关键词: 腰痛, 椎间盘退变, 养肝柔筋汤, 双氯芬酸钠, 网络药理学, 丝裂原活化蛋白激酶(MAPK)信号通路, 肿瘤坏死因子(TNF)信号通路, 缺氧诱导因子1(HIF-1)信号通路

Abstract:

BACKGROUND: The clinical efficacy of Yanggan Roujin Decoction in delaying intervertebral disc degeneration is significant; however, its underlying mechanism remains unclear.

OBJECTIVE: To validate the potential mechanisms by which Yanggan Roujin Decoction delays intervertebral disc degeneration using network pharmacology techniques and animal experiments.
METHODS: (1) The effective components of Yanggan Roujin Decoction and their action targets were screened using the Traditional Chinese Medicine Systems Platform and High-Throughput Experiment- and Reference-Guided Database of Traditional Chinese Medicine databases. Gene sets related to intervertebral disc degeneration were obtained from the Genecard, CTD, and DisGeNet databases. A protein-protein interaction network was constructed using the STRING database, and core targets were identified. The drug-component-target-disease network was constructed with Cytoscape 3.9.1 software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of the core targets were performed using the Ouyi Biological Cloud platform to identify potential therapeutic mechanisms. (2) A rat model of intervertebral disc degeneration was established through percutaneous puncture of the annulus fibrosus, followed by intervention with varying doses of Yanggan Roujin Decoction and diclofenac sodium. Changes in intervertebral disc tissue structure before and after intervention were assessed through histopathological staining, and the expression levels of core targets and relevant pathways were analyzed using western blot assay and quantitative PCR.
RESULTS AND CONCLUSION: (1) Network pharmacological analysis revealed that the active components of Yanggan Roujin Decoction consisted of 187 primary compounds, including beta-sitosterol, sitosterol, stigmasterol, quercetin, and kaempferol. The potential targets for treating intervertebral disc degeneration identified included 298 key proteins, primarily tumor protein 53, transcription factor c-Jun, interleukin-6, tumor necrosis factor-α, protein kinase B, and insulin. These targets were primarily involved in enzyme binding, nitric oxide synthase regulatory activity, and signaling pathways such as mitogen-activated protein kinase, hypoxia-inducible factor 1, tumor necrosis factor, interleukin-17. (2) Animal experiments demonstrated that Yanggan Roujin Decoction effectively alleviated the pathological changes in the intervertebral disc tissue structure caused by annulus fibrosus puncture. Compared with the normal group, the expression levels of tumor protein 53, transcription factor c-Jun, interleukin-6, and tumor necrosis factor-α were significantly elevated in the intervertebral disc tissues of rats in the model group (P < 0.01), whereas the expression levels of protein kinase B and insulin were significantly decreased (P < 0.01). The protein and mRNA levels of representative genes in the mitogen-activated protein kinase signaling pathway (38 and extracellular regulated protein kinases) and representative genes in the tumor necrosis factor pathway (interleukin-1β and tumor necrosis factor-α) were elevated, while the protein and mRNA levels of representative genes in the hypoxia-inducible factor-1α pathway (hypoxia-inducible factor-1α and vascular endothelial growth factor 1A) were reduced. After intervention with Yanggan Roujin Decoction at different doses, the protein and mRNA expression levels of tumor protein 53, transcription factor c-Jun, interleukin-6, tumor necrosis factor-α were significantly decreased (P < 0.01), while the protein and mRNA expression levels of protein kinase B and insulin were significantly increased (P < 0.01). Meanwhile, the protein and mRNA levels of representative genes in the mitogen-activated protein kinase signaling pathway (38 and extracellular regulated protein kinases) and representative genes in the tumor necrosis factor pathway (interleukin-1β and tumor necrosis factor-α) were reduced, while the protein and mRNA levels of representative genes in the hypoxia-inducible factor-1α pathway (hypoxia-inducible factor-1α and vascular endothelial growth factor 1A) were increased. After diclofenac sodium intervention, the expressions of interleukin 6 and tumor necrosis factor α at protein and mRNA levels were decreased (P < 0.01), and the protein and mRNA expressions of representative genes in the tumor necrosis factor signaling pathway (interleukin 1β and tumor necrosis factor α) decreased. However, there was no significant difference in the other indicators. To conclude, Yanggan Roujin Decoction effectively delays the progression of intervertebral disc degeneration, possibly through the regulation of tumor protein 53, transcription factor c-Jun, interleukin 6, tumor necrosis factor-α, protein kinase B, and insulin target genes, the inhibition of mitogen-activated protein kinase and tumor necrosis factor signaling pathways, and the activation of hypoxia-inducible factor 1 signaling pathway.

Key words: low back pain, intervertebral disc degeneration, Yanggan Roujin Decoction, diclofenac sodium, network pharmacology, mitogen-activated protein kinase (MAPK) signaling pathway, tumor necrosis factor (TNF) signaling pathway, hypoxia-inducible factor 1 (HIF-1) signaling pathway