摘要
目的:观察犀角地黄汤合方含药血清(XJDH)对体外诱导的树突状细胞(DC)相关功能的影响,探讨犀角地黄汤合方治疗ITP的作用机制。方法:采集6名健康志愿者外周血样本,利用密度梯度离心法分离出单个核细胞,利用磁珠分选出CD14+单核细胞,用重组人粒细胞⁃巨噬细胞集落刺激因子(GM⁃CSF)及重组人白介素4(IL⁃4)诱导成DC。将DC分为3组:对照组、模型组和XJDH组。通过CCK⁃8法确定含药血清干预浓度及时间,模型组和XJDH组分别加入终浓度为1μg/mL的脂多糖(LPS)刺激24 h后诱导DC成熟,对照组、模型组加入正常大鼠血清,XJDH组加入犀角地黄汤合方含药血清均干预24 h。利用流式细胞术(FCM)检测DC表面分子CD80,CD83,HLA⁃DR的水平,利用蛋白印迹法(WB)检测细胞TLR4和NF⁃κB表达情况,用酶联免疫吸附测定(ELISA)检测细胞上清液中IL⁃6,IL⁃12,TNF⁃α的水平。结果:与对照组相比,LPS刺激能够使DC表面分子CD80、CD83、HLA⁃DR表达增加,TLR4和NF⁃κB表达随之上升,分泌的相关促炎因子IL⁃6、IL⁃12、TNF⁃α增加(P<0.05)。相比于模型组,经XJDH干预后DC表面分子CD80、CD83、HLA⁃DR表达减少(P<0.05),TLR4、NF⁃κB蛋白表达下降(P<0.05),DC细胞上清液中IL⁃6、IL⁃12、TNF⁃α水平明显降低(P<0.05)。结论:犀角地黄汤合方含药血清可下调TLR4/NF⁃κB信号通路相关蛋白表达,抑制DC成熟,减少促炎因子的分泌,这可能是犀角地黄汤合方治疗免疫性血小板减少症的机制之一。
Objective:To observe the effects of drug⁃containing serum of Xijiao Dihuang combined prescription(XJDH)on the related functions of dendritic cells(DCs)induced in vitro,and to explore the mechanisms underlying the effectiveness of XJDH treatment on primary immune thrombocytopenia(ITP).Methods:Peripheral blood samples were collected from 6 healthy volunteers.Mononuclear cells were isolated by density gradient centrifugation,and CD14+mononuclear cells were collected by the magnetic separation technique.CD14+mononuclear cells were induced into immature DCs by recombinant human granulocyte⁃macrophage colony stimulating factor(GM⁃CSF)and recombinant human interleukin 4(IL⁃4).Immature DCs were divided into three groups:control group,model group and XJDH group.CCK⁃8 assay was used to determine the intervention concentration and time of drug⁃containing serum.Lipopolysaccharide(LPS)with the final concentration of 1μg/ml was added to model group and XJDH group respectively for 24 h to induce DCs maturation.Normal rat serum was added to control group and model group,and XJDH was added to XJDH group for 24 h.Flow cytometry was used to detect the levels of CD80,CD83 and HLA⁃DR on the surface of DCs.Western blot was used to detect the expression of TLR4 and NF⁃κB,and levels of IL⁃6,IL⁃12 and TNF⁃αin cell supernatant was detected by ELISA.Results:Compared with the control group,LPS stimulation increased the expression of CD80,CD83 and HLA⁃DR,with subsequent increasing expression of TLR4 and NF⁃κB,as well as IL⁃6,IL⁃12 and TNF⁃αincreased(P<0.05).In comparison with model group,the expression of DCs surface molecules CD80,CD83 and HLA⁃DR,DCs’expression of TLR4 and NF⁃κB protein,and the levels of IL⁃6,IL⁃12 and TNF⁃αin the cell supernatant of XJDH group decreased after the intervention of XJDH(P<0.05).Conclusion:Drug⁃containing serum of Xijiao Dihuang combined prescription can down⁃regulate TLR4/NF⁃κB signaling pathway related protein expression,inhibit DCs maturation,and red
作者
杨武霞
吴玉红
王梦晓
倪润丰
范栗玮
李润杰
李萌
王爱迪
刘宝山
YANG Wu-Xia;WU Yu-Hong;WANG Meng-Xiao;NI Run-Feng;FAN Li-Wei;LI Run-Jie;LI Meng;WANG Ai-Di;LIU Bao-Shan(Tianjin Medical University;General Hospital of Tianjin Medical University,Tianjin 300041,China)
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2022年第4期1176-1181,共6页
Journal of Experimental Hematology
基金
国家自然科学基金(82004334)。
关键词
犀角地黄汤合方
免疫性血小板减少症
树突细胞
炎症因子
脂多糖
Xijiao Dihuang combined prescription
primary immune thrombocytopenia
dendritic cells
inflammatory factor
lipopolysaccharide
