基于转录组测序探讨金天格胶囊调控SMSC外泌体miRNA和ACs mRNA治疗大鼠骨关节炎的作用机制

Exploring the Mechanism of Action of JinTianGe Capsules in Regulating SMSC-Exos miRNA and ACs mRNA for the Treatment OA in rats based on Transcriptome

关键词:  骨关节炎  转录组测序  金天格胶囊  滑膜间充质干细胞外泌体  关节软骨细胞

Key words:  OA  transgenic sequencing  jintiange capsules  SMSC-Exos  ACs

摘要:

目的 对金天格胶囊治疗骨关节炎(OA)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(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、双足平衡差异实验、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SMSCs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 OA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OA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(JTG) on osteoarthritis (OA) and the potential mechanism of synovial mesenchymal stem cell-derived 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 JTG, 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 SMSCs 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 JTG. 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, Il11, Cd40, Slc15a3, Pou2f2 and other genes.