文章摘要
陈思思,钱丽梅,陈艳春.防风提取物对IgE致敏肥大细胞的改善作用及机制研究[J].浙江中医药大学学报,2024,48(2):138-146.
防风提取物对IgE致敏肥大细胞的改善作用及机制研究
Study of Improvement Effects and Mechanism of Saposhnikovia Divaricata Extract on IgE Sensitized RBL-2H3 Cells
DOI：10.16466/j.issn1005-5509.2024.02.002
中文关键词: 防风提取物  RBL-2H3细胞  细胞脱颗粒  F-肌动蛋白  组胺  炎症因子
英文关键词: Saposhnikovia divaricata extract  RBL-2H3 cell  cell degranulation  F-actin  histamine  inflammatory factors
基金项目:浙江省中医药管理局项目(2021ZA104)；杭州市科技发展计划项目(20201203B216)；湖州市科学技术局公益性应用研究项目(2019GY06)
作者单位
陈思思 湖州市第三人民医院 浙江，湖州 313000 
钱丽梅 杭州市第七人民医院 
陈艳春 杭州市中医院 
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中文摘要:
      [目的] 探究防风(Saposhnikovia divaricata，SD)提取物对大鼠嗜碱性白血病细胞RBL-2H3脱颗粒的影响及作用机制。 [方法] 采用噻唑蓝(methylthialazole tetrazolium，MTT)比色法检测，根据5、25、50、100、200、400 μg·mL-1 SD提取物对RBL-2H3细胞活性的影响，确定后续实验浓度。以免疫球蛋白E(immunoglobulinE，IgE)诱导建立RBL-2H3细胞脱颗粒模型。设立空白对照组、模型组、低剂量SD提取物组(5 μg·mL-1)、中剂量SD提取物组(25 μg·mL-1)、高剂量SD提取物组(50 μg·mL-1)和地塞米松(dexamethasone，DXMS)组(100 μg·mL-1)，干预30 min。MTT法检测低、中、高剂量SD提取物对RBL-2H3细胞脱颗粒模型活性的影响。甲苯胺蓝染色观察脱颗粒细胞形态，计算细胞脱颗粒率。免疫荧光染色测定细胞F-肌动蛋白(F-actin)表达。酶联免疫吸附试验(enzyme linked immunosorbent assay，ELISA)检测细胞β-氨基己糖苷酶、组胺、白细胞介素-4(interleukin-4，IL-4)、白细胞介素-6(interleukin-6，IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)、干扰素-γ(interferon-γ，IFN-γ)水平。免疫印迹法检测细胞磷脂酰肌醇-3-羟基激酶(phosphoinositide-3 kinase，PI3K)、磷酸化-PI3K(phosphorylation-PI3K，p-PI3K)、蛋白激酶B(protein kinase B，AKT)、磷酸化-AKT(phosphorylation-AKT，p-AKT)、p38丝裂原活化蛋白激酶(p38 mitogen activited protein kinaseelisa，p38MAPK)、磷酸化-p38MAPK(phosphorylation-p38MAPK，p-p38MAPK)、核因子-κB(nuclear factor-κB，NF-κB)、磷酸化-NF-κB(phosphorylation-NF-κB，p-NF-κB)、细胞外调节激酶(extracellular regulated kinases，ERK)、磷酸化-ERK(phosphorylation-ERK，p-ERK)蛋白表达。[结果] 低、中、高剂量防风提取物(5、25、50 μg·mL-1)对RBL-2H3细胞活性无显著影响(P>0.05)。与空白对照组比较，模型组甲苯胺蓝染色细胞数量减少、形态变圆，细胞脱颗粒率显著上升，F-actin表达下降，β-氨基己糖苷酶、组胺、IL-4、IL-6、TNF-α水平升高，IFN-γ水平降低，p-PI3K/PI3K、p-AKT/AKT、p-p38MAPK/p38MAPK、p-NF-κB/NF-κB、p-ERK/ERK表达升高(P<0.01)。与模型组比较，低、中、高剂量SD提取物组和DXMS组细胞F-actin表达增加，β-氨基己糖苷酶、组胺、IL-4、IL-6、TNF-α释放显著下降(P<0.05，P<0.01)，IFN-γ释放显著增加(P<0.01)，p-PI3K/PI3K、p-AKT/AKT、p-p38MAPK/p38MAPK、p-NF-κB/NF-κB、p-ERK/ERK表达降低(P<0.05，P<0.01)；中、高剂量SD提取物组和DXMS组细胞数量增加，形态多呈梭形，细胞脱颗粒率显著下降(P<0.01)。与低剂量SD提取物组比较，高剂量SD提取物组和DXMS组细胞脱颗粒率下降(P<0.01)、F-actin表达增加(P<0.05)、p-p38MAPK/p38MAPK表达降低(P<0.01)。[结论] SD提取物可抑制IgE致敏的RBL-2H3细胞脱颗粒，降低炎性介质水平，其作用机制可能与抑制PI3K/AKT、p38MAPK/NF-κB、ERK蛋白磷酸化有关。
英文摘要:
      [Objective] To explore the degranulation effect and mechanism of Saposhnikovia divaricata(SD) extract on rat basophilic leukemia cell line RBL-2H3 cell. [Methods] Methylthialazole tetrazolium(MTT) test was used to select the concentrations in the subsequent experiments based on impact of 5，25，50，100，200，400 μg·mL-1 SD extract on the activity of RBL-2H3 cells. Immunoglobulin E(IgE) induction was used to establish RBL-2H3 cell degranulation model. Blank control group， model group， low dose SD extract group(5 μg·mL-1)， medium dose SD extract group(25 μg·mL-1)， high dose SD extract group (50 μg·mL-1) and dexamethasone(DXMS) group(100 μg·mL-1) were set up， with intervention for 30 minutes. MTT test was used to detect the effect of low， medium， high-dose SD extract on activity of RBL-2H3 cell degranulation model. Toluidine blue staining was used to observe the morphology of degranulation cells and calculate degranulation rates. Immunofluorescence staining was used to detect expression of F-actin. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of β-aminohexosidase， histamine， interleukin-4(IL-4)， interleukin-6(IL-6)， tumor necrosis factor-α(TNF-α)， interferon-γ (IFN-γ). Western blot was used to detect the expressions of phosphatidylinositide-3 kinase(PI3K)， phosphorylation-PI3K(p-PI3K)， protein kinase B(AKT)， phosphorylation-AKT(p-AKT)， p38 mitogen activated protein kinase(p38MAPK)， phosphorylation-p38MAPK(p-p38MAPK)， nuclear factors-κB(NF-κB)， phosphorylation-NF-κB(p-NF-κB)， extracellular regulated kinases(ERK) and phosphorylation-ERK(p-ERK) protein. [Results] The low， medium， high doses of SD extract (5，25，50 μg·mL-1) had no significant effects on the activity of RBL-2H3 cells (P>0.05). Compared with blank control group， the number of toluidine blue stained cells of model group was decreased， cells shape rounded， degranulation rate was increased， expression of F-actin was decreased， the levels of β- aminohexosidase， histamine， IL-4， IL-6， TNF-α were increased， IFN-γ level was decreased， the expressions of p-PI3K/PI3K， p-AKT/AKT， p-p38MAPK/p38MAPK， p-NF-κB/NF-κB and p-ERK/ERK were increased(P<0.01). Compared with model group， the F-actin expression of low， medium， high doses of SD extract groups and DXMS group was increased， levels of β-aminohexosidase， histamine， IL-4， IL-6 and TNF-α were decreased， IFN-γ level was increased(P<0.01)， the expressions of p-PI3K/PI3K， p-AKT/AKT， p-p38MAPK/p38MAPK， p-NF-κB/NF-κB and p-ERK/ERK were decreased(P<0.05， P<0.01); the number of toluidine blue stained cells in medium， high dose SD extract groups and DXMS group was increased with spindle cell shape， degranulation rate was decreased(P<0.01). Compared with low dose SD extract group， degranulation rate of high dose SD extract group and DXMS group was decreased(P<0.01)， F-actin expression was increased(P<0.05)， p-p38MAPK/p38MAPK expressions were decreased(P<0.01). [Conclusion] SD extract inhibited degranulation of IgE sensitized RBL-2H3 cell and decreased the levels of inflammatory mediators， its mechanism may be related to the inhibition the phosphorylation expression of PI3K/AKT， p38MAPK/NF-κB and ERK.
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