Abstract: BACKGROUND: Yougui Pill is a famous formula of the Chinese traditional medicine, which has good efficacy for lumbar disc herniation due to kidney yang insufficiency. 
OBJECTIVE: To investigate the potential targets and mechanism of action of Yougui Pill in the treatment of lumbar disc herniation by using network pharmacology and molecular docking technology, and verified by animal experiments.
METHODS: (1) Network pharmacological analysis: We obtained the active ingredients and targets of Yougui Pill from TCMSP and other databases, collected genes related to lumbar disc herniation from GeneCards database, and took the intersection of the two for the topological analysis to derive the main active ingredients and core therapeutic targets. Gene ontology function analysis and Kyoto encyclopedia of genes and genomes pathway enrichment analysis were performed using R software. (2) Molecular docking: Autodock and Pymol software were utilized for the prediction of molecular binding energy of TCM active ingredients to core therapeutic targets. (3) Animal experiments: Eighteen Sprague-Dawley rats were randomly divided into a control group, a degeneration group and a Yougui Pill group, with 6 rats in each group. A rat model of intervertebral disc degeneration was prepared by fiber puncture method in the degeneration and Yougui Pill groups. At 2 weeks after modeling, Yougui Pill was given by gavage in the Yougui Pill group, once a day for 2 consecutive weeks. The level of tumor necrosis factor-α in serum was detected by the ELISA method, and morphological changes of the annulus fibrosus and nucleus pulposus cells were observed using hematoxylin-eosin staining.
RESULTS AND CONCLUSION: There were 90 active ingredients and 64 targets, and the main active ingredients were found to be quercetin, kaempferol, β-carotene, soybean flavonoid, and 4'-O-methylnyasol. The core targets of Yougui Pill for the treatment of lumbar disc herniation were interleukin 6, tumor necrosis factor-α, AKT1, interleukin 1B, and vascular endothelial growth factor A. Enrichment analysis revealed that the intersecting genes might be expressed through the interleukin-17 signaling pathway, tumor necrosis factor signaling pathway, MAPK signaling pathway, PI3K-AKT signaling pathway, and other signaling pathways to improve intervertebral disc degeneration. The molecular docking test verified that quercetin, kaempferol, and β-carotene had strong binding ability to the core targets. Animal experiments showed that the level of serum tumor necrosis factor α in the degeneration group was higher than that in the control group (P < 0.05), and the level of serum tumor necrosis factor α in the Yougui Pill group was lower than that in the degeneration group (P < 0.05). Hematoxylin-eosin staining showed that the fibrous annulus of the intervertebral discs and the structure of the nucleus pulposus in the degeneration group were destroyed, and the number of nucleus pulposus cells was reduced; there was a tendency to reconstructing the fibrous annulus of the intervertebral discs in the Yougui Pill group, and the number of nucleus pulposus cells increased compared with the degeneration group. To conclude, Yougui Pill may treat lumbar disc herniation by improving disc degeneration through the effects of quercetin, kaempferol, beta-carotene and other key active ingredients on core targets such as tumor necrosis factor.

Key words: Yougui Pill, lumbar disc herniation, network pharmacology, molecular docking, signaling pathway, inflammatory factor, potential target, annulus fibrosus puncture