转录组测序与定量蛋白质组学分析医用臭氧治疗兔骨骼肌损伤的分子机制

RNA sequencing and quantitative proteomics analyses of molecular mechanism of medical ozone therapy for rabbit skeletal muscle injury

背景:医用臭氧局部注射作为一种新的治疗方法,对损伤的骨骼肌组织具有抗炎、镇痛及免疫调节等作用,但其作用机制尚缺乏系统的研究。目的:观察医用臭氧对兔骨骼肌挤压伤模型的干预效应,通过转录组测序与标记定量蛋白技术分析损伤的兔胫骨前肌在经过臭氧治疗后基因表达的差异情况,以期揭示医用臭氧治疗骨骼肌损伤的分子机制。方法:将18只日本大耳白兔随机分为空白组、模型组、臭氧组,每组6只,后两组构建兔胫前肌挤压伤模型。臭氧组在造模12 h后,损伤局部注射30μg/mL医用臭氧2 mL,臭氧治疗后3 d进行组织取材。采用苏木精-伊红染色观察骨骼肌的形态学变化,并应用转录组测序及串联质谱标签标记定量蛋白技术对兔胫骨前肌组织进行检测,通过生物信息学分析探索组间差异表达基因及蛋白参与的生物学过程,进而探究臭氧治疗骨骼肌损伤的分子机制。结果与结论:①经臭氧治疗后3 d,与空白组相比,模型组细胞肿胀,可见浸润的炎症细胞,部分肌纤维溶解;相对于模型组,臭氧组细胞水肿有所减轻,炎症细胞减少;②转录组测序及生物信息学分析结果显示:模型组与空白组相比,筛选出596个差异基因;臭氧组与模型组相比,筛选出405个差异基因;取其交集,共筛选出194个共有差异表达的基因;③定量蛋白质分析结果:模型组与空白组相比共有138个差异表达蛋白质,臭氧组与模型组相比共有242个差异表达蛋白,共有的差异表达蛋白有66个;④对共有差异表达的基因/蛋白进行基因本体论及京都基因与基因组百科全书富集分析,发现其主要富集在PI3K-Akt信号通路、Ras信号通路、MAPK信号通路等;⑤提示医用臭氧可能通过作用于Syk、FGF16、CSF-1、MRAS这些关键靶点来调控PI3K/Akt/NF-κB信号通路及Ras/MAPK/NF-κB信号通路,进而促进损伤肌肉组织的修复。

BACKGROUND:As a new treatment method,local injection of medical ozone has anti-inflammatory,analgesic and immunomodulatory effects on injured skeletal muscle tissue,but its mechanism of action is still lacking systematic research.OBJECTIVE:To observe the intervention effect of medical ozone on skeletal muscle crush injury of rabbit models,and to analyze the differential gene expression of injured rabbit tibialis anterior muscle after ozone treatment through RNA-sequencing and tandem mass tag labeled quantitative proteomic technology,in order to partially reveal the molecular mechanism of medical ozone treatment for skeletal muscle injury.METHODS:Eighteen Japanese white rabbits were randomly divided into a blank group,a model group,and an ozone group,with 6 rabbits in each group.Rabbit tibialis anterior muscle compression injury models were established in the latter two groups.After 12 hours of model establishment,2 mL of medical ozone with a mass concentration of 30μg/mL was injected into the injured muscle of rabbits in the ozone group.Tissue samples were taken 3 days after ozone treatment.Hematoxylin-eosin staining was used to observe the morphological changes of skeletal muscle.RNA sequencing and tandem mass spectrometry tag were used to detect rabbit tibialis anterior muscle tissue.Bioinformatics analysis was used to explore the biological processes of differentially expressed genes and proteins between groups and then to investigate the molecular mechanism of ozone treatment of skeletal muscle injury.RESULTS AND CONCLUSION:After 3 days of ozone treatment,compared with the blank group,the model group showed swelling of cells,infiltration of inflammatory cells,and partial dissolution of muscle fibers.Compared with the model group,cell edema was alleviated and the number of inflammatory cells was reduced in the ozone group.(2)The results of RNA sequencing and bioinformatics analysis showed that compared with the blank group,596 differentially expressed genes were screened in the model group.Compared with the model group,405 differentially expressed genes were screened in the ozone group.There were a total of 194 differentially expressed genes shared among these differentially expressed genes.Proteomic analysis results exhibited that compared with the blank group,the model group contained 138 differentially expressed proteins.Compared with the model group,242 differentially expressed proteins were determined in the ozone group.There were 66 differentially expressed proteins shared between the two comparison groups.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed on differentially expressed genes/proteins,and it was found that they were mainly enriched in the PI3K-Akt signaling pathway,Ras signaling pathway,and MAPK signaling pathway.It is indicated that medical ozone may promote the repair of injured muscle through regulating PI3K/Akt/NF-κB and Ras/MAPK/NF-κB signaling pathway by acting on target genes,such as Syk,FGF16,CSF-1,and MRAS.