双氢青蒿素对类风湿关节炎患者外周血单个核细胞TLR/MyD88信号通路的影响研究

doi:10.12114/j.issn.1007-9572.2021.00.061

摘要/Abstract

摘要： 背景　类风湿关节炎（RA）的发病机制复杂，已有研究证实Toll样受体（TLR）在RA的发病中起关键作用，其表达于树突状细胞、巨噬细胞、T细胞、B细胞等细胞表面，识别病原体不同的分子结构，进而上调炎性细胞因子和趋化因子，激活细胞内的信号转导通路，引起宿主细胞发生自身炎性反应，在自身免疫性疾病中发挥重要作用。目的　观察双氢青蒿素（DHA）对RA患者外周血单个核细胞（PBMCs）TLR4/MyD88信号通路及蛋白表达的影响。方法　选取2017年10月—2018年12月于内蒙古科技大学包头医学院第二附属医院就诊的活动期RA患者10例为观察组，同时随机选取本院体检中心健康体检者10例为对照组。取两组外周血PBMCs分离培养，将PBMCs分为5组，每组设5个复孔。（1）对照组：健康志愿者外周血PBMCs，不给予干预TLR介导的信号通路；（2）RA组：RA患者外周血PBMCs，不给予干预TLR介导的信号通路；（3）TLR2抑制剂组：RA患者外周血PBMCs，给予100 μg/L的TLR2抑制剂干预TLR介导的信号通路；（4）DHA低剂量组：RA患者外周血PBMCs，给予200 μmol/L的DHA干预TLR介导的信号通路；（5）DHA高剂量组：RA患者外周血PBMCs，给予1 000 μmol/L的DHA干预TLR介导的信号通路。检测并比较5组TLR2阳性率、TLR2 mRNA、MyD88 mRNA、TLR2、MyD88、肿瘤坏死因子α（TNF-α）及白介素6（IL-6）水平。结果　RA组TLR2阳性率高于对照组、TLR2抑制剂组（P<0.05）；DHA低剂量组TLR2阳性率高于DHA高剂量组（P<0.05）。RA组TLR2 mRNA、MyD88 mRNA高于对照组、TLR2抑制剂组（P<0.05）；DHA低剂量组TLR2 mRNA、MyD88 mRNA高于DHA高剂量组（P<0.05）。TLR2抑制剂组、RA组、DHA低剂量组、DHA高剂量组TLR2、MyD88高于对照组（P<0.05）。RA组TLR2、MyD88高于TLR2抑制剂组（P<0.05）；DHA低剂量组TLR2、MyD88高于DHA高剂量组（P<0.05）。TLR2抑制剂组、RA组、DHA低剂量组、DHA高剂量组TNF-α、IL-6水平高于对照组（P<0.05）。RA组TNF-α、IL-6水平高于TLR2抑制剂组（P<0.05）；DHA低剂量组TNF-α、IL-6水平高于DHA高剂量组（P<0.05）。结论　DHA可抑制RA患者的PBMCs中TLR2、MyD88表达和上清液中TNF-α及IL-6水平，可能与其可抑制TLR4/MyD88信号通路介导的炎性反应有关。

关键词: 关节炎, 类风湿；双氢青蒿素；Toll样受体；MyD88；肿瘤坏死因子α；白细胞介素6

Abstract: Background　The pathogenesis of rheumatoid arthritis（RA） is complicated.Previous studies have proved that toll-like receptor（TLR） plays a key role in the development of RA，which is expressed on the surface of cells such as dendritic cells，macrophages，T cells，B cells，identifies different molecular structures of pathogens，and then raises the inflammatory cytokines and chemokines，activates intracellular signal transduction pathways，causes an autoinflammatory response in the host cell，playing an important role in autoimmune diseases.Objective　To observe the effect of dihydroartemisinin （DHA） on TLR-mediated MyD88-dependent signaling pathway of peripheral blood mononuclear cells（PBMCs） and the express of protein.Methods　From the Second Affiliated Hospital of Baotou Medical College，Inner Mongolia University of Science & Technology from October 2017 to December 2018，10 active RA patients and 10 randomly selected physical examinees from the medical examination center were enrolled.PBMCs of two groups were isolated，cultured and divided into 5 groups with 5 multiple pores in each group.（1）control group：the PBMCs of the healthy volunteers were not interfered with TLR-mediated signaling pathway.（2）RA group：the PBMCs of RA patients were not interfered with TLR-mediated signal pathway.（3）TLR2 inhibitors group：the PBMCs of RA patients were treated with TLR2 inhibitors（100 μg/L） to interfere with TLR-mediated signaling pathway.（4）Low-dose DHA group：the PBMCs of RA patients were treated with DHA（200 μmol/L） to interfere with TLR-mediated signaling pathway.（5）High-dose DHA group：the PBMCs of RA patients were treated with DHA（1 000 μmol/L） to interfere with TLR-mediated signaling pathway.The positive expression rate of TLR2，and levels of TLR2 mRNA，MyD88 mRNA，TLR2，MyD88，TNF-α and IL-6 were detected and compared among the five groups.Results　（1）The positive expression rate of TLR2 in RA group was higher than that in control or TLR2 inhibitors group （P<0.05）.The positive expression rate of TLR2 in low-dose DHA group was higher than that in high-dose DHA group （P<0.05）.（2）RA group had higher average levels of TLR2 mRNA and MyD88 mRNA than control group or TLR2 inhibitors group（P<0.05）.Low-dose DHA group had higher average levels of TLR2 mRNA and MyD88 mRNA than high-dose DHA group （P<0.05）.（3） The control group had lower average levels of TLR2 and MyD88 than each of other groups（P<0.05）.The average levels of TLR2 and MyD88 in RA group were higher than those of TLR2 inhibitors group（P<0.05）.The average levels of TLR2 and MyD88 in low-dose DHA group were higher than those of high-dose DHA group（P<0.05）.（4） The control group had lower average levels of TNF-α and IL-6 than each of other groups（P<0.05） .The average levels of TNF-α and IL-6 in RA group were higher than those of TLR2 inhibitors group（P<0.05）.The average levels of TNF-α and IL-6 in low-dose DHA group were higher than those of high-dose DHA group（P<0.05）.Conclusion　DHA may inhibit the expression of TLR2 and MyD88 in mononuclear cells of RA patients，and the levels of TNF-αand IL-6 in the supernatant，which may be related to the inhibition of the inflammatory response by the TLR4-mediated MyD88-dependent signaling pathway.

Key words: Arthritis, rheumatoid；Dihydroartemisinin；Toll-Like Receptors；MyD88；Tumor Necrosis Factor-alpha；Interleukin-6

闫慧明,安燕,张雪,等. 双氢青蒿素对类风湿关节炎患者外周血单个核细胞TLR/MyD88信号通路的影响研究[J]. 中国全科医学, 2021, 24(14): 1780-1784. DOI: 10.12114/j.issn.1007-9572.2021.00.061.
YAN Huiming,AN Yan,ZHANG Xue, et al. Effect of Dihydroartemisinin on TLR-mediated MyD88-dependent Signaling Pathway in Peripheral Blood Mononuclear Cells in Patients with Rheumatoid Arthritis[J]. Chinese General Practice, 2021, 24(14): 1780-1784. DOI: 10.12114/j.issn.1007-9572.2021.00.061.

参考文献

［1］张洪长，刘明昕，张莹，等．青藤碱对类风湿关节炎成纤维样滑膜细胞MyD88、TRAF-6表达的影响［J］．中国免疫学杂志，2015，31（4）：485-489．DOI：10.3969/j.issn.1000-484X.2015.04.011.
ZHANG H C，LIU M X，ZHANG Y，et al．Effect of sinomenine on the expression of MyD88 and TRAF-6 in fibroblast like synoviocytes of rheumatoid arthritis［J］．Chin J Immunol，2015，31（4）：485-489．DOI：10.3969/j.issn.1000-484X.2015.04.011.
［2］HOU L F，HUANG H C.Immune suppressive properties of artemisinin family drugs［J］．Pharmacol Ther，2016，166：123-127．DOI：10.1016/j.pharmthera.2016.07.002.
［3］吴言为.青蒿素类衍生物SM934对MRL/Ipr狼疮小鼠的疗效作用及机制研究［C］//中国药理学会抗炎免疫药理专业委员会第十一届全国学术会议论文集．苏州，2014：162-163.
［4］蔡辉，姚茹冰.新编类风湿关节炎手册［M］．北京：人民军医出版社，2013：80-81.
［5］MCINNES I B，SCHETT G.The pathogenesis of rheumatoid arthritis［J］．N Engl J Med，2011，365（23）：2205-2219．DOI：10.1056/nejmra1004965.
［6］GOH F G，MIDWOOD K S.Intrinsic danger：activation of Toll-like receptors in rheumatoid arthritis［J］．Rheumatology （Oxford），2012，51（1）：7-23．DOI：10.1093/rheumatology/ker257.
［7］王圆圆.青藤碱对类风湿关节炎患者外周血单核细胞TLR4/MyD88/NF-κB信号通路的影响［D］．合肥：安徽中医药大学，2015.
WANG Y Y.Effect of sinomenine on TLR4/MyD88/NF-κ B signaling pathway in peripheral blood mononuclear cells of patients with rheumatoid arthritis［D］．Hefei：Anhui University of traditional Chinese Medicine，2015.
［8］钱雷，汪晓莺，吕丽君，等．类风湿性关节炎患者早期外周血单个核细胞Toll样受体2、4表达及意义［J］．检验医学，2012，8（27）：665-669．DOI：10.3969/j.issn.1673-8640.2012.08.011.
QIAN L，WANG X Y，LYU L J，et al．Expression and significance of Toll like receptor 2 and 4 in peripheral blood mononuclear cells of patients with rheumatoid arthritis［J］．Laboratory Medicine，2012，8（27）：665-669．DOI：10.3969/j.issn.1673-8640.2012.08.011.
［9］ADHIKARY R，MAJHI A，MAHANTI S，et al．Protective effects of methanolic extract of Adhatoda vasica Nees leaf in collagen-induced arthritis by modulation of synovial toll-like receptor-2 expression and release of pro-inflammatory mediators［J］．Journal of Nutrition &Intermediary Metabolism，2016，10：1-11．DOI：10.1016/j.jnim.2015.11.001.
［10］蔡辉，张群燕，姚茹冰.脂多糖对类风湿关节炎患者外周血单核细胞 TLR2、TLR4 mRNA及其蛋白表达的影响［J］．贵州医科大学学报，2017，42（1）：68-70．DOI：10.19367/j.cnki.1000-2707.2017.01.015.
CAI H，ZHANG Q Y，YAO R B.Effect of lipopolysaccharide on the expression of TLR2，TLR4 mRNA in peripheral blood mononuclear cells of patients with rheumatoid arthritis［J］．Journal of Guizhou Medical University，2017，42（1）：68-70．DOI：10.19367/j.cnki.1000-2707.2017.01.015.
［11］XU H，HE Y，YANG X，et al．Anti-malarial agent artesunate inhibits TNF-αlpha-induced production of proinflammatory cytokines via inhibition of NF-kappaB and PI3 kinase/Akt signal pathway in human rheumatoid arthritis fibroblast-like synoviocytes［J］．Rheumatology（Oxford），2007，46（6）：920-926．DOI：10.1093/rheumatology/kem014.
［12］FENG F B，QIU H Y.Effects of Artesunate on chondrocyte proliferation，apoptosis and autophagy through the PI3K/ AKT/mTOR signaling pathway in rat models with rheumatoid arthritis［J］．Biomed Pharmacother，2018，102：1209-1220．DOI：10.1016/j.biopha.2018.03.142.

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