摘要
目的研究调衡方多糖(Tiaoheng prescription polysaccharides,ThPPs)对巨噬细胞的调节作用及相关信号通路的影响,探讨其调节机体免疫的作用机制。方法获取小鼠腹腔巨噬细胞(macrophage,Mφ)并体外培养,随机分为空白对照组,LPS模型组(1μg·mL^(-1)),ThPPs小剂量(50μg·mL^(-1))、中剂量(150μg·mL^(-1))、大剂量(250μg·mL^(-1))组。干预24 h后收集细胞,采用CCK-8法考察其增殖活性;采用格里斯试剂法(Griess)检测细胞上清液中一氧化氮(NO)的分泌量;用免疫组化法测巨噬细胞表面白介素-12(IL-12)的表达量;采用实时荧光定量PCR测定Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核因子-κB(NF-κB)、肿瘤坏死因子受体相关因子-6(TRAF-6)及肿瘤坏死因子-α(TNF-α)的mRNA表达情况。Western blotting法检测TLR4、NF-κB、TNF-α的蛋白表达情况。结果ThPPs中、高剂量组与空白组比较,可显著增强Mφ对NO、IL-12分泌的功能(P<0.01);Real-time PCR与免疫印迹法显示ThPPs中、高剂量组与空白组比较,可显著上调TLR4、TNF-α、NF-κB、MyD88与TRAF-6的基因表达及TLR4、TNF-α、NF-κB的蛋白表达(P<0.01)。结论实验结果显示ThPPs可增强Mφ对NO、IL-12分泌的功能,上调TLR4、TNF-α、NF-κB、MyD88与TRAF-6的基因表达及TLR4、TNF-α、NF-κB的蛋白表达,而IL-12及TNF-α又是调控辅助性T细胞(Th)及Mφ分型的关键细胞因子。ThPPs激活Mφ信号通路可能与增强Mφ对NO、IL-12分泌的功能及活化NF-κB通路有关。
Objective To study the regulatory effect of Tiaoheng prescription polysaccharides(ThPPs)on macrophages and related signal pathways,and to explore the mechanism of its regulatory effect on immune system.Methods The peritoneal macrophages(Mφ)were obtained and cultured in vitro.They were randomly divided into three groups:blank control group,LPS stimulation group(1μg/mL),small Th PPs group(50μg/mL),medium ThPPs group(150μg/mL)and large ThPPs group(250μg/mL).After 24 hours of intervention,cells were collected and their proliferative activity was examined by CCK-8 method and Griess reagent method the secretion of NO,the expression of IL-12 on the surface of macrophages was measured by immunohistochemistry,TLR4,MyD88,NF-κB,TRAF-6 and TNF-αmRNA were detected by real-time quantitative PCR condition.Western blotting was used to detect the expression of TLR4,NF-κB and TNF-α.Results Compared with the blank group,the medium and high dose of ThPPs could significantly enhance the function of NO and IL-12 secretion(P<0.01);real time PCR and Western blotting showed that the medium and high dose of ThPPs could significantly increase the gene expression of TLR4,TNF-α,NF-κB,MyD88,TRAF-6 and the expression of TLR4,TNF-α,NF-κB(P<0.01).Conclusion ThPPs can enhance the secretion of NO and IL-12,up regulate the gene expression of TLR4,TNF-α,NF-κB,MyD88 and TRAF-6,and the expression of TLR4,TNF-α,NF-κB,while IL-12 and TNF-αare the key cytokines to regulate Th and Mφtyping.ThPPs activation of the Mφsignal pathway is related to the enhancement of the function of Mφin NO and IL-12 secretion,activating NF-κB pathway.
作者
张宏方
李文侠
刘洋
徐洋洋
谭芷芬
王媛媛
张怡敏
ZHANG Hongfang;LI Wenxia;LIU Yang;XU Yangyang;TAN Zhifen;WANG Yuanyuan;ZHANG Yimin(Shaanxi University of Traditional Chinese Medicine,Xianyang 712046,Shaanxi,China)
出处
《辽宁中医药大学学报》
CAS
2021年第7期9-14,共6页
Journal of Liaoning University of Traditional Chinese Medicine
基金
陕西省科技厅自然基础科研基金(2019JM-551)
陕西省中医管理局科研基金(JCP002)
关键词
调衡方多糖
巨噬细胞
TOLL样受体4
核因子-ΚB
细胞因子
Tiaoheng prescription polysaccharides
macrophages
toll like receptor 4
nuclear factor kappa B
cytokine
