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.%背景:腰突颗粒是课题组治疗腰椎间盘退行性疾病的经验方,一直有较好的临床疗效,但其作用机制一直未明确.目的:基于转录组测序(RNA-Seq)技术探讨临床经验方腰突颗粒防治腰椎间盘退变的可能分子机制.方法:将10只新西兰大白兔按随机数字表随机分为生理盐水组和腰突颗粒组(n=5),生理盐水组予以生理盐水20 mL/次灌胃,腰突颗粒组予以灌服腰突颗粒水煎剂20 mL/次,均2次/d,连续灌胃1周.于最后一次灌胃完成2 h后经颈总动脉采血收集兔血清分别制作为10%DMEM培养液;分别使用2种血清培养液培养人髓核细胞,镜下观察2组细胞形态,锥虫蓝染色后测定2组细胞活力;另取第3代髓核细胞,干预48 h后采用RNA-Seq技术对2组细胞中的mRNA进行检测,进行转录组测序筛选差异mRNA;将得到测序结果进行差异基因表达、GO功能显著性富集及Pathway显著性富集分析.结果与结论:①腰突颗粒组髓核细胞贴壁后以圆形或梭形多见,胞浆充足,胞核清晰.细胞核核表面光滑,核膜完整,长条状粗面内质网排列整齐,线粒体少,胞质内可见散在溶酶体及纤丝;生理盐水组细胞呈梭形和多角形,细胞核大,核仁多为2或3个,细胞核表面稍粗糙且质网可见轻微扩张,线粒体少且有部分膜不完整;②腰突颗粒组髓核P1、P2、P3代细胞贴壁速度、传代时间及细胞活力均优于生理盐水组(P<0.05);③比较2组测序结果发现,差异表达基因共有464个,其中上调基因有143个,下调基因有321个.差异基因的GO功能显著性富集分析显示,差异基因主要集中在生物过程部分的细胞调控与代谢过程中.差异基因的Pathway显著性富集分析结果发现,主要集中在代谢相关通路;④综上,通过RNA-Seq技术获得了腰突颗粒防治椎间盘退变作用的差异基因表达谱,腰突颗粒防治椎间盘退变的分子机制主要通过调节上调细胞外基质代谢相关基因及下调多糖合成相关基因实现,可为进一步研究提供基础.
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