基于RNA-Seq技术探讨青藤碱干预膝骨关节炎大鼠的作用机制

Study on the Mechanism of Sinomenine in the Treatment of Knee Osteoarthritis Based on RNA-Seq Technique

目的观察青藤碱对于碘乙酸钠诱导的膝骨关节炎(KOA)大鼠关节软骨的转录组影响。方法按随机数表法将30只SD大鼠分为空白(CK)组、模型(M)组和治疗(T)组3组,每组10只。M、T组大鼠右下肢关节腔内单次注射碘乙酸钠(1 mg/50μL生理盐水)建立KOA模型,注射后每周测量全部大鼠右下肢膝关节直径。造模21天后T组开始注射青藤碱(60 mg/kg),每天1次,连续10天。末次给药后8 h,对3组大鼠进行取材,麻醉后剖开大鼠右下肢关节,取下一部分关节后固定脱钙进行组织病理学分析;一部分关节软骨进行总RNA提取后测序,检测3组大鼠软骨组织中转录组基因表达情况,再对测序数据进行分析,包括差异基因表达分析、基因本体论联合会建立的数据库(GO)功能富集分析以及京都基因与基因组百科全书(KEGG)通路富集分析。结果青藤碱干预能够使大鼠膝关节直径减小(P<0.05),减轻关节软骨的损伤,降低Mankin’s评分(P<0.05);3组大鼠共有24个具有显著性差异的基因,显著富集于GO功能分类中的单核细胞趋化、CCR趋化因子受体结合和KEGG富集的细胞因子受体相互作用、趋化因子信号通路等,其中主要作用的靶点基因为C-C基序趋化因子配体(CCL)4、CCL9、Prkag3等。结论青藤碱可通过多途径、多靶点对KOA发挥疗效,关键靶点CCL4、CCL9、Prkag3及其相关的信号通路可能是青藤碱干预KOA的主要作用机制。

Objective To observe the effect of sinomenine on the transcriptome of articular cartilage in rats with knee osteoarthritis(KOA)induced by sodium iodoacetate(MIA).Methods Totally 30 SD rats were randomly divided into three groups:blank group(CK),model group(M)and treatment group(T),10 in each group.The osteoarthritis model was established by intra-articular injection of sodium iodoacetate(2 mg/50μL normal saline)into the right lower limb of the M group and T group.The knee joint diameter of the right lower limb of all rats was measured every week after injection.After 21 days of modeling,rats in the T group began to be injected with sinomenine(60 mg/kg)once a day for 10 consecutive days.Eight h after the last administration,the right lower limb joints were cut open after anesthesia.Part of the joints were removed and fixed and decalcified for histopathological analysis.Part of the articular cartilage was removed and the total RNA was extracted and sequenced.The gene expressions in the cartilage tissues of the three groups were detected,and the sequencing data were analyzed,including differential expression analysis,gene ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Results Sinomenine reduced the diameter of knee joint,attenuated the injury of articular cartilage and lowered Mankin's score(P<0.05).There were 24 genes with significant differences among the three groups of experimental rats,which were significantly enriched in monocyte chemotaxis,CCR chemokine receptor binding,KEGG-enriched cytokine receptor interaction and chemokine signal pathway in the functional classification of GO.Of them,the main target groups were C-C motif chemokine ligand(CCL)4,CCL9 and Prkag3.Conclusions Sinomenine exerted its therapeutic effect on osteoarthritis through multiple pathways and multiple targets.The key targets CCL4,CCL9,Prkag3 and their related signal pathways may be the main mechanism of sinomenine in the treatment of osteoarthritis.