摘要
旨在探讨火把花根片抗类风湿关节炎(RA)骨破坏的潜在作用及其分子机制。采用超高效液相色谱-质谱联用技术分析火把花根片的主要成分,并基于主要成分预测其候选靶标基因集。通过GeneCards和遗传学与医学文献数据库(OMIM)获取RA骨破坏的关键靶点,构建蛋白-蛋白相互作用(PPI)网络,并通过拓扑分析确定关键靶点。进一步利用基因本体论(GO)和京都基因与基因组百科全书(KEGG)富集分析揭示火把花根片抗RA骨破坏的分子机制。通过MTS实验评估火把花根片对巨噬细胞活力的影响,筛选无毒浓度。构建核因子κB受体活化因子配体(RANKL)诱导的体外破骨细胞分化模型。采用酒石酸酸性磷酸酶(TRAP)染色和纤维形肌动蛋白环(F-actin)染色观察破骨细胞形成和分化情况,同时通过免疫荧光法和Western blot法检测火把花根片对破骨细胞分化体系中核心靶点蛋白变化的影响。结果显示,火把花根片的主要成分包括雷公藤甲素、雷公藤红素、雷酚内酯等14种化合物。进一步网络分析表明,热休克蛋白90(HSP90)是火把花根片抗RA骨破坏的关键靶基因。TRAP染色和F-actin染色结果显示,火把花根片干预后,TRAP阳性多核细胞以及肌动蛋白环的数量和面积呈剂量依赖性降低(P<0.01)。Western blot结果表明,20、40μg·mL^(-1)火把花根片干预后,HSP90蛋白表达显著降低(P<0.01),10μg·mL^(-1)火把花根片可降低肿瘤坏死因子受体相关分子6(TRAF6)、活化T细胞核因子c1(NFATc1)蛋白表达(P<0.01),各剂量火把花根片可显著降低破骨分化标志性蛋白基质金属蛋白酶9(MMP9)、组织蛋白酶K(CTSK)表达(P<0.01)。免疫荧光结果进一步证实,火把花根片明显抑制破骨细胞中HSP90和CTSK水平,以及NFATc1的活化。综上,该研究证实火把花根片可能通过调控HSP90这一关键靶点抑制RANKL诱导破骨细胞分化从而发挥抗RA骨破坏作用,这将为火把花根片防治RA骨破坏提供新的思路。
This study aimed to investigate the potential role of Colquhounia Root Tablets against bone destruction in rheumatoid arthritis(RA)and its molecular mechanism.The study used ultra-performance liquid chromatography-mass spectrometry to analyze the major components of Colquhounia Root Tablets and predicted its candidate target gene set based on the major components.The key targets of RA bone destruction were obtained through GeneCards and the Database of Genetics and Medical Literature(OMIM),protein-protein interaction(PPI)network was constructed,and the key targets were identified by topological analysis.The molecular mechanism of Colquhounia Root Tablets against RA bone destruction was further revealed using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The effects of Colquhounia Root Tablets on macrophage viabilitywas assessed by MTS assay and screened for non-toxic concentrations. A model of receptor activator of nuclear factor-κB (RANKL)induced osteoclast differentiation in vitro was constructed. Colquhounia Root Tablets were used to observe the formation anddifferentiation of osteoclasts by tartrate-resistant acid phosphatase (TRAP) staining and fibrous actin(F-actin) staining, and the effectsof Colquhounia Root Tablets on the changes of core target proteins in the osteoclast differentiation system were detected byimmunofluorescence and Western blot. The results showed that the main components of Colquhounia Root Tablets included 14compounds such as triptolide, celastrol, and triptophenolide. Further network analysis revealed that heat-shock protein 90 (HSP90)was the key target gene of Colquhounia Root Tablets for anti-RA bone destruction. TRAP staining and F-actin staining showed that thenumber and area of TRAP-positive polymorphonuclear cells, as well as actin rings, were reduced in a dose-dependent manner after theintervention of Colquhounia Root Tablets ( P < 0. 01). Western blot results showed that the expression of HSP90 protein wassignificantly reduced after intervention with Colquhounia Root Tablets at 20 and 40 μg·mL^(-1) (P<0. 01);Colquhounia Root Tablets at10 μg·mL^(-1) could significantly decrease the expression of necrosis factor receptor associated molecule 6 (TRAF6) and nuclear factor ofactivated T cells 1 (NFATc1) proteins (P < 0. 01);moreover, all doses of Colquhounia Root Tablets significantly reduced theexpression of osteoclast differentiation marker proteins matrix metalloproteinase 9 (MMP9) and cathepsin K (CTSK) (P <0. 01).Immunofluorescence results further confirmed that Colquhounia Root Tablets significantly inhibited HSP90 and CTSK levels, as well asNFATc1 activation in osteoblasts. In conclusion, the present study confirmed that Colquhounia Root Tablets may inhibit RANKLinducedosteoclast differentiation by regulating the key target of HSP90, thus exerting an anti-RA bone destruction effect, which willprovide a new idea for Colquhounia Root Tablets to prevent and treat bone destruction in rheumatoid arthritis.
作者
陈沛萍
汪倩
黄凤玉
孔祥英
林娜
苏晓慧
CHEN Pei-ping;WANG Qian;HUANG Feng-yu;KONG Xiang-ying;LIN Na;SU Xiao-hui(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2024年第23期6389-6398,共10页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(82274176)。
关键词
火把花根片
热休克蛋白90
类风湿关节炎
骨破坏
Colquhounia Root Tablets
heat shock protein 90
rheumatoid arthritis
bone destruction
