当归多糖作为潜在佐剂对细胞的免疫激活及机制研究  

Research on immune activation and mechanism of Angelica sinensis polysaccharide as potential adjuvant in cells

作　　者：何沅聪 尤朋涛 黄雅宁[1] 张剑锋 苟君波 王建 HE Yuan-cong;YOU Peng-tao;HUANG Ya-ning;ZHANG Jian-feng;GOU Jun-bo;WANG Jian(School of Pharmacy,Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei Key Laboratory of Resources and Chemistry of Chinese Medicine,Hubei University of Chinese Medicine,Wuhan 430065,China;Xuancheng Institutes of Food and Drug Control,Xuancheng 242000,China;Hubei Shizhen Laboratory,Wuhan 430061,China)

机构地区：[1]湖北中医药大学药学院,湖北武汉430065 [2]湖北中医药大学中药资源与中药化学湖北省重点实验室,湖北武汉430065 [3]宣城市食品药品检验中心,安徽宣城242000 [4]湖北时珍实验室,湖北武汉430061

出　　处：《药学学报》2025年第3期637-645,共9页Acta Pharmaceutica Sinica

基　　金：湖北省科技厅自然科学基金青年项目(2023AFB389);湖北省药品监督检验研究院重点实验室开放课题项目(2023HBKFZ002);湖北中医药大学重大科技攻关项目(2023ZDXM007);湖北中医药大学博士启动项目(2023ZXB012)。

摘　　要：本研究旨在探讨当归多糖(Angelica sinensis polysaccharide,ASP)作为潜在疫苗佐剂对巨噬细胞RAW264.7免疫激活作用及其释放细胞因子的影响,同时阐明其潜在的信号通路作用机制。采用细胞计数试剂(cell counting kit-8,CCK-8)法检测细胞活力,流式细胞术检测5个浓度梯度的ASP对RAW264.7细胞表面分子白细胞分化抗原(cluster of differentiation,CD)80、CD86和主要组织相容性复合物II(major histocompatibility complex II,MHC II)表达的影响,ELISA法测定细胞上清液中白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)等细胞因子的含量;quantitative polymerase chain reaction(qPCR)检测Toll样受体4(Tolllikereceptor4,TLR4)、髓样分化因子88(myeloiddifferentiationfactor88,MyD88)、TNF受体相关因子6(TNF receptor associated factor 6,TRAF6)、核因子κB(nuclear factor kappa-B,NF-κB)信号通路的mRNA表达水平,Western blot检测TRAF6、IκB激酶(IκB kinase,IKK)和p65蛋白磷酸化水平。结果显示,不同浓度的ASP对RAW264.7无细胞毒性。ASP均能浓度依赖性地促进RAW264.7细胞表面分子CD80、CD86和MHC II的表达上调(P<0.05)。不同浓度的ASP组均使得IL-6和TNF-α等的分泌水平有显著的提升(P<0.05),且在一定范围内存在量-效关系。qPCR结果显示,不同浓度的ASP均使得TLR4、MyD88、TRAF6和NF-κB的mRNA表达水平上调。不同浓度的ASP均能提高TRAF6、p-IKK和p-p65的表达水平。TLR4受体抑制剂TAK-242能显著降低ASP诱导的细胞因子分泌量(P<0.05)。本研究揭示了ASP可通过TLR4等信号通路显著增强共刺激分子和细胞因子的表达,显示出作为疫苗佐剂的潜力。The purpose of this study is not only to investigate the effects of Angelica sinensis polysaccharide(ASP)as a potential vaccine adjuvant on immune activation and cytokine release in RAW264.7 macrophages,but also to elucidate its underlying involved signaling mechanisms.Cell viability was evaluated by the CCK-8 assay.Flow cytometry was used to analyze the influence of ASP at five distinct concentration gradients on the expression of cluster of differentiation(CD)80,CD86,and major histocompatibility complex II(MHC II)on RAW264.7 cell surfaces.The levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in cell culture supernatant were determined by enzyme-linked immunosorbent assay(ELISA)method.Molecular techniques,including quantitative polymerase chain reaction(qPCR)were utilized to assess the mRNA expression levels of Toll-like receptor 4(TLR4)-myeloid differentiation factor 88(MyD88)-TNF receptor associated factor 6(TRAF6)-nuclear factor kappaB(NF-κB)signaling pathway.The levels of TRAF6,and the phosphorylation levels of IκB kinase(IKK)and p65proteins were detected by Western blot.The results show that ASP at varying concentrations promote the proliferation of RAW264.7 cells without cytotoxicity.Surface molecules CD80,CD86,and MHC II on RAW264.7cells showed statistically significant up-regulation in response to ASP compared to the blank control(P<0.05),with a dose-dependent effect within an optimal range.Furthermore,ASP also elevated cytokines IL-6 and TNF-αsecretion levels by RAW264.7 cells compared to the normal control(P<0.05),exhibiting a dose-response relationship within a specific concentration span.The qPCR results indicated that ASP groups at different concentrations all led to upregulation of mRNA expression levels of TLR4,MyD88,TRAF6,and NF-κB signaling pathway.The expression levels of TRAF6,p-IKK and p-p65 were increased by different concentrations of ASP.The TLR4 inhibitor TAK-242 significantly reduced the secretion of cytokines induced by APS(P<0.05).This study highlights the immunostim

关 键 词：当归多糖 佐剂 免疫活性 TLR4信号通路 RAW264.7细胞 

分 类 号：R966[医药卫生—药理学]