衰老细胞促进病毒感染诱导的炎症并抑制病毒复制

Senescent Cells Promote Viral Infection-induced Inflammation and Inhibit Viral Replication

目的:探究衰老对病毒感染和病毒复制的影响及机制。方法:利用阿霉素(DOX)诱导人非小细胞肺癌细胞(A549)衰老(衰老组),常规培养的A549细胞为对照组。利用带绿色荧光蛋白标签的水疱性口炎病毒(VSV)和单纯疱疹病毒(HSV)感染对照组和衰老组A549细胞。选取无特定病原体(SPF)级C57BL/6小鼠,8周龄为年轻组(n=3),15月龄以上为年老组(n=3),腹腔注射VSV病毒1×10~7PFU/只,24h后采集肝、脾、肺组织。应用实时荧光定量PCR(RT-PCR)检测干扰素-β(IFN-β)、白介素-6(IL-6)、白介素-1β(IL-1β)以及病毒复制;蛋白质印迹法(WB)检测核因子κB(NF-κB)信号通路关键蛋白核因子κB p65蛋白、磷酸化核因子κB p65(p-p65)蛋白表达量;荧光倒置显微镜观察VSV病毒和HSV病毒复制。结果:与对照组相比,衰老组细胞周期阻滞蛋白P16、P21显著升高;β-半乳糖苷酶染色阳性细胞也明显增多;两组细胞同时感染VSV病毒,衰老组细胞IFN-β显著降低,而IL-6、IL-1β显著升高;同时NF-κB通路关键蛋白p65、p-p65表达较对照组明显增加。两组细胞感染VSV及HSV病毒,衰老组细胞相应的病毒复制水平也显著降低(P<0.05或P<0.01)。与年轻组小鼠相比,年老组小鼠感染VSV病毒后脾和肺中炎症反应显著增强,肝中无明显变化。结论:DOX可以诱导细胞衰老。衰老细胞和衰老个体感染病毒后表现为较高的炎症反应,衰老细胞感染病毒还表现较低的抗病毒反应,这些可能是衰老导致病毒疾病进程延长的潜在原因。

Objective:To investigate the role and mechanism of senescence in viral infection and viral replication.Method:Cell senescence was established by doxorubicin(DOX)stimulation on human non-small cell lung cancer cells(A549).These A549 cells and A549 cells in natural state were infected with vesicular stomatitis virus(VSV)or herpes simplex virus(HSV)at the same time.C57BL/6 mice of specific pathogen free(SPF)were divided into two groups,8 weeks old as young group(n=3),15 months old as old group(n=3).All mice were infected with VSV(1×107 pfu/mouse)for 24 h through intraperitoneal injection.Interferon-β(IFN-β),Interleukin-6(IL-6),Interleukin-1β(IL-1β)and viruses replication were detected by RT-PCR.The protein levels of nuclear factor-κB p65(p65)and phosphorylated nuclear factor-κB p65(p-p65),key proteins of the nuclear factor-κB(NF-κB)pathway,were detected by Western blot.Differences in viruses replication were also observed by a fluorescent inverted microscope.Results:Cell cycle arrest proteins P16,P21 and the number of positive cells inβ-galactosidase staining in the DOX-induced senescent A549 cells were significantly increased compared to the other group.After infected with the VSV at the same time,the IFN-βin the senescent cell group was significantly reduced,while the IL-6 and IL-1βwere significantly increased.At the same time,the expressions of key proteins p65 and p-p65 in the NF-κB pathway in the senescent cell group were significantly higher than those in the normal cell group.When infected with VSV and HSV,the corresponding viral replication level of aging cell group was also significantly reduced(P<0.05 or P<0.01).Compared with young mice,the inflammatory response in the spleen,lung of the elderly mice was significantly enhanced after virus infection,but that in liver was not changed significantly.Conclusion:DOX treatment can induce cell senescent.Senescent cells and individuals infected with virus showed higher inflammatory response,and aged cells infected with virus also showed lower interferon response,which may be the underlying reason for the prolongation of viral disease process caused by aging.