The therapeutic efficacy of Gancao Fuzi decoction through the "Gut-joint" axis in knee osteoarthritis and its impact on purine signaling

Abstract Ethnopharmacological relevance: During the Eastern Han Dynasty, the renowned physician Zhang Zhongjing initially documented Gancao Fuzi decoction(GCFZD) in his book "Synopsis of Golden Chamber". This formulation has been extensively employed in clinical practice by subsequent generations of physicians as an efficacious and safe treatment for knee osteoarthritis. However, its mechanism of action remains somewhat unclear, and to date, there have been no studies investigating the mechanism underlying GCFZD's therapeutic effects on knee osteoarthritis through the "Gut-joint" axis or its impact on purine signaling. Aims of the study: The aim of this study was to investigate the therapeutic effects of GCFZD on Knee osteoarthritis(KOA) via the "Gut-joint" axis, and the effects of GCFZD on purine signals P2X7 and P2Y14. Materials and methods: 18 Sprague-Dawley rats were divided into six groups, including a blank control group, KOA group, celecoxib group, and high, medium, and low dose groups of GCFZD. Each group consisted of 3 rats that received oral administration of GCFZD.The blank control group and KOA group were administered saline in the corresponding volume. The KOA rats model were established, and drug administration started in the 2 week after modeling at a frequency of once per day for 4 weeks. After 4 weeks of treatment, the arthritis index scores of the rats in each group were evaluated along with imaging and histopathological changes in the intestinal tract. Additionally, levels of inflammatory factors in serum as well as expression levels of P2X7 and P2Y14 in knee joints were determined using Western Blot method. Results: Through experimental comparison, it was observed that the joint inflammation index score of each group exhibited a significant reduction, accompanied by varying degrees of decrease in inflammatory factors. After GCFZD treatment, the levels of IL-1α, IL-1, IL-1β, IL-6, IL-17, IL-18, IL-23, and TNF-α in the serum exhibited varying degrees of reduction, with particularly notable decreases observed for IL-1α and IL-17; nevertheless, the therapeutic effect on IL-18 was notably superior to that of GCFZD in the celecoxib group. Immunofluorescence analysis in this study revealed varying degrees of changes in the expression of CD4, CD8, CD39, CD73, and P2X7 following treatment, with a notable increase observed in the expression of P2X7. Additionally, Western blot assay detected visible purine signals P2X7 and P2Y14 expression. Conclusion: The findings of this study have validated the therapeutic efficacy of GCFZD through the "Gut-joint" axis in KOA rats, with its mechanism being associated with alterations in intestinal permeability. Furthermore, GCFZD exhibits distinct effects on purine signals P2X7 and P2Y14.Investigating the functions and regulatory mechanisms of the GCFZD will enhance our comprehension of the pathogenesis of KOA and provide theoretical support for innovative treatment strategies. The future research on the P2X7 and P2Y14 receptors holds promise for discovering more potent drugs that specifically target these receptors, thereby offering renewed optimism for the management of inflammatory diseases.