Cheng's Juanbi Decoction Inhibits Rheumatoid Arthritis Pathology by Blocking the WTAP-Wnt7b-Wnt/β-Catenin Signaling Axis

Abstract

Objective 

 Cheng's Juanbi Decoction (CSJBD) is a classic traditional Chinese medicine formula for treating rheumatoid arthritis (RA), exhibiting significant clinical efficacy, but the underlying mechanisms remain unclear. We investigated whether CSJBD inhibited RA pathology by blocking the WTAP-Wnt7b-Wnt/β-catenin signaling axis using a collagen-induced arthritis (CIA) mouse model and fibroblast-like synoviocytes (FLSs) derived from RA patients (RA FLSs) and examined the underlying mechanisms.

Methods 

 We conducted in vivo experiments. Male C57BL/6 mice weighing 17 to 20 g were used to establish the CIA model. The mice were assigned to 6 groups, including the normal group, the model (CIA) group, the model + CSJBD-L (8.1 g/kg) group, the model + CSJBD-M (16.2 g/kg) group, the model + CSJBD-H (32.4 g/kg) group, and the model + leflunomide (LEF) (0.05 mg/10 g) group, with 10 mice in each group. CSJBD was administered twice daily via gastric gavage, while LEF was administered once daily via gastric gavage, for a duration of 28 days. We also conducted in vitro experiments. RA FLSs were assigned to 4 groups, including the RA FLSs + CSJBDS-L group receiving 10% CSJBDS-containing serum, the RA FLSs + CSJBDS-M group receiving 15% CSJBDS-containing serum, the RA FLSs + CSJBDS-H group receiving 20% CSJBDS-containing serum, and the RA FLSs + NC group (negative control). To study whether WTAP regulated Wnt7b, RA FLSs were divided into the RA FLSs group, the RA FLSs + si-WTAP#3 group, the RA FLSs + si-WTAP#3 + Wnt7b-OE group, and the RA FLSs + si-WTAP#3 + Wnt7b-NC group. To study the underlying mechanism by which CSJBT affected RA FLSs, RA FLSs were divided into the RA FLSs group, the RA FLSs + CSJBDS-M group, the RA FLSs+CSJBDS-M + Wnt7b-OE group, and the RA FLSs+CSJBDS-M + NC group. We used ultra-high performance liquid chromatography (UPLC) to identify and quantify key monomer compounds from CSJBD as quality criteria for CSJBD preparation. Bioinformatics, CCK-8, RT-qPCR, Western blot, immunofluorescence, and related methods were employed to assess the therapeutic efficacy and underlying mechanisms of CSJBD in treating RA.

Results 

 According to the UPLC analysis, ferulic acid, osthole, mulberroside A, notopterol, and gentiopicroside were identified as quality control standards for the preparation of CSJBD formula. CSJBD improved RA pathology in CIA mice, reduced the levels of interleukin (IL)-6, IL-1β, IL-8, and tumor necrosis factor-α (TNF-α) in their serum, and decreased the expression of RA pathological genes MMP3 and fibronectin, with the difference between groups being statistically significant. Bioinformatics analysis suggested that CSJBD might inhibit RA pathology by suppressing the Wnt/β-catenin signaling pathway through Wnt7b. Experimental results showed that the expression of WTAP and Wnt7b was significantly increased in RA. After knocking down WTAP, the expression of Wnt7b was significantly reduced, and the Wnt/β-catenin signaling pathway was also inhibited, with the difference between groups being statistically significant (P < 0.05), confirming that WTAP regulated the pathway via Wnt7b. According to experimental verification, CSJBD significantly inhibited the Wnt/β-catenin signaling pathway and the proliferation of RA FLSs. Wnt7b overexpression reversed the inhibitory effect of CSJBD on the Wnt/β-catenin signaling pathway and the proliferation of RA FLSs, indicating that Wnt7b is the direct target of CSJBD.

Conclusion 

 CSJBD inhibits RA pathology by blocking the WTAP-Wnt7b-Wnt/β-catenin signaling axis, with Wnt7b identified as a direct therapeutic target of CSJBD.

Keywords: Rheumatoid arthritis, Cheng's Juanbi Decoction, WTAP, Wnt7b, Wnt/β-catenin signaling pathway