Therapeutic Effect and Mechanism of Shanyu Pills in Treating "Arthromyodynia" Model Rats Based on Network Pharmacology and Experiment

OBJECTIVE To investigate the therapeutic effects of Shanyu pills on ankle arthrogryposis in adjuvant rats through network pharmacology and animal experiments and to explore the theoretical basis of using Shanyu pills to treat rheumatoid arthritis(RA) from the perspective of "arthromyodynia" and "strengthening vital Qi to eliminate pathogenic factor" in traditional Chinese medicine.

METHODS  The active ingredients of Dioscoreae Rhizoma, Atractylodis Macrocephalae Rhizoma, and Ginseng Radix et Rhizoma were screened and target predicted using the TCMSP database. RA targets were searched and screened in GeneCards, OMIM, and DrugBank. A "drug-active ingredient-disease" network diagram was constructed using Cytoscape 3.9.1. The intersection targets were input into the STRING database for analysis to obtain protein interaction networks and then imported into Cytoscape 3.9.1 for visualization. Metascape was used to perform GO and KEGG enrichment analyses on intersecting targets, and then a microbiome platform was used for enrichment analysis and visualization. In addition, molecular docking was performed using AutoDock, and the results were visualized using Pymol. A rat model of adjuvant arthritis was established using Freund’s complete adjuvant. Thirty-six SD rats were randomly divided into the normal control group, the model group, the positive drug group, the low-dose group of Shanyu pills(4.61 g·kg⁻¹), the medium-dose group of Shanyu pills(9.22 g·kg⁻¹), and the high-dose group of Shanyu pills(18.43 g·kg⁻¹). The diameter of the ankle joint of rats was measured, and the swelling degree of the ankle joint was calculated. The ankle joint of rats was removed for histopathological observation, and the TUNEL reagent was used to detect the degree of the apoptosis of synovial cells in rats and to determine the level of IL-6 and TNF-α. Moreover, the content of MDA was measured by taking the spleen and calculating the spleen index. The expression level of MAPK3 and Caspase 3 mRNA in synovial tissue was detected using qRT-PCR.

RESULTS A total of 54 effective ingredients were selected, corresponding to 325 targets of Shanyu pills. In addition, 845 genes were found to be closely related to RA, of which 86 overlapped with the target of Shanyu pills. Functional enrichment analysis indicated that Shanyu pills exert its therapeutic effect in RA by regulating multiple pathways. The swelling degree of the medication group was significantly reduced compared with the model group(P<0.05). HE staining results indicated that Shanyu pills could inhibit the degree of synovial swelling. The results of TUNEL staining indicated that the administration of Shanyu pills could inhibit synovial cell apoptosis. Compared with the model group, the level of IL-6, TNF-α, and MDA, as well as the spleen index, decreased in the medication group(P<0.05). Furthermore, the protein expression levels of MAPK3, Caspase 3 mRNA, and MAPK3 in the ankle joint tissue of rats were significantly reduced(P<0.05).

CONCLUSION Shanyu pills can attenuate ankle joint destruction in adjuvant model rats, which may help "strengthening vital Qi to eliminate pathogenic factor" and then treat arthromyodynia.