Yanggan Roujin Decoction delays intervertebral disc degeneration: network pharmacological analysis and experimental validation in rat models

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 Pharmacology Database and High-Throughput Experiment- and Reference-Guided Database of Traditional Chinese Medicine. 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 using Cytoscape 3.9.1 software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed on the Ouyi Biological Cloud Platform to obtain potential therapeutic mechanisms. (2) A rat model of intervertebral disc degeneration was established by percutaneous puncture of the annulus fibrosus. Different doses of Yanggan Roujin Decoction and diclofenac sodium were administered. Histopathological staining was used to assess structural changes in intervertebral disc tissue before and after intervention. Western blot and quantitative real-time PCR were used to detect the expression of core targets and related pathways. RESULTS AND CONCLUSION: Network pharmacology analysis identified 187 active components of Yanggan Roujin Decoction, mainly including β-sitosterol, sitosterol, stigmasterol, quercetin, and kaempferol. There were 298 potential targets for treating intervertebral disc degeneration, mainly including tumor protein p53, transcription factor c-Jun, interleukin-6, tumor necrosis factor α, protein kinase B, and insulin. These targets were mainly 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, and interleukin-17. Animal experiments showed that Yanggan Roujin Decoction effectively alleviated pathological structural changes in intervertebral disc tissue caused by annulus fibrosus puncture. Compared with the blank control group, the model group showed increased protein and mRNA expression of tumor protein p53, transcription factor c-Jun, interleukin-6, and tumor necrosis factor α (P < 0.01), while protein kinase B and insulin protein and mRNA expression decreased (P < 0.01). Meanwhile, the expression of representative genes in the mitogen-activated protein kinase signaling pathway (P38, extracellular regulated protein kinase) and tumor necrosis factor signaling pathway (interleukin-1β, tumor necrosis factor α) increased, while the expression of representative genes in the hypoxia-inducible factor 1 signaling pathway (hypoxia-inducible factor 1α, vascular endothelial growth factor A) decreased. After intervention with different doses of Yanggan Roujin Decoction, the protein and mRNA expression of tumor protein p53, transcription factor c-Jun, interleukin-6, and tumor necrosis factor α decreased (P < 0.01), while protein kinase B and insulin protein and mRNA expression increased (P < 0.01). Simultaneously, the expression of representative genes in the mitogen-activated protein kinase signaling pathway (P38, extracellular regulated protein kinase) and tumor necrosis factor signaling pathway (interleukin-1β, tumor necrosis factor α) decreased, while the expression of representative genes in the hypoxia-inducible factor 1 signaling pathway (hypoxia-inducible factor 1α, vascular endothelial growth factor A) increased. After diclofenac sodium intervention, the protein and mRNA expression of interleukin-6 and tumor necrosis factor α decreased (P < 0.01), and the protein and mRNA expression of representative genes in the tumor necrosis factor signaling pathway (interleukin-1β, tumor necrosis factor α) decreased, but there were no significant differences in other indicators. These results indicate that Yanggan Roujin Decoction can effectively delay the progression of intervertebral disc degeneration, and the mechanism may be related to the regulation of tumor protein p53, transcription factor c-Jun, interleukin-6, tumor necrosis factor α, protein kinase B, insulin target genes, inhibition of mitogen-activated protein kinase and tumor necrosis factor signaling pathways, and activation of hypoxia-inducible factor 1 signaling pathway.

1. Introduction

Intervertebral disc degeneration is a major cause of low back pain, with complex pathological mechanisms characterized by progressive structural and functional deterioration of the intervertebral disc [1-4]. The degenerative process involves the regulation of multiple factors and signaling pathways, including inflammatory responses, apoptosis, extracellular matrix metabolic imbalance, and oxidative stress. Current clinical treatments for intervertebral disc degeneration are mainly divided into surgical and non-surgical approaches. Non-surgical treatments include medication, physical therapy, and rehabilitation [5-6]. However, intervertebral disc degeneration is prone to recurrence and high costs, and most current treatments aim to alleviate symptoms without fundamentally reversing the degenerative process [7-8]. Therefore, identifying key therapeutic targets to delay or even reverse intervertebral disc degeneration has become a priority.

In traditional Chinese medicine, the pathological state of pain, swelling, and limited movement caused by chronic strain leading to blood stasis, dampness retention, and cold accumulation is classified under the category of 'tendon injury' [9-10]. As stated in the Suwen: 'When food qi enters the stomach, it disperses essence to the liver, and moistens the tendons.' When liver blood is sufficient, the tendons are properly nourished, maintaining normal motor function. Conversely, if liver yang qi is insufficient, the tendons lose warmth and nourishment, leading to flaccidity and weakness, manifesting as excessive 'ba' (relaxation) and pathological fatigue. Yanggan Roujin Decoction, an empirical formula for treating low back pain, is composed of 13 herbs including Cornus officinalis, Radix Paeoniae Alba, Lycium barbarum, Angelica sinensis, Bupleurum chinense, Cyperus rotundus, Dipsacus asper, Radix Angelicae Pubescentis, Eucommia ulmoides, Acanthopanax gracilistylus, Achyranthes bidentata, Piper kadsura, and Poria cocos. The formula nourishes yin, softens the liver, and strengthens the spleen, achieving the effect of nourishing the liver and relaxing tendons.