摘要
目的观察木犀草素对流感病毒感染引起的细胞炎症反应的影响,探讨木犀草素调控流感病毒引发的免疫反应的作用机制。方法①用细胞计数试剂盒(CCK-8)法检测不同浓度木犀草素(5、10、25、30μmol/L)对小鼠巨噬细胞RAW 264.7存活率的影响。②咪喹莫特(R837)刺激RAW 264.7或原代腹腔巨噬细胞的同时,用不同浓度木犀草素(5、10、20μmol/L)干预细胞3、6、18 h后,用荧光定量逆转录聚合酶链式反应(RT-qPCR)法和酶联免疫吸附测定(ELISA)法检测白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)、IL-1β、β干扰素(IFN-β)、干扰素诱导蛋白-10(IP-10)以及血红素加氧酶-1(HO-1)基因及TNF-α、IL-6的表达水平。③R837刺激RAW 264.7细胞的同时,用不同浓度木犀草素(5、10、20μmol/L)干预细胞1、3 h,用Western blot法检测细胞核因子-κB(NF-κB)p65蛋白、磷酸化p65(p-p65)蛋白、磷酸化糖原合成酶激酶3β(p-GSK3β)蛋白、Kelch样ECH关联蛋白1(Keap1)和核内核因子红细胞2相关因子(Nrf2)蛋白的表达水平。④用Nrf2抑制剂(ML385)处理RAW 264.7细胞12 h后,加入R837和木犀草素(5μmol/L)继续培养6 h,用RT-qPCR法检测细胞IL-6、TNF-α、IL-1β、IFN-β、IP-10 mRNA的表达水平。⑤流感病毒A/PR/8(PR8)感染A549细胞2 h,同时用不同浓度木犀草素(5、10、20μmol/L)处理细胞10 h,用RT-qPCR法检测细胞IL-6、TNF-α、IL-1β、IFN-β、IP-10和单核细胞趋化蛋白-1(MCP-1)mRNA的表达水平。结果①30μmol/L以内各浓度木犀草素对RAW 264.7细胞存活率没有明显影响(P>0.05)。②木犀草素能够显著降低R837诱导的RAW 264.7细胞IL-6、TNF-α的表达水平和IL-1β、IFN-β、IP-10 mRNA表达水平,且木犀草素也能显著降低原代腹腔巨噬细胞IL-6、TNF-α的表达水平。③木犀草素可以促进Nrf2入核,上调HO-1 mRNA表达并抑制p-p65表达,促进糖原合成酶激酶3β(GSK3β)的磷酸化。④在R837诱导的巨噬细胞炎症反应中,ML385可恢复木犀草素抑制的RAW 264.7细胞炎症因子IL-6、IL-1β、IFN-β和IP-10 mRNA的表达水平。⑤木犀草素抑制PR8感染引起的A549细胞炎症因子的产生。结论木犀草素可通过抑制GSK3β活性促进Nrf2/HO-1通路,从而抑制流感病毒感染引起的炎症反应。
Objective To observe the effects of luteolin on cell inflammatory response induced by influenza virus infection and to explore the mechanisms of luteolin regulating the immune response triggered by influenza virus.Methods①Cell Counting Kit-8(CCK8)assay was used to observe luteolin(5,10,25,30μmol/L)on the viability of macrophages in mouse macrophage RAW 264.7.②RAW 264.7 cells or mouse peritoneal macrophages were stimulated by imiquimod(R837)and cells were treated with luteolin(5,10,20μmol/L)for 3,6 or 18 h,the expression levels of interleukin(IL)-6,tumor necrosis factor-α(TNF-α),IL-1β,interferon-β(IFN-β),interferon gamma-induced protein 10(IP-10)and heme oxygenase-1(HO-1)were detected using fluorescent quantitative reverse transcription-polymerase chain reaction(RT-qPCR)and enzyme-linked immunosorbent assay(ELISA)methods.③RAW 264.7 cells stimulated by R837 were treated with different concentrations of luteolin(5,10,20μmol/L)for 1 or 3 h.The expression levels of nuclear factor-κB(NF-κB)p65 protein,phosphorylation p65(p-p65)protein,phosphorylated glycogen synthase kinase-3β(p-GSK3β),kelch-like ECH-associated protein 1(Keap1)and nuclear factor erythroid 2-related factor 2(Nrf2)levels in nuclear were detected using Western blot method.④RAW 264.7 cells were pretreated with Nrf2 inhibitor(ML385)for 12 h,and cells were treated with R837 and luteolin(5μmol/L)for another 6 h.The mRNA expression levels of IL-6,TNF-α,IL-1β,IFN-βand IP-10 were detected using RT-qPCR method.⑤A549 cells were infected with influenza virus A/PR/8C(PR8)for 2 h,and cells were treated with different concentrations of luteolin(5,10,20μmol/L)for 10 h.The mRNA expression levels of IL-6,TNF-α,IL-1β,IFN-β,IP-10 and monocyte chemoattractant protein-1(MCP-1)were detected using RT-qPCR method.Results①Different concentrations of luteolin under 30μmol/L had no effect on the viability of RAW 264.7 cells(P>0.05).②Luteolin significantly reduced the expression levels of IL-6 and TNF-α.Luteolin also reduced the mRNA expression levels of IL-1β,IFN-βand IP-10 in RAW 264.7 cells.Meanwhile,luteolin reduced the secretion of IL-6 and TNF-αin primary peritoneal macrophages under stimulation of R837.③Luteolin could promote Nrf2 into nuclear,up-regulate HO-1 mRNA expression,inhibit p-p65 expression,and promote the phosphorylation of glycogen synthase kinase 3β(GSK3β).④In the R837-induced macrophage inflammation,ML385 could restore the mRNA expression levels of IL-6,IL-1β,IFN-βand IP-10 inhibited by luteolin in RAW 264.7 cells.⑤Luteolin inhibited the production of inflammatory factors in A549 cells induced by PR8 infection.Conclusion Luteolin could inhibit inflammation caused by influenza virus infection by inhibiting GSK3βactivity and promoting the Nrf2/HO-1 pathway.
作者
陶艺文
许利荣
胡友
郑月娟
周春仙
TAO Yiwen;XU Lirong;HU You;ZHENG Yuejuan;ZHOU Chunxian(School of Integrative Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;The Research Center for Traditional Chinese Medicine,Shanghai Institute of Infectious Diseases and Biosecurity,Shanghai 201203,China;Shanghai Key Laboratory of Health Identification and Evaluation,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)
出处
《上海中医药杂志》
CSCD
2024年第9期61-69,共9页
Shanghai Journal of Traditional Chinese Medicine
基金
国家重点研究与发展计划项目(2021YFE0200900)
国家自然科学基金项目(82073901)
上海市市级科技重大专项(ZD2021CY001)。
关键词
流感病毒
炎症反应
免疫反应
木犀草素
作用机制
中药研究
influenza virus
inflammatory response
immunological reaction
luteolin
mechanism of action
traditional Chinese herbal medicine research
