Shaoyao Gancao Decoction ameliorates rheumatoid arthritis via inhibition of TNF-α/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shaoyao (root of Paeonia lactiflora Pall.) Gancao (root and rhizome of Glycyrrhiza uralensis Fisch.) Decoction (SGD) originated from Shanghan Lun during the Eastern Han Dynasty. As a classical prescription commonly used in the treatment of Bi syndrome-including diseases such as rheumatoid arthritis (RA), osteoarthritis, and ankylosing spondylitis, SGD has demonstrated notable efficacy in alleviating joint swelling and pain. However, its active components and mechanism of action against RA have not been fully elucidated.

AIM OF THE STUDY: To investigate the active components in SGD and to explore its underlying mechanism of action against RA.

MATERIALS AND METHODS: A collagen-induced arthritis (CIA) rat model and an MH7A cell model were used to investigate the therapeutic effects and underlying molecular mechanism of SGD in vivo and in vitro. Evaluated parameters included hind paw swelling, ankle circumference, arthritis index (AI) score, mechanical withdrawal threshold (MWT), pain pressure threshold (PPT), and ankle joint histopathology. The chemical profile of SGD and synovial tissue was analyzed using UHPLC-QE Plus-MS/MS. The levels of TNF-α, IL-1β, IL-6, and COX-2 were measured in rat serum and in MH7A cells following treatment with SGD and its key constituents-paeoniflorin, liquiritin, glycyrrhizic acid, and glycyrrhetinic acid (PLGGS). Protein expression of TNF-α, p-IKK, p-IκBα, NF-κB p-p65, and COX-2 was determined using Western blotting. Molecular docking and cellular thermal shift assay (CETSA) were performed to verify the binding of PLGGS components to IKK, IκBα, and NF-κB p65 and, thereby, to investigate the modulation of the TNF-α/NF-κB signaling pathway. This integrated approach was used to systematically elucidate the pharmacological effects and potential mechanisms of SGD.

RESULTS: Using UHPLC-QE Plus-MS/MS analysis, we characterized 59 distinct chemical constituents in SGD formula granules. Synovial tissue analysis further identified four specific compounds: paeoniflorin, liquiritin, glycyrrhizic acid, and glycyrrhetinic acid. In CIA rats, SGD alleviated hind paw swelling, reduced AI score and local temperature, increased MWT and PPT, decreased thymus and spleen indices, and mitigated ankle joint pathological damage. Both SGD and PLGGS significantly suppressed IL-1β-induced MH7A cell proliferation, reduced serum and cellular levels of inflammatory factors (TNF-α, IL-1β, IL-6, and COX-2), and downregulated the protein expression of TNF-α, p-IKK, p-IκBα, NF-κB p-p65 and COX-2. Molecular docking and CETSA results confirmed that PLGGS components exhibit substantial binding affinity to IKK, IκBα, and NF-κB p65.

CONCLUSIONS: SGD alleviated RA by inhibiting the TNF-α/NF-κB pathway, with its efficacy attributed to four active components (paeoniflorin, liquiritin, glycyrrhizic acid, and glycyrrhetinic acid) targeting IKK, IκBα, and NF-κB p65. These findings collectively suggest that SGD is a promising candidate for RA treatment. However, a potential study limitation is the use of a single batch of SGD. Future studies with multiple batches are warranted to confirm the reproducibility of these effects.