RIP3在H1N1流感病毒感染中发挥促炎症病理作用

Rip3 plays a role in the inflammatory responses during H1N1 influenza virus infection

目的研究调控细胞程序性坏死(necroptosis)的关键分子——受体相互作用蛋白激酶3(receptor-interacting protein 3, RIP3)在甲型流感病毒H1N1 PR8感染中的作用。方法用5.25×10~3半数组织细胞感染剂量(50%tissue culture infective dose, TCID_(50))的流感病毒H1N1 PR8通过滴鼻方式感染RIP3敲除(RIP3^(-/-))小鼠和野生型(WT)C57BL/6小鼠,连续14 d每天称量小鼠体重,观察小鼠生存状态,并记录死亡情况。分别在感染后第3天(day post infection, d. p. i)和第7天处死解剖小鼠。取整个肺称重,左叶肺用4%多聚甲醛固定后进行HE染色,检测感染后肺组织的病理变化;通过实时荧光定量PCR(qRT-PCR)检测肺组织病毒载量;流式微珠阵列术(CBA)检测肺匀浆上清中部分炎症相关细胞因子。结果 5.25×10^(3 )TCID_(50)流感病毒感染后,各组小鼠均出现一定程度临床症状:RIP3^(-/-)组小鼠感染后死亡率(50%)较WT组小鼠(87.5%)显著降低(P<0.05)。分别对比每天两组小鼠体重,发现从第3天开始,WT组小鼠的体重降低比率大于RIP3^(-/-)组,但两组小鼠体重总体改变趋势无统计学差异。病毒学方面,两组小鼠在相同时间点(3、7 d. p. i)病毒载量差异均无显著性。炎症应答方面:两组小鼠肺指数(肺重与体重比值)差异无显著性。病理方面:肉眼观察大体病理及HE病理切片显示:RIP3^(-/-)组小鼠肺组织损伤较轻,炎症浸润较少;小鼠肺部炎症细胞因子也较WT组相对减少。结论 RIP3敲除的条件下,流感病毒H1N1 PR8感染小鼠时因减弱了炎症应答导致肺部病理损伤减轻,提示RIP3可能在H1N1 PR8流感病毒感染中发挥促炎症病理作用。

Objective To investigate the role of RIP3(receptor-interacting protein3),which is a key molecule in regulating programmed cell necrosis,in H1N1influenza virus infection.Methods RIP3knockout(RIP3-/-)and wild-type(WT)mice were infected by influenza virus H1N1PR8at a dose of5.25×10^3TCID50(50%tissue culture infective dose).Changes of clinical signs,survival,and body weight of those mice were monitored daily for14consecutive days.Six mice from each group were sacrificed at3and7days post-infection(d.p.i.),from which whole lungs were harvested.Some of the lobes were fixed in4%paraformaldehyde for histopathological assessment and the rest were split and stored at-80℃for further analysis.Real-time quantitative PCR and cytometric bead array(CBA)were performed to detect viral loads in lungs and inflammatory cytokines in supernatants of lung homogenates.Results Both groups showed severe symptoms after the infection of PR8.The RIP3-/-mice with PR8infection showed a high survival rate(50%)compared with the control group(12.5%)(P<0.05).The body weight loss of WT was greater than that of RIP3mice from3d.p.i.,but there was no significant difference between these two groups.No significant differences in viral loads and lung weight to body weight ratio were also observed between the two groups at3and7d.p.i.Pathological changes in RIP3-/-mice were less severe than those in WT mice.CBA detection assay revealed that the levels of proinflammatory cytokines in the lungs of RIP3-/-mice were lower than those in WT mice.Conclusion RIP3plays a pathogenic role in mice infected with5.25×10^3TCID50of influenza virus H1N1PR8by promoting inflammatory responses.