基于PI3K/AKT/NF-κB信号通路调控细胞凋亡探讨扶正解毒化瘀方抗人呼吸道合胞病毒的作用机制  

Study on the Mechanism of Fuzheng Jiedu Huayu Formula Against Human Respiratory Syncytial Virus Based on the Regulation of Apoptosis by PI3K/AKT/NF-κB Signal Pathway

作　　者：张亚楠 晏军[1] 宋金华[2] 王明哲[1] 宋利琼[3] 李金桐 翟志光[4] 王成祥 班承钧 程淼[1] ZHANG Yanan;YAN Jun;SONG Jinhua;WANG Mingzhe;SONG Liqiong;LI Jintong;ZHAI Zhiguang;WANG Chengxiang;BAN Chengjun;CHENG Miao(Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine,Beijing 100700,China;NHC Key Laboratory of Medical Virology and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing,102206,China;State Key Laboratory of Infectious Disease Prevention and Control,National institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Institute of Basic Theory for Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China;Beijing University of Chinese Medicine Third Affiliated Hospital,Beijing 100029,China)

机构地区：[1]北京中医药大学东直门医院,北京100700 [2]中国疾病预防控制中心,病毒病预防控制所,国家卫生健康委医学病毒学与病毒疾病重点实验室,北京102206 [3]中国疾病预防控制中心,传染病预防控制所,传染病预防控制国家重点实验室,北京102206 [4]中国中医科学院中医基础理论研究所,北京100700 [5]北京中医药大学第三附属医院,北京100029

出　　处：《病毒学报》2023年第5期1217-1226,共10页Chinese Journal of Virology

基　　金：北京中医药大学2019年度青年教师项目(项目号:2019-JYB-JS-040),题目:“三阳合治”法通过内质网应激干预流感病毒感染宿主细胞凋亡机制研究;国家自然科学基金青年科学基金项目(项目号:81403371),题目:扶正解毒化瘀方及最佳有效部位群干预呼吸道合胞病毒融合性病变形成期的作用机制研究;国家自然科学基金面上项目(项目号:81973784),题目:基于“状态理论”的开痹补肺汤通过肠道微生态-胆碱能抗炎通路减轻流感病毒致小鼠急性肺损伤的机制研究;北京自然科学基金面上项目(项目号:7232293),题目:三阳合治法调控IL6-STAT3-SOCS3减轻流感病毒诱导肺损伤的免疫机制研究~~。

摘　　要：观察扶正解毒化瘀方对人呼吸道合胞病毒(Human respiratory syncytial virus,HRSV)感染相关细胞凋亡的影响,探究其抗病毒作用机制。体外实验确定扶正解毒化瘀方对人喉癌上皮细胞(Hep-2)的细胞毒作用,选取最大无毒浓度(TC0)进行干预实验;以HRSV A亚型感染Hep-2细胞,设细胞对照组、病毒对照组、利巴韦林药物对照组及扶正解毒化瘀方组,显微镜下观察细胞病变,通过酶联免疫吸附测定(ELISA)检测炎性因子调节的正常T细胞表达和分泌(RANTES)水平,Western Blot检测磷脂酰肌醇3-激酶(PI3K)/苏氨酸激酶(AKT)/核因子κB(NF-κB)通路相关蛋白表达,反转录聚合酶链反应(RT-PCR)检测凋亡相关蛋白的mRNA相对水平。与细胞对照组相比,病毒对照组细胞病变明显,细胞上清RANTES水平升高,细胞PI3K/AKT/NF-κB通路相关蛋白AKT、糖原合成酶激酶-3(GSK-3β)、NF-κBp65表达增加,凋亡相关蛋白Fas、Bax、TRAIL的mRNA水平升高,两组间差异具有统计学意义(P<0.05)。而扶正解毒化瘀方能改善细胞病变,显著降低RANTES水平,抑制AKT、GSK-3β、NF-κBp65蛋白表达,降低Fas、Bax、TRAIL mRNA水平(P<0.05)。扶正解毒化瘀方可抑制HRSV感染所致细胞病变,减轻炎症反应,其机制可能与抑制PI3K/AKT/NF-κB通路激活,减少细胞凋亡有关。To investigate the effect of Fuzheng Jiedu Huayu Formula on apoptosis related to human respiratory syncytial virus(HRSV)infection and explore its antiviral mechanism.The cytotoxic effect of Fuzheng Jiedu Huayu Formula on human laryngeal cancer epithelial cells(Hep-2)was determined in vitro,and the maximum nontoxic concentration(TC_0)was selected for intervention experiment.Hep-2 cells were infected with subtype HRSV A and divided into Normal control,Viral control,Ribavirin and Fuzheng Jiedu Huayu Formula group.Cell pathological effects were observed through microscope.The regulated upon activation normal T cell expressed and secreted(RANTES)was detected by enzyme-linked immunosorbent assay(ELISA).The level of phosphatidylinositol 3-kinase(PI3K)/threonine kinase(AKT)/nuclear factorκB(NF-κB)pathway-related protein expression were detected by Western Blot,and the relative mRNA level of apoptosis-related protein were tested by reverse transcription-polymerase chain reaction(RT-PCR).Compared with the Normal control group,the Viral control group showed obvious cell pathological effects,increased expression of RANTES in cell supernatant,and increased expression of PI3K/AKT/NF-κB-Pathway-associated proteins AKT,glycogen synthase kinase-3(GSK-3β)and NF-κBp65,and increased mRNA levels of apoptosis-related proteins Fas,Bax,and TRAIL.The difference between the two groups was statistically significant(P<0.05).While Fuzheng Jiedu Huayu Formula can improve the cytopathic effect,significantly reduce the level of RANTES,inhibit the expression of AKT,GSK-3βand NF-κBp65,and reduce mRNA levels of Fas,Bax and TRAIL(P<0.05).Fuzheng Jiedu Huayu Recipe can inhibit the cytopathic effect caused by HRSV infection and reduce the inflammatory reaction.Its mechanism may be related to inhibiting the activation of PI3K/AKT/NF-κB pathway,and inhibiting cell apoptosis.

关 键 词：呼吸道合胞病毒 扶正解毒化瘀方 PI3K/AKT/NF-κB信号通路 细胞凋亡 作用机制 

分 类 号：R373.1[医药卫生—病原生物学]