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

doi:10.12307/2025.636

Abstract

Abstract: 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 30 μg/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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: skeletal muscle injury, medical ozone, RNA sequencing, proteomics, differential gene expression

CLC Number:

Ruan Zhen, Kou Jiushe. RNA sequencing and quantitative proteomics analyses of molecular mechanism of medical ozone therapy for rabbit skeletal muscle injury[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(18): 3767-3774.

Figures/Tables 9

主成分分析结果显示，空白组和模型组之间的转录本差异较大，臭氧组和空白组之间的差异较小，见图2A。根据基因表达的相似性，主成分分析表明了两组之间不同的方向性，结果表明，臭氧治疗减少了肌肉挤压损伤引起的转录变化。对差异基因进行统计发现，模型组与空白组相比，共有596个差异基因，其中485个基因表达水平上调，111个基因下调；臭氧组与模型组相比，共有405个差异基因，其中64个基因表达水平上调，341个基因下调，见图2B。火山图结果更直接地显示了组间差异基因的分布，见图2E，F。韦恩图显示，模型组/空白组和臭氧组/模型组比较中，有194个共有差异表达的基因，其中180个基因在模型组/空白组中上调，而在臭氧组/模型组中下调，14个基因在模型组/空白组中下调，而在臭氧组/模型组中上调，见图2C，D，表明医用臭氧治疗影响了骨骼肌损伤中的194个基因的表达，这194个基因可能是臭氧治疗骨骼肌损伤的关键基因。对194个共有差异基因双向聚类分析显示：每组内的3个生物重复首先聚在一起，聚类成不同的区间；不同组的组内表达情况基本一致，表明组内相关性强，生物重复性好。相较于空白组和臭氧组，模型组有更多的基因上调；臭氧组和空白组的相似度较高，聚类分层接近，见图3A。 2.3.2 差异基因的富集分析对模型组/空白组与臭氧组/模型组重叠的194个差异表达基因做GO 富集分析，分析结果见图4A，显示共有表达差异基因主要参与细胞表面受体信号通路、细胞黏附生物附着、免疫系统过程等生物学过程；主要富集于细胞表面、细胞外区域、细胞外间隙、膜筏、整合素复合物等细胞组分；参与信号受体结合、细胞黏附分子结合、整合素结合、钙离子结合、磷酸甘油酸酯激酶活性和生长因子受体结合等分子功能。KEGG pathway富集结果见图4B，显示这些差异基因主要富集于肌动蛋白细胞骨架调节、Rap1信号通路、细胞黏附分子、白细胞的跨内皮迁移、趋化因子信号通路、磷脂酰肌醇3激酶/蛋白激酶B(phosphoinositide 3-kinase/protein kinase B，PI3K-Akt)信号通路、核因子κB信号通路等。 2.4 蛋白质组学结果分析 2.4.1 差异蛋白的分析模型组与空白组相比，共有 138 个差异表达蛋白质，其中上调的蛋白有 87 个，下调的蛋白有 51 个；臭氧组与模型组相比，共有 242 个差异表达蛋白，其中上调的蛋白有106 个，下调的蛋白有 136 个。蛋白质定量结果统计见图5A。韦恩图显示4组间共有66个差异表达蛋白，44个蛋白在模型组/空白组中上调，而在臭氧组/模型组中下调，10个蛋白在模型组/空白组中下调，而在臭氧组/模型组中上调(图5B，C)。对组间的66个共有差异蛋白进行双向聚类分析显示，组内聚类较好，组内各样本间相关性较好，3组间呈现明显差异，"

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