| 引用本文: |
黄家望,王康宇,马心悦,刘卓琳,冯芷莹,尹抗抗,李玲.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[J].湖南中医药大学学报,2023,43(9):1584-1590[点击复制] |
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| 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 |
| 黄家望,王康宇,马心悦,刘卓琳,冯芷莹,尹抗抗,李玲 |
| (湖南中医药大学中西医结合学院, 湖南 长沙 410208;湖南中医药大学中医学院, 湖南 长沙 410208;湖南中医药大学科技创新中心, 湖南 长沙 410208;湖南中医药大学中西医结合学院, 湖南 长沙 410208;中西医结合病原生物学湖南省重点实验室, 湖南 长沙 410208) |
| 摘要: |
| 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7.204 μmol/L，因此，将后续实验中的木犀草素药物浓度定为5 μmol/L和2.5 μmol/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。 |
| 关键词: 木犀草素 A型流感病毒 作用机制 VEGF/PI3K/Akt/eNOS信号通路 |
| DOI：10.3969/j.issn.1674-070X.2023.09.006 |
| 投稿时间：2023-03-25 |
| 基金项目:国家自然科学基金项目（81973670）；湖南省自然科学基金项目（2020JJ5418）；中药粉体与创新药物省部共建国家重点实验室培育基地开放基金项目（21PTKF1007）；湖南省大学生创新创业培训项目（A202210541108）；湖南中医药大学中西医结合病原生物学湖南省重点实验室开放基金项目（2022KFJJ02）；湖南中医药大学研究生创新项目（2022CX63）。 |
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| Mechanism of action of luteolin against influenza A virus based on the VEGF/PI3K/Akt/eNOS signaling pathway |
| HUANG Jiawang,WANG Kangyu,MA Xinyue,LIU Zhuolin,FENG Zhiying,YIN Kangkang,LI Ling |
| (School of Integrated Chinese and Western Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;School of Chinese Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;Science & Technology Innovation Center, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;School of Integrated Chinese and Western Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;Hunan Key Laboratory of Pathogeny Biology of Integrated Chinese and Western Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| 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 7.204 μmol/L. Therefore, the drug concentrations of luteolin in subsequent experiments were set at 5 μmol/L and 2.5 μmol/L. Compared with the normal group, the model group exhibited increased secretion of TNF-α and 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-α and 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. |
| Key words: luteolin influenza A virus mechanism of action VEGF/PI3K/Akt/eNOS signaling pathway |
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