雷帕霉素在甲型H1N1流感病毒感染中对糖酵解和炎性因子的调节作用  

Regulatory role of rapamycin in glycolysis and inflammatory cytokines response during H1N1 influenza virus infection

作　　者：宋融融 徐九洋 刘智博[2] 曹彬 Song Rongrong;Xu Jiuyang;Liu Zhibo;Cao Bin(Peking University China-Japan Friendship School of Clinical Medicine,Beijing 100029,China;Department of Pulmonary and Critical Care Medicine,China-Japan Friendship Hospital,Beijing 100029,China)

机构地区：[1]北京大学中日友好临床医学院,北京100029 [2]中日友好医院呼吸与危重症医学科,北京100029

出　　处：《国际呼吸杂志》2024年第2期154-161,共8页International Journal of Respiration

基　　金：国家自然科学基金(82200010);中国医学科学院重大协同创新项目(2021-I2M-1-048);中国医学科学院呼吸病学研究院青年科学基金(2023-ZF-13)。

摘　　要：目的探讨免疫抑制剂雷帕霉素对流感病毒复制的影响及在流感病毒感染后调节糖酵解和炎性因子的作用。方法本研究为实验研究,采用甲型H1N1流感病毒株A/PR/8分别感染人肺泡上皮细胞系A549和永生化小鼠骨髓源巨噬细胞(iBMDM),构建流感病毒感染细胞模型。使用人非小细胞肺癌细胞系A459设置空白对照组、感染对照组、感染雷帕霉素组、感染二甲亚砜组,在流感病毒感染及雷帕霉素处理48 h后,使用空斑法和血凝法测定上清液病毒滴度,定量聚合酶链反应法检测细胞内病毒基因、糖酵解相关基因(PDK3、PKM、GAPDH、LDHA、HK2、PGAM1、PGK1)和炎性因子干扰素α(IFN-α)、Caspase-1、白细胞介素(IL)-1β、IL-18、IL-6、IL-8、凋亡相关斑点样蛋白(ASC)]基因相对量的表达。设置巨噬细胞空白对照组,巨噬细胞感染对照组和巨噬细胞感染雷帕霉素组,使用小鼠永生化骨髓巨噬细胞(iBMDM)在流感病毒感染及雷帕霉素处理24 h后,酶联免疫吸附测定法检测上清肿瘤坏死因子(TNF)-α浓度。进行3次平行重复实验。结果感染雷帕霉素组A549细胞上清中的病毒滴度与感染对照组、感染二甲亚砜组比较差异均无统计学意义(均P>0.05)。感染雷帕霉素组NP基因CT值较感染对照组和感染二甲亚砜组升高,分别为[(17.50±0.35)比(16.43±0.12)和(16.52±0.27),差异有统计学意义,均P<0.05];感染对照组A549细胞上清中炎性因子基因GAPDH、LDHA、HK2、PGAM1表达量均较空白对照组上调,分别为[(2.34±0.32)比(1.01±0.16)、(2.43±0.18)比(1.01±0.18)、(2.63±0.48)比(1.00±0.06),(17.97±1.13)比(1.00±0.09)差异有统计学意义,均P<0.05];与感染对照组相比,感染雷帕霉素组下调了GAPDH、LDHA、HK2、PGAM1的相对表达量,分别为[(1.48±0.19)比(2.34±0.32)、(1.79±0.09)比(2.43±0.18)、(1.65±0.28)比(2.63±0.48)、(10.48±0.81)比(17.97±1.13),差异有统计学意义,均P<0.05];感染雷帕霉素组基因PDK3�Objective To investigate the effect of the immunosuppressant rapamycin on viral replication in influenza virus-infected cell,and to determine its role in regulating glycolysis pathway and inflammatory response during influenza virus infection.Methods This was an experimental study.Human alveolar epithelial cell A549 and iBMDM(immortalized murine bone marrow-derived macrophages)cell lines were infected with influenza virus A/H1N1/PR/8 to construct a cell model of influenza virus infection.Human non-small cell lung cancer cell line A459 was used to set up a blank control group,an infection control group,a rapamycin infection group,and a dimethyl sulfoxide infection group.At 48 hours post infection and rapamycin treatment,the cell culture supernatant virus titer was determined by plaque assay and hemagglutination assay;the expression levels of viral gene,glycolysis genes(PDK3,PKM,GAPDH,LDHA,HK2,PGAM1,and PGK1),and inflammatory factor interferonα,Caspase-1,Interleukin(IL)-1β,IL-18,IL-6,IL-8,and apoptosis associated speckle like protein(ASC)genes were determined with real time quantitative PCR(RQ-PCR).A macrophage blank control group,a macrophage infection control group,and a macrophage rapamycin infection group were set up.At 24 hours post mouse iBMDM infection and rapamycin treatment,the supernatant cytokine tumor necrosis factor(TNF)-αin iBMDM culture was measured by ELISA.Three parallel replicate experiments were performed.Results There was no significant difference in viral titer in the supernatant of A549 cells between the rapamycin infection group and infection control group,rapamycin infection group and dimethyl sulfoxide infection group(both P>0.05).The CTvalue of influenza virus NP gene in the rapamycin infection group was higher than that in the infection control group(17.50±0.35 vs 16.43±0.12)and in the dimethyl sulfoxide infection group(17.50±0.35 vs 16.52±0.27),with significant differences(both P<0.05).The expressions of GAPDH,LDHA,HK2,and PGAM1 in the infection control group were up-regulated com

关 键 词：流感病毒A型 雷帕霉素 糖酵解 炎性因子 

分 类 号：R511.7[医药卫生—内科学]