摘要
目的 对金天格胶囊治疗骨关节炎的药效进行确证,基于高通量测序技术探讨滑膜间充质干细胞外泌体(synovial mesenchymal stem cell exosomes,SMSC-Exos)与关节软骨细胞(articular chondrocytes,ACs)治疗骨关节炎的潜在作用机制.方法 采用Ⅱ型胶原酶诱导大鼠骨关节炎,通过一般行为学观察、双足平衡差异试验、机械足反射阈值、Micro-CT观察与番红固绿染色进行药效验证.将SMSC、ACs在适宜浓度的含药血清中培养,对照组、模型组和金天格组ACs进行mRNA测序以及SMSC-Exos进行miRNA测序.筛选差异表达mRNA与差异表达miRNA,并进行靶基因预测.通过Venn图对SMSC与ACs的差异表达基因求交集得到共同差异表达基因,运用Cytoscape软件构建miRNA-mRNA调控网络.对共同差异表达基因进行表达趋势分析以及对差异表达基因mRNA与miRNA、Micro-CT药效指标与差异表达基因mRNA进行相关性分析.结果 骨关节炎病理状态下,miRNA-23a-3p、miRNA-342-3p、miRNA-146b-5p、miRNA-501-3p、miRNA-214-3p表达下调,miRNA-222-3p、miRNA-30e-3p、miRNA-676-3p、miRNA-192-5p表达上调(P＜0.05),这些miRNA的表达均在金天格胶囊含药血清干预后逆转表达,且趋势明显.Micro-CT药效指标、mRNA与miRNA之间存在一定的相关性.结论 金天格胶囊治疗骨关节炎的药效作用明显,其作用机制可能与促进SMSC-Exos靶向 ACs 转运 miRNA 进而调控 Serpinb10、Ntn1、Il1b、Tgm2、Megf10、Il11、Cd40、Slc15a3、Pou2f2 等基因有关.
Abstract
OBJECTIVE To confirm the therapeutic effect of Jintiange capsules on osteoarthritis(OA)and the potential mechanism of synovial mesenchymal stem cell exosomes(SMSC-Exos)and articular chondrocytes(ACs)in the treatment of OA based on high-throughput sequencing technology.METHODS Type Ⅱ collagenase-induced OA rats were used for efficacy verification through general behavioral observation,bipedal balance difference experiment,mechanical foot reflex threshold,Micro-CT observation,and Safranin O-Fast Green staining.SMSCs and ACs were cultured in suitable concentration of drug-containing serum,and mRNA sequencing was performed on ACs in the control,model,and Jintiange capsules groups,as well as miRNA sequencing on SMSC-Exos.Differential expressed mRNAs and miRNAs were screened and target genes were predicted.The common differential expressed genes between SMSC and ACs were obtained by intersecting the differential expressed genes,and a miRNA-mRNA regulatory network was constructed using Cytoscape software.The expression trend analysis of common differential expressed genes was conducted,as well as the correlation analysis between differential expressed gene mRNA and miRNA,Micro-CT efficacy indicators,and differential expressed gene mRNA.RESULTS Under the pathological state of OA,the expression of miRNA-23a-3p,miRNA-342-3p,miRNA-146b-5p,miRNA-501-3p,and miRNA-214-3p were down-regulated,while miRNA-222-3p,miRNA-30e-3p,miRNA-676-3p,and miRNA-192-5p were up-regulated(P＜0.05).The expressions of these miRNAs were significantly reversed after intervention with drug-containing serum of Jintiange capsules.There was a certain correlation between Micro-CT efficacy indicators,mRNA and miRNA.CONCLUSION Jintiange capsule has obvious efficacy in the treatment of OA,and its mechanism may be related to the promotion of SMSC-Exos targeting ACs to transport miRNA and then regulate Serpinb10,Ntn1,Il1b,Tgm2,Megf10,1111,Cd40,Slc15a3,Pou2f2 and other genes.
基金项目
国家中医药管理局青年岐黄学者培养项目(国中医药人教函2022256号)
陕西省重点研发计划资助项目(2022SF-238)
陕西省中医药管理局中医药重点学科建设项目(2017001)
陕西省中医药管理局中医药"双链融合"中青年科研创新团队项目(2022-SLRH-LJ-001)
陕西中医药大学学科创新团队项目(2019-YL11)
NETL
NSTL
万方数据