基于转录组探讨蠲痹颗粒治疗寒证类风湿关节炎的作用机制

Mechanism of Juanbi Granule in the Treatment of Cold Syndrome Rheumatoid Arthritis Based on Transcriptomics

目的通过对蠲痹颗粒治疗前后的寒证类风湿关节炎(rheumatoid arthritis,RA)患者和健康人的外周血CD4^(+)T淋巴细胞进行转录组测序及差异基因表达分析,探讨蠲痹颗粒治疗RA的作用机制。方法2021年7月至2022年6月期间,在云南中医药大学第一附属医院风湿科住院部选取20例类风湿关节炎患者(RA组)作为研究对象,其中女性13例(65.0%),男性7例(35.0%),平均年龄(45.7±10.8)岁。随机分为2组,对照组予甲氨蝶呤口服,观察组在对照组治疗基础上加服蠲痹颗粒。同时选取云南中医药大学第一附属医院体检中心健康供者10例作为健康对照组(HC组)。分别抽取2组RA患者治疗前后以及健康对照组的静脉血样,分离出外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中的CD4^(+)T淋巴细胞,进行转录组测序。通过差异基因表达分析挖掘蠲痹颗粒治疗RA的潜在机制。结果RNA-seq测序结果显示,与健康对照组相比,RA患者共筛得2053个差异表达基因。这些差异基因GO分析与已知GO数据库中RA疾病显著相关的12个生物学过程显著重合。仅经甲氨蝶呤治疗的RA患者,主要影响炎症反应和肽段酪氨酸的磷酸化;同时使用甲氨蝶呤加蠲痹颗粒治疗1个月后的RA患者,在RA疾病显著相关生物学过程的富集明显减少,仅有2个富集,治疗3个月后不再有相关生物学过程富集。结论蠲痹颗粒可能通过干预CD4^(+)T淋巴细胞中与RA疾病高度相关的生物学过程达到高效治疗RA的目的。

Objective To investigate the effective mechanism of Juanbi Granule in treating cold syndrome RA,we performed the transcriptome sequencing and differential expression genes analysis of peripheral blood CD4^(+)T lymphocytes of patients with cold syndrome Rheumatoid arthritis(RA)before and after being treated with Juanbi Granule and healthy people.Methods Between July 2021 and June 2022,a total of 20 rheumatoid arthritis patients(RA group)were selected from the inpatient rheumatology department of the First Affiliated Hospital of Yunnan University of Chinese medicine as the study subjects,of which 13(65.0%)were female and 7(35.0%)were male,with a mean age of(45.7±10.8)years old.They were allocated into two groups randomly.The comparison group was treated with methotrexate by mouth,and the intervention group received Juanbi granules on the basis of the comparison group's treatment.At the same time,10 healthy donors from the physical examination Center of the First Affiliated Hospital of Yunnan University of Chinese Medicine were selected as the healthy control group(HC group).CD4^(+)T lymphocytes of peripheral blood mononuclear cells(PBMC)were isolated by venous blood samples of the two groups of RA patients before and after treatment and of the healthy control group,and transcriptome sequencing was performed.Differential gene expression analysis explored the potential mechanism of Juanbi Granule's prescription in treating RA.Results RNA-seq sequencing results showed that 2053 differentially expressed genes were detected in RA patients compared with healthy controls.These differential gene GO analysis coincide significantly with 12 biological process associated with RA disease in the known GO database.The inflammatory response and the peptidyl-tyrosine phosphorylation were mainly affected in RA patients treated only with methotrexate.However,in RA patients treated with the drug methotrexate and Juanbi Granule for one month,enrichment in these related biological process was significantly reduced,and only two related biological process were enriched.After three months of treatment,there was no enrichment in related biological process.Conclusion Juanbi Granule can achieve effective treatment of RA by blocking the biological process highly related to RA disease in CD4^(+)T lymphocytes.