基于VEGF/PI3K/Akt/eNOS信号通路探讨木犀草素干预A型流感病毒的作用机制

Mechanism of action of luteolin against influenza A virus based on the VEGF/PI3K/Akt/eNOS signaling pathway

目的探讨木犀草素对A型流感病毒(influenza A virus,IAV)感染的小鼠肺上皮细胞损伤模型的作用机制。方法将小鼠肺上皮MLE-12细胞分为正常组、模型组、奥司他韦组、高剂量组和低剂量组,除正常组外,其余4组均使用IAV感染,正常组加入等量病毒稀释液;2 h后弃病毒工作液,正常组和模型组加入病毒维持液,药物组加入用病毒维持液稀释的药物工作液,干预8 h后收集细胞。使用CCK-8检测细胞增殖情况;ELISA实验检测细胞上清液中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-6(interleukin-6,IL-6)的表达;免疫荧光检测细胞内血管内皮生长因子(vascular endothelial growth factor,VEGF)蛋白表达情况;RT-qPCR技术检测细胞内VEGF、蛋白激酶B(protein kinase B,Akt)、3-磷酸肌醇激酶(phosphoinositide 3-kinase,PI3K)和内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)mRNA表达情况;Western blot法检测细胞内VEGF、Akt、PI3K和eNOS蛋白表达情况。结果CCK-8结果显示,不同浓度的木犀草素均能降低IAV感染所致的细胞损伤(P<0.01),其EC50值为7.204μmol/L,因此,将后续实验中的木犀草素药物浓度定为5μmol/L和2.5μmol/L。与正常组相比,模型组细胞上清液中TNF-α和IL-6升高(P<0.01),细胞内VEGF的荧光蛋白强度增强(P<0.01),细胞内VEGF、Akt、PI3K和eNOS的mRNA和蛋白的表达升高(P<0.01);与模型组相比,高剂量组和低剂量组细胞上清液中TNF-α和IL-6降低(P<0.01),细胞内VEGF的荧光蛋白强度减弱(P<0.01),细胞内VEGF、Akt、PI3K和eNOS的mRNA和蛋白的表达降低(P<0.01)。结论木犀草素能抑制炎性细胞因子的分泌,缓解IAV感染所致的细胞损伤,其过程可能与VEGF/PI3K/Akt/eNOS信号通路密切相关。

Objective To explore the mechanism of action of luteolin on lung epithelial cell injury model of mice infected with influenza A virus(IAV).Methods Mouse lung epithelial cells(MLE-12)were divided into normal group,model group,oseltamivir group,high-dose group,and low-dose group.Except for the normal group,the other four groups were infected with IAV.The normal group received an equal amount of virus diluent,and the virus working solution was discarded after 2 h.The normal group and the model group were added with virus maintenance solution,and the medication groups were added with drug working solution diluted with virus maintenance solution.After 8 hours of intervention,the cells were collected.Cell proliferation was evaluated using the CCK-8 assay.The expressions of tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)in cell supernatant were measured by ELISA.Immunofluorescence was used to determine protein expression of intracellular vascular endothelial growth factor(VEGF).RT-qPCR was performed to examine the mRNA expressions of VEGF,protein kinase B(Akt),phosphoinositide 3-kinase(PI3K),and endothelial nitric oxide synthase(eNOS)in cells.Western blot analysis was conducted to measure the protein expressions of VEGF,Akt,PI3K,and eNOS in cells.Results CCK-8 assay showed that different concentrations of luteolin could alleviate cell damage caused by IAV infection(P<0.01),with an EC50 of 7.204μmol/L.Therefore,the drug concentrations of luteolin in subsequent experiments were set at 5μmol/L and 2.5μmol/L.Compared with the normal group,the model group exhibited increased secretion of TNF-αand IL-6 in cell supernatant(P<0.01),increased fluorescence intensity of VEGF in cells(P<0.01),and up-regulated mRNA and protein expressions of VEGF,Akt,PI3K,and eNOS in cells(P<0.01).Compared with the model group,the high-and low-dose groups showed reduced secretion of TNF-αand IL-6 in cell supernatant(P<0.01),decreased fluorescence intensity of VEGF in cells(P<0.01),and down-regulated mRNA and protein expressions of VEGF,Akt,PI3K,and eNOS in cells(P<0.01).Conclusion Luteolin can inhibit the secretion of inflammatory cytokines and alleviate cell damage caused by IAV infection,which may be closely related to the VEGF/PI3K/Akt/eNOS signaling pathway.