麻杏石甘汤通过外泌体miR-1249-5p靶向SLC4A1抑制流感病毒细胞级联损伤效应研究  

Maxing Shigan Decoction inhibits influenza virus cells cascade damage by targeting SLC4A1 with exosome miR-1249-5p

作　　者：马心悦 黄家望 冯芷莹 刘卓琳 王康宇 卢芳国[3] 朱梦晨 李玲 MA Xin-Yue;HUANG Jia-wang;FENG Zhi-ying;LIU Zhuo-lin;WANG Kang-yu;LU Fang-guo;ZHU Meng-chen;LI Ling(College of Integrated Traditional Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha 410208,China;College of Traditional Chinese Medicine,Hunan University of Chinese Medicine,Changsha 410208,China;Medical School of Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学中西医结合学院,湖南长沙410208 [2]湖南中医药大学中医学院,湖南长沙410208 [3]湖南中医药大学医学院,湖南长沙410208

出　　处：《中草药》2023年第9期2832-2840,共9页Chinese Traditional and Herbal Drugs

基　　金：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)。

摘　　要：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);qRTPCR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.Results CCK-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

关 键 词：麻杏石甘汤 A型流感病毒 功能性外泌体 miR-1249-5p 肺泡上皮细胞 肺泡巨噬细胞 

分 类 号：R285.5[医药卫生—中药学]