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

作　　者：陈思思[1] 钱丽梅 陈艳春[3] CHEN Sisi;QIAN Limei;CHEN Yanchun(Huzhou Third People's Hospital,Huzhou(313000),China;Hangzhou Seventh People's Hospital;Hangzhou Hospital of Traditional Chinese Medicine)

机构地区：[1]湖州市第三人民医院,浙江湖州3130002 [2]杭州市第七人民医院 [3]杭州市中医院

出　　处：《浙江中医药大学学报》2024年第2期138-146,共9页Journal of Zhejiang Chinese Medical University

基　　金：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)。

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、400μg·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(5μg·mL^(-1))、中剂量SD提取物组(25μg·mL^(-1))、高剂量SD提取物组(50μg·mL^(-1))和地塞米松(dexamethasone,DXMS)组(100μg·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、50μg·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-�[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 5,25,50,100,200,400μg·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(5μg·mL^(-1)),medium dose SD extract group(25μg·mL^(-1)),high dose SD extract group(50μg·mL^(-1))and dexamethasone(DXMS)group(100μg·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 ofβ-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(5,25,50μg·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 ofβ-aminohexosidase,histamine,IL-4,IL-6,TNF-αwere increased,IFN-γlevel was decreased,the expressions of p-PI3K/PI3K,p-AKT/

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 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]