和Robo4表达和分布中起着重要作用。该研究结果将为进一步探究H9N2 AIVs及其点突变毒株介导宿主病理发生的分子机制和抗炎策略的选择提供基础数据。

The site 627 of PB2 protein has been confirmed to be a key factor in determining the host range and pathogenicity of influenza A virus(IAV).To further investigate the effects of E627K mutation in the PB2 protein of H9N2 Avian Influenza Virus(AIV)on the expression and distribution of RIP3,Slit2 and Robo4 in the lungs of BALB/C mice,in this study,BALB/C mice were infected with H9N2 AIV(VK627)and its amino acid substitution in PB2 residue 627 strain(rVK627E).At 1,3,5,and 6 days post⁃inoculation,the lungs of three euthanized mice in each group were collected,respectively.The specific expression and histological distribution of RIP3,Slit2 and Robo4 in lung tissue were detected by qRT⁃PCR and immunohistochemistry.The results showed that the expression of RIP3 in the lungs significantly increased in challenge group compared with the control group,meanwhile,the expression level of RIP3 in VK627 group was significantly higher than that in rVK627E group.These two H9N2 AIV strains inhibited the expression of Slit2 and Robo4 in lung at 1⁃day post infection.After that,the expression level of Slit2 and Robo4 increased dramatically in rVK627E group than VK627 infected group and the control.Immunohistochemical detection showed that the epithelial cells of alveolar wall and pulmonary arterial vascular endothelial cells exhibited mild positive staining for RIP3,and it was also moderately to strongly expressed in the submucosa cells of trachea of the challenge group.The alveolar epithelial cells and mucosal epithelial cells of trachea were medium to strong positive staining for Slit2 protein expression in the control group and rVK627E group,while there was relatively weak positive staining of the same cells in VK627 groups.For Robo4,the location of positive cells in the lung had consistency to Slit2.In summary,H9N2 AIVs infected mice significantly affected the expression and distribution of RIP3 and Slit2/Robo4,which were closely related to the pathogenicity of H9N2 AIVs in mice.Mutations in PB2 protein locus 627 play an important role in the influence of H9N2 AIVs on RIP3,Slit2 and Robo4 expression and distribution.This study will provide basic data for further exploration of the molecular mechanism of H9N2 AIVs and its point mutant strain⁃mediated host pathogenesis and the selection of anti⁃inflammatory strategies.