基于PI3K/Akt/mTOR信号通路探讨玉屏风散对肥大细胞脱颗粒的影响  

Study on the effect of Yupingfeng powder on mast cell degranulation based on PI3K/Akt/mTOR pathway

作　　者：韦薇 覃骊兰[2] 李莉[2] 郑伟灏 WEI Wei;QIN Lilan;LI Li;ZHENG Weihao(Guangxi University of Traditional Chinese Medicine,Nanning 530001,China)

机构地区：[1]广西中医药大学药学院,南宁530001 [2]广西中医药大学中药学教研室,南宁530001

出　　处：《环球中医药》2021年第9期1580-1586,共7页Global Traditional Chinese Medicine

基　　金：国家自然科学基金(81560755);广西中医药大学引进博士科研启动基金(2018BS021);广西研究生教育创新计划(YCSY20190031)。

摘　　要：目的探讨玉屏风散及其有效组分抑制肥大细胞活化脱颗粒的过程是否与PI3K/Akt/mTOR信号通路相关。方法在体外用细胞计数CCK-8(cell counting kit-8)方法观察玉屏风散对P815肥大细胞是否有抑制其存活性的作用;用胰蛋白酶(1μg/mL)刺激P815肥大细胞的方法建立肥大细胞脱颗粒模型,收集细胞上清液,采用酶联免疫吸附法检测细胞上清液中β-氨基己糖苷酶(β-hexosaminidase,β-Hex)、组胺、白细胞介素(interleukin,IL)-4、IL-13、血小板激活因子(platelet activating factor,PAF)等相关细胞因子释放浓度变化;蛋白质印迹法(Western blot)检测相关蛋白磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinases,PI3K)、蛋白激酶B(protein kinase B,PKB/Akt)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)及其磷酸化水平的表达;实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)检测PI3K、Akt、mTOR在mRNA水平上的表达量变化。结果玉屏风散浓度为25μg/mL以下时对细胞活性无明显抑制作用,在浓度为12.5μg/mL、25μg/mL时玉屏风散能有效抑制β-Hex、组胺的释放及IL-4、IL-13、PAF的分泌(P<0.05);Western blot结果显示玉屏风散有一定抑制PI3K、Akt、mTOR的磷酸化作用(P<0.05);qRT-PCR结果显示其能降低PI3K、Akt、mTOR的表达(P<0.05)。结论玉屏风散对肥大细胞脱颗粒活化的抑制作用可能是通过抑制PI3K/Akt/mTOR信号通路实现的。Objective To investigate whether the mechanism of Yupingfeng powder and its effective components inhibiting the activation and degranulation of mast cells is related to PI3K/Akt/mTOR signaling pathway.Methods CCK-8 method was used to observe whether Yupingfeng powder can inhibit the viability of P815 mast cells in vitro.Mast cell degranulation model was established by stimulating P815 mast cells with trypsin(1μg/mL),and the supernatant of cells was collected.ELISA method was used to detect the changes of the release concentration ofβ-hexosaminidase(β-Hex),histamine,interleukin(IL)-4,IL-13,platelet activating factor(PAF)and other related cytokines in cell supernatant;Western blot was used to detect the expression of related proteins phosphatidylinositol-3-kinase(PI3K),protein kinase B(PKB/Akt),mammalian target of rapamycin(mTOR)and its phosphorylation levels;real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the changes of PI3K,Akt,mTOR,and mRNA levels.Results At the concentration of 12.5μg/mL and 25μg/mL,Yupingfeng powder can effectively inhibit the release ofβ-Hex,histamine and the secretion of IL-4,IL-13 and PAF(P<0.05).Western blot results showed that Yupingfeng powder could inhibit the phosphorylation of PI3K,Akt and mTOR(P<0.05);qRT-PCR results showed that it can reduce the expression of PI3K,Akt and mTOR(P<0.05).Conclusion The inhibitory effect of Yupingfeng powder on the degranulation activation of mast cells may be achieved through inhibition of PI3K/Akt/mTOR signaling pathway.

关 键 词：玉屏风散 肥大细胞 脱颗粒活化 PI3K/Akt/mTOR信号通路 过敏性疾病 β-氨基己糖苷酶 白细胞介素 血小板激活因子 

分 类 号：R285.5[医药卫生—中药学]