Mechanism of Huayu Qiangshen Tongbi Decoction combined with methotrexate in treatment of rheumatoid arthritis based on exosomal microRNA research

NIU Yunbao; LIANG Huasheng; XIAO Yunting; WU Xiaodong; CHEN Xianghong; WANG Maojie; MEI Liyan; GAO Kaixin; HUANG Runyue; CHEN Xiumin

doi:10.16305/j.1007-1334.2024.z20240522001

您当前的位置：

首页 >

文章列表页 >

Mechanism of Huayu Qiangshen Tongbi Decoction combined with methotrexate in treatment of rheumatoid arthritis based on exosomal microRNA research

更新时间：2024-12-23

- Mechanism of Huayu Qiangshen Tongbi Decoction combined with methotrexate in treatment of rheumatoid arthritis based on exosomal microRNA research
- Shanghai Journal of Traditional Chinese Medicine Vol. 58, Issue 12, Pages: 137-144(2024)
- 作者机构：
  
  1.广州中医药大学第二临床医学院（广东广州 510120）
  2.广州中医药大学第二附属医院（广东省中医院）风湿科（广东广州 510120）
  3.广州中医药大学第二附属医院（广东省中医院）省部共建中医湿证国家重点实验室（广东广州 510120）
  4.广州中医药大学第二附属医院（广东省中医院）粤港澳中医药与免疫疾病研究联合实验室（广东广州 510120）
  5.广州中医药大学第二附属医院（广东省中医院）广东省中医药防治难治性慢病重点实验室（广东广州 510120）
- 作者简介：
- 基金信息：
- DOI：10.16305/j.1007-1334.2024.z20240522001
  CLC：
- Published：10 December 2024，
  
  Received：22 May 2024，
- 稿件说明：
移动端阅览
牛运宝,梁华胜,萧韵婷,等.基于外泌体微小RNA研究化瘀强肾通痹方联合甲氨蝶呤治疗类风湿关节炎的作用机制[J].上海中医药杂志,2024,58(12):137-144.

NIU Yunbao,LIANG Huasheng,XIAO Yunting,et al.Mechanism of Huayu Qiangshen Tongbi Decoction combined with methotrexate in treatment of rheumatoid arthritis based on exosomal microRNA research[J].Shanghai Journal of Traditional Chinese Medicine,2024,58(12):137-144.
牛运宝,梁华胜,萧韵婷,等.基于外泌体微小RNA研究化瘀强肾通痹方联合甲氨蝶呤治疗类风湿关节炎的作用机制[J].上海中医药杂志,2024,58(12):137-144. DOI： 10.16305/j.1007-1334.2024.z20240522001.

NIU Yunbao,LIANG Huasheng,XIAO Yunting,et al.Mechanism of Huayu Qiangshen Tongbi Decoction combined with methotrexate in treatment of rheumatoid arthritis based on exosomal microRNA research[J].Shanghai Journal of Traditional Chinese Medicine,2024,58(12):137-144. DOI： 10.16305/j.1007-1334.2024.z20240522001.

摘要

目的

从血清外泌体微小RNA（miRNA）角度研究化瘀强肾通痹方联合甲氨蝶呤（MTX）治疗类风湿关节炎（RA）的作用机制，为RA的临床治疗及新药研发提供思路。

方法

选取12例采用化瘀强肾通痹方联合MTX治疗后疗效较为明显的RA患者，收集治疗前后的全血样本，采用外泌体提取试剂盒提取血清外泌体，借助外泌体miRNA表达测序技术检测患者血清外泌体miRNA表达谱，通过数据库预测差异表达miRNA的靶基因，然后利用基因本体（GO）和京都基因和基因组百科全书（KEGG）富集分析解析差异表达miRNA的靶基因功能。

结果

①共构建24个miRNA文库，测序结果显示，治疗前后RA患者血清外泌体中有19个miRNA表达下调（

0.05）、1个miRNA表达上调（

0.05）。②实时荧光定量逆转录聚合酶链式反应（RT-qPCR）法检测结果显示，经治疗，RA患者血清外泌体中

hsa⁃miR⁃15b⁃3p

、

hsa⁃miR⁃16⁃5p

、

hsa⁃miR⁃199b⁃5p

、

hsa⁃miR⁃223⁃5p

、

hsa⁃miR⁃532⁃5p

、

hsa⁃miR⁃589⁃5p

、

hsa⁃miR⁃619⁃5p

相对表达量降低（

0.05），与miRNA测序结果一致。③靶基因预测涉及SMAD家族成员2基因（

SMAD2

）、成纤维细胞生长因子受体3基因（

FGFR3

）、C-C基序趋化因子配体2基因（

CXCL2

）和丝氨酸/苏氨酸蛋白激酶Pim-1原癌基因（

PIM1

）等。④大多数靶基因富集的通路包括机体系统、代谢、人类疾病和环境信息处理等，均与骨和软骨的破坏以及炎症有关。

结论

化瘀强肾通痹方联合MTX治疗RA的作用机制可能与调节血清外泌体miRNA及其靶基因来预防RA诱导的骨和软骨破坏以及炎症有关。

Abstract

Objective

To investigate the mechanism of the Huayu Qiangshen Tongbi Decoction （HQTD） combined with methotrexate （MTX） in the treatment of rheumatoid arthritis （RA） from the perspective of serum exosomal microRNAs （miRNAs）， providing new insights into the clinical treatment of RA and the development of new drugs.

Methods

Twelve RA patients who exhibited significant therapeutic responses to HQTD combined with MTX were selected for the study. Whole blood samples were collected from each patient both before and after treatment. Serum exosomes were isolated using commercially available exosome extraction kits， and the exosomal miRNA expression profiles were analyzed via miRNA sequencing technology. Differentially expressed miRNAs were identified， and their target genes were predicted using established databases. Functional enrichment analyses， including Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG）， were performed to elucidate the biological functions of the target genes associated with the differentially expressed miRNAs.

Results

①A total of 24 miRNA libraries were successfully constructed. Sequencing analysis revealed that 19 miRNAs were significantly downregulated （

0.05） and 1 miRNA was upregulated （

0.05） in the serum exosomes of RA patients after treatment. ②Real-time quantitative reverse transcription polymerase chain reaction （RT-qPCR） confirmed that after treatment， the relative expression levels of

hsa⁃miR⁃15b⁃3p， hsa⁃miR⁃16⁃5p， hsa⁃miR⁃199b⁃5p， hsa⁃miR⁃223⁃5p， hsa⁃miR⁃532⁃5p， hsa⁃miR⁃589⁃5p

， and

hsa⁃miR⁃619⁃5p

in serum exosomes of RA patients were reduced （

0.05）， aligning with the miRNA sequencing results. ③Target gene predictions identified genes such as SMAD family member 2 （

SMAD2

）， fibroblast growth factor receptor 3 （

FGFR3

）， C-C motif chemokine ligand 2 （

CXCL2

）， and serine/threonine-protein kinase Pim-1 proto-oncogene （

PIM1

）. ④Most of the pathways enriched for these target genes were associated with body systems， metabolism， huma

n diseases， and environmental information processing， implicating bone and cartilage destruction and inflammation in the disease process.

Conclusion

The mechanism by which HQTD combined with MTX treats RA may involve the regulation of serum exosomal miRNAs and their target genes， potentially preventing RA-induced bone and cartilage destruction and inflammation.

关键词

类风湿关节炎外泌体微小RNA中药复方甲氨蝶呤中西医结合疗法

Keywords

rheumatoid arthritisexosomesmicroRNAtraditional Chinese medicine compoundmethotrexateintegrated Chinese and Western medicine therapy

references

SCOTT D L， WOLFE F， HUIZINGA T W. Rheumatoid arthritis［J］. Lancet， 2010， 376（9746）： 1094-1108.

KERSCHBAUMER A， SEPRIANO A， BERGSTRA S A， et al. Efficacy of synthetic and biological DMARDs： a systematic literature review informing the 2022 update of the EULAR recommendations for the management of rheumatoid arthritis［J］. Ann Rheum Dis， 2023， 82（1）： 95-106.

MCINNES I B， SCHETT G. The pathogenesis of rheumatoid arthritis［J］. N Engl J Med， 2011， 365（23）： 2205-2219.

SCHAFFER D， FLORIN T， EAGLE C， et al. Risk of serious NSAID-related gastrointestinal events during long-term exposure：a systematic review［J］. Med J Aust， 2006， 185（9）： 501-506.

KATTURAJAN R， S V， RASOOL M， et al. Molecular toxicity of methotrexate in rheumatoid arthritis treatment：A novel perspective and therapeutic implications［J］. Toxicology， 2021， 461： 152909.

DIXON W G， HYRICH K L， WATSON K D， et al. Drug-specific risk of tuberculosis in patients with rheumatoid arthritis treated with anti-TNF therapy： results from the British Society for Rheumatology Biologics Register（BSRBR）［J］. Ann Rheum Dis， 2010， 69（3）： 522-528.

SZOSTAK B， MACHAJ F， ROSIK J， et al. Using pharmacogenetics to predict methotrexate response in rheumatoid arthritis patients［J］. Expert Opin Drug Metab Toxicol， 2020， 16（7）： 617-626.

WANG Y， CHEN S， DU K， et al. Traditional herbal medicine： Therapeutic potential in rheumatoid arthritis［J］. J Ethnopharmacol， 2021， 279： 114368.

时一鸣，常岑，姜平，等. 中药单体化合物调控NF⁃κB信号通路干预类风湿关节炎的研究进展［J］. 上海中医药杂志， 2023， 57（10）： 89-94.

陈秀敏，黄闰月，晏菁遥，等. 化瘀通痹方联合甲氨蝶呤治疗难治性类风湿关节炎临床观察［J］. 中国中西医结合杂志， 2015， 35（11）： 1326-1330.

WU J Q， CHEN X G， LV Y， et al. Chinese herbal formula Huayu-Qiangshen-Tongbi Decoction compared with leflunomide in combination with methotrexate in patients with active rheumatoid arthritis：an open-label， randomized， controlled， pilot study［J］. Front Med（Lausanne）， 2020， 7： 484.

吕媛. 化瘀强肾通痹方联合MTX治疗类风湿关节炎的临床疗效及骨保护研究［D］. 广州：广州中医药大学， 2020.

BARTEL D P. MicroRNAs： target recognition and regulatory functions［J］. Cell， 2009， 136（2）： 215-233.

HUANG R Y， WU J Q， LIU Z H， et al. MicroRNAs in rheumatoid arthritis： what is the latest with regards to diagnostics？［J］. Expert Rev Mol Diagn， 2019， 19（5）： 363-366.

CHEN X M， GAO K X， WU X D， et al. Chinese herbal formula Huayu-Qiangshen-Tongbi Decoction attenuates rheumatoid arthritis through upregulating miR-125b to suppress NF‑κB‑induced inflammation by targeting CK2［J］. J Immunol Res， 2022， 2022： 2836128.

QIN Q， SONG R， DU P， et al. Systemic proteomic analysis reveals distinct exosomal protein profiles in rheumatoid arthritis［J］. J Immunol Res， 2021， 2021： 9421720.

NI Z， ZHOU S， LI S， et al. Exosomes：roles and therapeutic potential in osteoarthritis［J］. Bone Res， 2020， 8： 25.

于娟，张立平，孙宁，等. 外泌体与自身免疫病的研究进展［J］. 医学研究生学报， 2018， 31（9）： 972-976.

ARNETT F C， EDWORTHY S M， BLOCH D A， et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis［J］. Arthritis Rheum， 1988， 31（3）： 315-324.

ALETAHA D， NEOGI T， SILMAN A J， et al. 2010 Rheumatoid arthritis classification criteria：an American College of Rheumatology/European League Against Rheumatism collaborative initiative［J］. Arthritis Rheum， 2010， 62（9）： 2569-2581.

SHEEHY C， EVANS V， HASTHORPE H， et al. Revising DAS28 scores for remission in rheumatoid arthritis［J］. Clin Rheumatol， 2014， 33（2）： 269-272.

CHEN X M， YAO D N， WANG M J， et al. Deep sequencing of plasma exosomal microRNA level in psoriasis vulgaris patients［J］. Front Med（Lausanne）， 2022， 9： 895564.

XING Q， FU L， YU Z， et al. Efficacy and safety of integrated traditional Chinese medicine and western medicine on the treatment of rheumatoid arthritis：a meta-analysis［J］. Evid Based Complement Alternat Med， 2020， 2020： 4348709.

孙青，谢瑶，赵卫红，等. 大剂量甲氨蝶呤治疗毒副作用系统分析［J］. 中国当代儿科杂志， 2017， 19（7）： 781-785.

朱露林，张奇，刘梦琴，等. 中药及天然药物对甲氨蝶呤致肝损伤保护作用的研究进展［J］. 中国中药杂志， 2021， 46（7）： 1727-1737.

王妍. 雷公藤与甲氨蝶呤、黄芪与当归联用治疗RA机制的生物信息学分析［D］. 北京：中国中医科学院， 2012.

谢志忠. 三妙散联合西药治疗类风湿关节炎（湿热痹阻）随机平行对照研究［J］. 实用中医内科杂志， 2018， 32（10）： 64-67.

马永池. 独活寄生汤加针刺联合西药治疗类风湿关节炎（肝肾亏虚）随机平行对照研究［J］. 实用中医内科杂志， 2019， 33（3）： 42-44.

WANG Z， LINGHU K G， HU Y， et al. Deciphering the pharmacological mechanisms of the Huayu-Qiangshen-Tongbi Formula through integrating network pharmacology and in vitro pharmacological investigation［J］. Front Pharmacol， 2019， 10： 1065.

YE P， MI Z， WEI D， et al. MiR-3960 from mesenchymal stem cell-derived extracellular vesicles inactivates SDC1/Wnt/β-Catenin axis to relieve chondrocyte injury in osteoarthritis by targeting PHLDA2［J］. Stem Cells Int， 2022， 2022： 9455152.

KIM H J， KIM I S， LEE S G， et al. MiR-144-3p is associated with pathological inflammation in patients infected with Mycobacteroides abscessus［J］. Exp Mol Med， 2021， 53（1）： 136-149.

JIAO P， WANG X P， LUORENG Z M， et al. MiR-223： an effective regulator of immune cell differentiation and inflammation［J］. Int J Biol Sci， 2021， 17（9）： 2308-2322.

NICOLIN V， DAL PIAZ F， NORI S L， et al. Inhibition of bone resorption by Tanshinone Ⅵ isolated from Salvia miltiorrhiza Bunge［J］. Eur J Histochem， 2010， 54（2）： e21.

NI Q， LU K， LI J， et al. Role of TGFβ signaling in maternal ethanol-induced fetal articular cartilage dysplasia and adult onset of osteoarthritis in male rats［J］. Toxicol Sci， 2018， 164（1）： 179-190.

ZHOU S R， XIE Y L， LI W， et al. Conditional deletion of Fgfr3 in chondrocytes leads to osteoarthritis-like defects in temporomandibular joint of adult mice［J］. Sci Rep， 2016， 6： 24039.

KIM S D， KIM H N， LEE J H， et al. Trapidil，a platelet-derived growth factor antagonist， inhibits osteoclastogenesis by down-regulating NFATc1 and suppresses bone loss in mice［J］. Biochem Pharmacol， 2013， 86（6）： 782-790.

BECKMANN J， DITTMANN N， SCHUTZ I， et al. Effect of M3 muscarinic acetylcholine receptor deficiency on collagen antibody-induced arthritis［J］. Arthritis Res Ther， 2016， 18： 17.

ZHONG H， FAN X L， FANG S B， et al. Human pluripotent stem cell-derived mesenchymal stem cells prevent chronic allergic airway inflammation via TGF‑β1‑Smad2/Smad3 signaling pathway in mice［J］. Mol Immunol， 2019， 109： 51-57.

FOTH M， ISMAIL N F B， KUNG J S C， et al. FGFR3 mutation increases bladder tumourigenesis by suppressing acute inflammation［J］. J Pathol， 2018， 246（3）： 331-343.

DE FILIPPO K， DUDECK A， HASENBERG M， et al. Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation［J］. Blood， 2013， 121（24）： 4930-4937.

LIM R， BARKER G， LAPPAS M. Inhibition of PIM1 kinase attenuates inflammation-induced pro-labour mediators in human foetal membranes in vitro［J］. Mol Hum Reprod， 2017， 23（6）： 428-440.

KOMLA-EBRI D， DAMBROISE E， KRAMER I， et al. Tyrosine kinase inhibitor NVP-BGJ398 functionally improves FGFR3-related dwarfism in mouse model［J］. J Clin Invest， 2016， 126（5）： 1871-1884.

HENLEY Z A， BAX B D， INGLESBY L M， et al. From PIM1 to PI3Kdelta via GSK3β： target hopping through the Kinome［J］. ACS Med Chem Lett， 2017， 8（10）： 1093-1098.

Views

下载量

CSCD

CNKI被引量

Alert me when the article has been cited

提交

Tools

Publicity Resources

Analysis of medication patterns in patented Chinese herbal formulas for treating gouty arthritis based on multivariate data mining

Jiang Quan's clinical experience in treating rheumatic and immune diseases complicated by pregnancy

Constructing syndrome differentiation evidence for dampness syndrome in rheumatoid arthritis based on association rules

Advances in mathematical model⁃based research on the composition， compatibility law and efficacy evaluation of traditional Chinese medicine compounds

Related Author

Li Ping

WANG Yinjie

HU Kexin

ZHU Yawen

CHENG Yuanyuan

LI Ming

HUANG Chuanbing

QIAN Ai

Related Institution

Department of Rehabilitation,Hanchuan People’s Hospital of Hubei Province

Preparation Center of Yinchuan traditional Chinese Medicine Hospital of Ningxia Hui Autonomous region

Center for Xin'an Medicine and Moderization of Traditional Chinese Medicine of IHM，The First Affiliated Hospital of Anhui University of Chinese Medicine

Guang'anmen Hospital， China Academy of Chinese Medical Sciences

Department of Rheumatology and Immunology， The Second Affiliated Hospital， Guangzhou University of Chinese Medicine

⁰