黄连素下调NRAV和PI3K/AKT通路减轻RSV感染致HEp-2细胞的损伤  

Berberine downregulates NRAV and PI3K/AKT pathways to alleviate damage to HEp-2 cells caused by RSV infection

作　　者：崔玉娟 赵辉 苏东霞 张莹 胡丹东 CUI Yu-juan;ZHAO Hui;SU Dong-xia;ZHANG Ying;HU Dan-dong(School of Life Science,Northwest Normal University,Lanzhou 730070,China;Beijing Yanqing Center for Disease Prevention and Control,Beijing 102100,China;Beijing Yanqing Market Supervision Inspection and Testing Monitoring Center,Beijing 102100,China)

机构地区：[1]西北师范大学生命科学学院,甘肃兰州730070 [2]北京市延庆区疾病预防控制中心,北京102100 [3]北京市延庆区市场监管检验检测监控中心,北京102100

出　　处：《中国药理学通报》2024年第4期747-755,共9页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 51873175)。

摘　　要：目的探讨长链非编码RNA NRAV(LncRNA NRAV)和PI3K/AKT通路在黄连素(berberine,BE)减轻RSV感染致HEp-2细胞损伤中的机制。方法将HEp-2细胞感染RSV,并用BE、PI3K激活剂740Y-P处理或过表达NRAV。qRT-PCR检测NRAV、RSV-F、NS2表达水平;CCK-8实验检测细胞存活率;流式细胞术检测细胞的凋亡率和线粒体膜电位;ATP检测试剂盒检测ATP水平;Western blot检测细胞PI3K、AKT、PINK1、Parkin、Beclin1、p62、LC3Ⅰ、LC3Ⅱ、BNIP3、NLRP3、ASC、caspase-1蛋白表达;MitoSOX染色检测细胞线粒体ROS(mtROS);ELISA检测细胞IL-1β、IL-6、IL-8、TNF-α分泌水平。结果过表达NARV细胞凋亡率、RSV活性增加,敲低后结果相反;BE能显著抑制NRAV和PI3K/AKT通路(P<0.05),改善线粒体功能、诱导线粒体自噬,提高细胞的活性、降低凋亡率(P<0.05),并降低NLRP3炎性小体活化水平和IL-1β、IL-6、IL-8、TNF-α水平(P<0.05)。过表达NRAV或740Y-P处理可逆转BE对RSV感染HEp-2细胞的改善作用。结论BE能够减轻RSV感染所致HEp-2细胞损伤,其机制可能与BE下调NRAV和PI3K/AKT通路,诱导线粒体自噬,进而减轻线粒体损伤和炎症反应有关。Aim To explore the mechanism of long non coding RNA NRAV(LncRNA NRAV)and PI3K/AKT pathway in berberine(BE)alleviating RSV infection induced damage to HEp-2 cells.Methods HEp-2 cells were infected with RSV and treated with BE,PI3K activator 740Y-P or overexpressed NRAV.QRT PCR was used to detect the expression levels of NRAV,RSV-F and NS2;CCK-8 experiment was performed to detect cell survival rate;flow cytometry was used to detect the apoptosis rate and mitochondrial membrane potential of cells;ATP detection kit was applied to detect ATP levels;Western blot was used to detect the protein expression of PI3K,AKT,PINK1,Parkin,Beclin1,p62,LC3 I,LC3 II,BNIP3,NLRP3,ASC,and caspase-1 in cells;MitoSOX staining was used to detect mitochondrial ROS(mtROS)in cells;ELISA was used to detect the secretion levels of IL-1β,IL-6,IL-8,and TNF-αin cells.Results Overexpression of NARV resulted in an increase in cell apoptosis rate and RSV activity,while knockdown resulted in the opposite effect;BE could significantly inhibit the NRAV and PI3K/AKT pathways(P<0.05),improve mitochondrial function,induce mitochondrial autophagy,increase cell activity,reduce apoptosis rate(P<0.05),and reduce NLRP3 inflammasome activation levels and IL-1β,IL-6,IL-8,and TNF-αlevels(P<0.05).Overexpression of NRAV or 740Y-P could reverse the improvement effect of BE on RSV infection in HEp-2 cells.Conclusion BE can alleviate the damage to HEp-2 cells caused by RSV infection,and its mechanism may be related to BE downregulating the NRAV and PI3K/AKT pathways,inducing mitochondrial autophagy,and thereby reducing mitochondrial damage and inflammatory response.

关 键 词：黄连素 RSV LncRNA NRAV PI3K/AKT通路 线粒体损伤 HEp-2细胞损伤 

分 类 号：R284.1[医药卫生—中药学] R329.24[医药卫生—中医学] R342.2R373.1R364.5