Shaoyao Gancao Decoction(SGD) is a classic formula used in the clinical treatment of joint diseases, such as rheumatoid arthritis(RA), though its mechanism of action remains unclear. This study aimed to explore the mechanism of SGD in treating RA through chemical and network pharmacology analyses, combined with cellular experiments. UPLC-Orbitrap-MS~2 was used to qualitatively analyze SGD and drug-containing serum of rats after oral administration of SGD, thereby identifying the chemical composition and plasma components of SGD. Potential targets for the plasma components in treating RA were identified using the SwissTargetPrediction, PharmMapper, GeneCards, and DrugBank databases, and a protein-protein interaction(PPI) network was constructed using the STRING data platform. GO functional enrichment and KEGG pathway enrichment analyses were conducted using the Metascape database. Molecular docking and lipopolysaccharide(LPS)-induced RAW264.7 cell experiments were utilized for in vitro validation. The results identified 95 compounds in SGD, including 15 prototypical absorbed components, i.e., 7 flavonoids, 5 terpenoids, 2 phenolic compounds, and 1 other compound. Network pharmacology analysis revealed that licoisoflavanone, liquiritin apioside, 5-hydroxyferulic acid, albiflorin, hederagenin, and paeoniflorin were the pharmacodynamic components of SGD for treating RA. The core targets of SGD for RA treatment were identified as SRC, MAPK, EGFR, HSP90AA1, and STAT3, with regulation of the NF-κB, PI3K-Akt, and MAPK signaling pathways identified as key mechanisms for anti-RA effects of SGD. Molecular docking results showed that the six core components exhibited high affinity with the key targets SRC, MAPK, and NF-κB. In vitro cellular experiments demonstrated that SGD down-regulated the expression of inflammatory factors, including interleukin-1β(IL-1β), cyclooxygenase-2(COX-2), and tumor necrosis factor-α(TNF-α), in LPS-induced RAW264.7 cells. Western blot analysis revealed that SGD significantly reduced the phosphorylation levels of NF-κB p65 and p38 MAPK proteins. This study provides a scientific basis for further research into the active components and mechanisms of action of SGD in treating RA.
Keywords: Shaoyao Gancao Decoction; UPLC-Oribtrap-MS~2; network pharmacology; plasma components; rheumatoid arthritis.