摘要
探究新风胶囊(XFC)通过调控lncRNA MAPKAPK5-AS1(MK5-AS1)对类风湿关节炎(RA)滑膜成纤维细胞(FLS)凋亡和炎症的影响。收集健康人(正常组)和RA脾虚湿盛证患者(RA组)各30例,RA患者入院后予XFC口服治疗(XFC组),提取外周血单个核细胞(PBMCs),观察MK5-AS1与临床指标的相关性,及XFC治疗前、后MK5-AS1的表达;建立RA-FLS细胞系,制备XFC含药血清,构建MK5-AS1过表达质粒,转染至RA-FLS中,观察XFC含药血清通过MK5-AS1对RA-FLS炎症和凋亡的影响。MK5-AS1在RA脾虚湿盛证患者PBMCs中表达降低(P<0.001),ROC曲线结果显示AUC为83.9%,相关性分析结果表明MK5-AS1与ESR、CRP、RF、CCP和脾虚湿盛证证候积分呈负相关,XFC治疗后MK5-AS1表达升高(P<0.001)。关联规则结果显示,XFC治疗与MK5-AS1、ESR、CRP、RF及脾虚湿盛证证候积分下降的支持度均大于83%、置信度均大于80%和提升度均大于1。RT-qPCR结果显示,经TNF-α刺激后RA-FLS中MK5-AS1 RNA表达降低(P<0.01);XFC含药血清干预后MK5-AS1 RNA表达升高(P<0.05);经转染pcDNA3.1-MK5-AS1后MK5-AS1 RNA表达升高(P<0.01)。ELISA结果显示,经TNF-α刺激后RA-FLS中促炎因子IL-17表达升高、抑炎因子IL-4表达降低(P<0.01);XFC含药血清干预后IL-17表达降低、IL-4表达升高(P<0.01);经转染pcDNA3.1-MK5-AS1后IL-17表达降低、IL-4表达升高(P<0.01)。免疫荧光(IF)结果显示,经TNF-α刺激后RA-FLS中促凋亡蛋白Bax表达降低、抑凋亡蛋白Bcl-2表达升高(P<0.01);XFC含药血清干预后Bax表达升高、Bcl-2表达降低(P<0.01);经转染pcDNA3.1-MK5-AS1后Bax表达升高、Bcl-2表达降低(P<0.05)。MK5-AS1在RA-PBMCs和RA-FLS中表达均降低,XFC可通过调控MK5-AS1,抑制RA炎症反应和促进细胞凋亡,治疗RA。
To explore the regulatory effects of Xinfeng Capsules(XFC) on the apoptosis of synovial fibroblasts(FLS) and inflammation in rheumatoid arthritis(RA) via lncRNA MAPKAPK5-AS1(MK5-AS1). Thirty healthy people and 30 patients with RA due to spleen deficiency and dampness exuberance were collected for extracting the peripheral blood mononuclear cells(PBMCs) before and after XFC treatment, which were used to observe the correlation between MK5-AS1 and clinical indicators as well as MK5-AS1 expression before and after XFC treatment. Following the establishment of RA-FLS cell line and the preparation of XFC-containing serum, MK5-AS1-overexpression plasmid was constructed and transfected into RA-FLS for investigating the efficacy of XFC-containing serum in regulating inflammation and apoptosis of RA-FLS via MK5-AS1. The expression of MK5-AS1 in PBMCs of patients with RA due to spleen deficiency and dampness exuberance was decreased(P<0.001). The ROC curve analysis revealed the AUC of 83.9%. Correlation analysis showed that MK5-AS1 was negatively correlated with ESR, CRP, RF, CCP, and spleen deficiency and dampness exuberance syndrome score. The expression of MK5-AS1 increased significantly after XFC treatment(P<0.001). As demonstrated by association analysis, XFC decreased MK5-AS1, ESR, CRP, RF, and spleen deficiency and dampness exuberance syndrome score, with the degree of support all greater than 83%, confidence greater than 80%, and lift greater than 1. The results of RT-qPCR showed that the MK5-AS1 RNA expression significantly decreased after TNF-α stimulation(P<0.01), which, however, increased significantly after the intervention with XFC-containing serum(P<0.05). Such expression rose again after the transfection of pcDNA3.1-MK5-AS1(P<0.01). ELISA results showed that TNF-α stimulation elevated the expression of pro-inflammatory factor IL-17 but lowered the expression of anti-inflammatory factor IL-4(P<0.01). After intervention with XFC-containing serum, the expression of IL-17 decreased while that of IL-4 increased(P<0.01). The transfection of pcDNA3.1-MK5-AS1 contributed to the reduction in IL-17 expression but the elevation in IL-4 expression(P<0.01). The immunofluorescence(IF) findings demonstrated that the expression of pro-apoptotic protein Bax was down-regulated, whereas that of the anti-apoptotic protein Bcl-2 was up-regulated after TNF-α stimulation(P<0.01). After the intervention with XFC-containing serum, the Bax expression was increased, while Bcl-2 expression was decreased(P<0.01), which were remarkably collaborated by the transfection of pcDNA3.1-MK5-AS1(P<0.05). The expression of MK5-AS1 is significantly decreased in both RA-PBMCs and RA-FLS, implying that XFC inhibits inflammatory reaction and promotes the apoptosis in RA by regulating the expression of MK5-AS1.
作者
文建庭
刘健
王馨
王杰
WEN Jian-ting;LIU Jian;WANG Xin;WANG Jie(Department of Rheumatology y First Affiliated Hospital of Anhui University of Chinese Medicine,Hefei 230031,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2021年第24期6542-6548,共7页
China Journal of Chinese Materia Medica
基金
国家自然科学基金面上项目(81973655,82074373)
国家重点研发计划“中医药现代化研究”重点专项(2018YFC1705204)
安徽省高校协同创新项目(GXXT-2020-025)
安徽省名中医刘健工作室建设项目(中医药发展秘[2018]11号)
安徽省“115”创新团队项目(皖人才办[2019]1号)
新安医学教育部重点实验室开放基金项目(2020xayx10)。
