目的 探究麻杏石甘汤通过外泌体miR-1249-5p靶向溶质载体家族4成员A1(solute carrier family 4 member 1,SLC4A1)抑制流感病毒诱导的肺上皮细胞损伤-巨噬细胞极化的级联损伤效应的作用机制。方法 以肺上皮细胞为研究对象,采用CCK-8检测麻杏石甘汤含药血清的安全剂量和对流感病毒刺激肺上皮细胞治疗剂量,NanoSight鉴定肺上皮细胞源外泌体的粒径和浓度,qRT-PCR检测外泌体中miR-1249-5p基因表达。以与肺上皮细胞源外泌体共培养的巨噬细胞为研究对象,采用qRT-PCR检测巨噬细胞中miR-1249-5p、SLC4A1和核因子-κB(nuclear factor-κB,NF-κB)通路相关基因表达;Western blotting检测巨噬细胞中SLC4A1和NF-κB通路相关蛋白表达;流式细胞术和qRT-PCR检测巨噬细胞M1/M2极化表型。结果 CCK-8结果显示,10%和20%的麻杏石甘汤含药血清对肺上皮细胞无细胞毒性,显著抑制流感病毒诱导的肺上皮细胞损伤(P<0.05、0.01);NanoSight检测各组肺上皮细胞源外泌体大小无明显变化,外泌体浓度有所改变;qRT-PCR结果显示流感病毒刺激的肺上皮源外泌体miR-1249-5p基因表达明显降低(P<0.01),各给药组miR-1249-5p基因表达显著升高(P<0.05、0.001)。肺上皮源外泌体共培养后的巨噬细胞中miR-1249-5p基因表达与外泌体相一致(P<0.05、0.001);qRT-PCR和Western blotting结果显示,模型组巨噬细胞SLC4A1和NF-κB通路基因和蛋白表达显著升高(P<0.01、0.001),各给药组SLC4A1和NF-κB通路基因和蛋白表达显著下降(P<0.05、0.01);流式细胞术结果显示模型组巨噬细胞CD86⁺比例明显增高(P<0.01),各给药组巨噬细胞CD86⁺比例显著降低(P<0.05);qRT-PCR结果显示共培养后模型组巨噬细胞肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)和IL-1β的表达显著升高(P<0.01、0.001),IL-10和转化生长因子-β1(transforming growth factor-β1,TGF-β1)的表达显著下降(P<0.05、0.01),各给药组以上因子表达趋势与之相反(P<0.05、0.01、0.001)。结论 麻杏石甘汤可能通过肺上皮细胞源外泌体递送miR-1249-5p到巨噬细胞中,并靶向巨噬细胞中SLC4A1调控NF-κB信号通路,抑制流感病毒的细胞级联损伤效应的作用机制。

Objective To explore the mechanism of Maxing Shigan Decoction (麻杏石甘汤) targeting SLC4A1 through miR-1249-5p to inhibit the cascade damage effect of influenza virus induced lung epithelial cells damage macrophage polarization. Methods Lung epithelial cells were taken as the research object, CCK-8 was used to detect the safe dose of Maxing Shigan Decoction drug containing serum and therapeutic dose of Maxing Shigan Decoction on lung epithelial cells stimulated by influenza virus; NanoSight was used to identify the particle size and concentration of pulmonary epithelial cell derived exosomes, qRT-PCR was used to detect the expression level of miR-1249-5p in the exosomes. The co-culture system of pulmonary epithelial cell derived exosomes and macrophages was taken as the research object, qRT-PCR was used to detect miR-1249-5p, SLC4A1, and nuclear factor-κB (NF-κB) pathway related factor gene expressions in macrophages; Western blotting was used to detect SLC4A1 and NF-κB pathway related protein expressions in macrophages; Flow cytometry and qRT-PCR were used to detect the M1/M2 polarization phenotype of macrophages. ResultsCCK-8 results showed that 10% and 20% of Maxing Shigan Decoction containing serum had no cytotoxicity on lung epithelial cells and significantly inhibited influenza virus induced lung epithelial cell damage (P ＜ 0.05, 0.01); NanoSight detection showed that there was no significant change in the size of lung epithelial cell derived exosomes in each group, but the concentration of exosomes changed; qRT-PCR results showed that the expression of miR-1249-5p gene in lung epithelial cell derived exosomes stimulated by influenza virus was significantly reduced (P ＜ 0.01), and the expression of miR-1249-5p gene was significantly increased in each treatment group (P ＜ 0.05, 0.001). The expression of miR-1249-5p gene in macrophages co-cultured with pulmonary epithelial derived exosomes was consistent with that of exosomes (P ＜ 0.05, 0.001); The results of qRT-PCR and Western blotting showed that in macrophages of model group, SLC4A1 and NF-κB pathway genes and proteins expressions were significantly increased (P ＜ 0.01, 0.001), and SLC4A1 and NF-κB pathway genes and proteins expression in each treatment group were significantly decreased (P ＜ 0.05, 0.01); Flow cytometry results showed that CD86⁺ ratio of macrophages in model group was significantly increased (P ＜ 0.01), while the CD86⁺ ratio of macrophages in each treatment group was significantly reduced (P ＜ 0.05); The qRT-PCR results showed that in macrophages of model group after co-culture, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β expressions were significantly increased (P ＜ 0.01, 0.001), IL-10 and transforming growth factor-β1 (TGF-β1) expressions were significantly decreased (P ＜ 0.05, 0.01), and the expression trend of above factors in each treatment group was opposite (P ＜ 0.05, 0.01, 0.001). Conclusion Maxing Shigan Decoction may deliver miR-1249-5p to macrophages through exosomes derived from lung epithelial cells, and target SLC4A1 to regulate NF-κB signaling pathway in macrophages, inhibits the cellular cascade damage effect of influenza viruses.

R285.5

国家自然科学基金资助项目（81973670）；国家自然科学基金资助项目（82074250）；湖南省自然科学基金资助项目（2020JJ5418，2020JJ4063）