Guizhi-Shaoyao-Zhimu Decoction regulates the IL-17R-MAPK pathway to alleviate rheumatoid arthritis

Guizhi-Shaoyao-Zhimu Decoction (GSZD), derived from Synopsis of the Golden Chamber, has been used for over two thousand years to treat joint swelling and pain disorders such as "Lijie Feng" (arthralgia due to cold-dampness). It is one of the most representative traditional Chinese medicine (TCM) formulas for treating rheumatoid arthritis (RA). However, its modern pharmacological mechanism lacks systematic elaboration, and new research will better explore the underlying mechanism of its action.

With the collagen-induced arthritis (CIA) mouse model as the core experimental platform, this study first performed comprehensive chemical composition identification and quality control of GSZD via ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) to clarify its pharmacodynamic material basis, combined transcriptomic profiling with network pharmacology approaches, and further implemented in vitro and in vivo experimental validation including lipopolysaccharide (LPS)-stimulated macrophages and IL-17RA blockade with Brodalumab to clarify whether GSZD exerts anti-rheumatoid arthritis (RA) activity by targeting the interleukin-17 receptor (IL-17R)-MAPK signaling axis.

Sixty male SPF-grade DBA/1 mice were randomly divided into five groups: Sham, CIA, low-dose GSZD (13.24 g crude drug/kg·d), high-dose GSZD (26.48 g crude drug/kg·d), and leflunomide (10 mg/kg). Intragastric administration began 7 days after the secondary immunization and continued for 28 days. Body weight, paw thickness, and arthritis scores were monitored; spleen and thymus indices were calculated. Serum and joint tissues were collected for ELISA, qPCR, Western blot, immunohistochemistry, and histopathological staining. The chemical profile of GSZD was established by UPLC-HRMS and matched against the LuMet-TCM database. Transcriptome sequencing was performed on knee joint homogenates (CIA vs. GSZD-H, n = 3 per group) and integrated with network pharmacology (TCMSP, SwissTargetPrediction, OMIM, GeneCards, STRING, Metascape) to predict core targets. RAW264.7 macrophages were pretreated with GSZD (10-100 μg/mL) for 2 h and then stimulated with LPS (100 ng/mL) for 24 h. For IL-17RA blockade, cells were pre-incubated with Brodalumab (10 μg/mL, 30 min), followed by GSZD (100 μg/mL, 1 h) and LPS (1 μg/mL, 24 h). Molecular docking (AutoDock Vina) was used to evaluate the binding of major components to IL-17R.

UPLC-HRMS identified 979 constituents in GSZD. Network pharmacology and transcriptomic analyses jointly predicted the IL-17R-MAPK axis as a central target. In vivo, GSZD dose-dependently attenuated arthritis symptoms, reduced spleen and thymus indices, and alleviated synovial hyperplasia and macrophage activation (decreased F4/80 and CD11b). GSZD lowered pro-inflammatory cytokines (IL-6, TNF-α, IL-1β) and chemokines (CCL2, CCL7) in serum and joints, suppressed matrix metalloproteinases (MMP3, MMP9), and reduced cartilage/bone erosion. Immunohistochemistry confirmed that GSZD inhibited IL-17RA, ERK1/2, and phosphorylated p38 in joint tissues. In vitro, GSZD suppressed IL-17R expression and the phosphorylation of p38, ERK, and JNK in LPS-stimulated macrophages. IL-17RA blockade with Brodalumab completely abrogated the inhibitory effect of GSZD on MAPK phosphorylation and IL-1β production, confirming IL-17RA dependency. Molecular docking showed that multiple GSZD components bind directly to IL-17R.

This study demonstrates that GSZD, a multi-component formula characterized by UPLC-HRMS, alleviates synovial inflammation and osteoarticular destruction by blocking the IL-17R-MAPK cascade in an IL-17RA-dependent fashion. These findings provide modern scientific evidence for the clinical application of GSZD in treating RA.