Molecular mechanism of Yaotu Granules in the prevention and treatment of lumbar disc degeneration based on RNA-seq technology / 中国组织工程研究

ABSTRACT

BACKGROUND:The Herbal Compound formula Yaotu Granules for lumbar disc degeneration has achieved satisfactory efficacy, but the underlying mechanism remains unclear. OBJECTIVE: To explore the possible molecular mechanisms ofYaotu Granules for lumbar disc degeneration based on transcriptome sequencing (RNA-seq). METHODS: Ten New Zealand white rabbits were randomly divided into control and experimental groups (n=5 per group), followed by intragastric injection of 20 mL of normal saline and 20 mL of water decoction of Yaotu Granules, respectively, twice daily for consecutive 1 week. The different drug serums were prepared using serum samples obtained from the common carotid artery at 2 hours after the last injection. Human nucleus pulposus cells were cultured in DMEM containing 10% different drug serums to observe the cellular morphology in each group. The cell viability was detected by trypan blue staining. The passage 3 human nucleus pulposus cells were pretreated for 48 hours, the mRNA level was detected using RNA-seq, and the differentially expressed mRNA was screened by RNA-seq. Subsequently, the differential gene expression, Gene Ontology (GO) enrichment analysis and Pathway enrichment analysis were performed. RESULTS AND CONCLUSION: In the experimental group, there were round or fusiform cells with abundant cytoplasm and clear nuclei that showed smooth surface and complete nuclear membrane; elongated rough endoplasmic reticulum arranged regularly, and less mitochondrion, scattered lysosomes and filaments were visible in the cytoplasm. In the control group, spindle and polygonal cells were found, and the large nuclei with 2 or 3 nucleoli and slightly rough surface were observed; there were mildly dilated endoplasmic reticulum, and few mitochondria with incomplete membrane. The adherent rate, generation time and cell viability of passage 1, 2 and 3 cells in the experimental group were significantly better than those in the control group (P < 0.05). Sequencing results found that there were 464 differentially expressed genes including 143 upregulated, and 321 downregulated genes. GO analysis revealed that the differentiated genes were mainly concentrated on cell regulation and metabolism. Pathway analysis found that mainly differentiated genes focused on the metabolism related pathways. These findings suggest that the differentially expressed gene profile of Yaotu Granules for lumbar disc degeneration is obtained by RNA-seq technology.Yaotu Granules mainly upregulate extracellular matrix metabolism-related genes and downregulate polysaccharide synthesis related genes in the prevention and treatment of lumbar disc degeneration, which provides basis for further research.