Jiawei Qingluo Yin alleviates rheumatoid arthritis in rats by inhibiting activation of the Hedgehog signaling pathway to promote apoptosis of synovial cells

doi:10.12122/j.issn.1673-4254.2026.10.13

Abstract

Abstract:

Objective To investigate the mechanism of Jiawei Qingluo Yin (JWQLY) for alleviating rheumatoid arthritis (RA) in rats. Methods Network pharmacology, molecular docking and dynamics analyses were performed to identify the core components, key targets, and pathways of JWQLY against RA and the core component-key target binding. In a rat model of antigen-induced arthritis (AIA) established using complete Freund's adjuvant, the effects of MTX (0.5 mg/kg) and low-, medium-, and high-dose JWQLY (1.03, 3.1, and 9.3 g/kg, respectively) were tested on paw swelling, arthritis index (AI), synovial histopathology, apoptosis, and serum IL-1β and VEGF levels. In the mechanistic investigation, the rat models were treated with high-dose (9.3 g/kg) JWQLY, vismodegib (a Hedgehog inhibitor, 10 mg/kg), or both, and the changes in paw swelling, AI, histopathology, and expressions of Bcl-2, Bax, Ihh, Smo, Gli1, cleaved caspase-3, and cleaved caspase-9 were examined. Results A total of 306 intersecting targets were identified between JWQLY and RA. GO and KEGG enrichment analyses revealed enrichment of the targets in negative regulation of apoptosis and inflammation and in Hedgehog and apoptosis pathways. Molecular docking studies confirmed stable binding of the core components in JWQLY to Shh and other Hedgehog targets. The rat models of AIA showed obvious paw swelling and significantly elevated AI, pathology scores, and serum IL-1β and VEGF levels, and these changes were dose-dependently alleviated by JWQLY treatment. The rat models receiving treatment with vismodegib or JWQLY+ vismodegib exhibited significantly reduced Ihh, Smo, Gli1, and Bcl-2 expression with increased expressions of Bax, cleaved- caspase-3, and cleaved-caspase-9 and apoptosis rates of the synovial cells. JWQLY and vismodegib synergistically suppressed Hedgehog signaling and enhanced synovial cell apoptosis. Conclusion JWQLY alleviates RA in rats by inhibiting Hedgehog pathway activation, down-regulating the Bcl-2/Bax ratio, and promoting synovial cell apoptosis.

Key words: Jiawei Qingluo Yin, rheumatoid arthritis, Hedgehog signaling pathway, synovial cells, apoptosis

Yifei HAO, Lianqing CAI, Siyu TANG, Xiaoxuan YU, Yan LI, Xiu WANG, Jie ZHU. Jiawei Qingluo Yin alleviates rheumatoid arthritis in rats by inhibiting activation of the Hedgehog signaling pathway to promote apoptosis of synovial cells[J]. Journal of Southern Medical University, 2026, 46(10): 2434-2446.

Figures/Tables 13

Fig.1 Venn diagram of the targets of components of Jiawei Qingluo Yin (JWQLY) and rheumatoid arthritis (RA) targets.

Fig.2 Component-Target-Pathway network diagram (A) and PPI network diagram (B).

Fig.3 Bar plot of GO enrichment analysis (A) and bubble plot of KEGG enrichment analysis (B).

Tab.1 Results of molecular docking binding energy

Constituent	GSK3B	ARRB1	CCND1	SHH	BCL2
Luteolin	-7.6	-7.1	-8.1	-8.8	-7.8
Quercetin	-7.2	-8.2	-7.2	-7.2	-7.7
Beta-sitosterol	-7.5	-7.1	-7.9	-9.0	-7.1
Kaempferol	-8.0	-7.2	-7.8	-7.5	-7.9
Stigmasterol	-7.8	-6.6	-7.6	-7.6	-7.9

Fig.4 Molecular docking results and molecular dynamics simulation of the key components of JWQLY and the core targets. A: Molecular docking binding between the key compounds (quercetin, beta-sitosterol, luteolin, and kaempferol) and the core targets (GSK3B, ARRB1, CCND1, SHH, and BCL2). B: RMSD fluctuation curve of the Shh-Beta-sitosterol complex during molecular dynamics simulation over time (ps). C. RMSF distribution of residues in the Shh-Beta-sitosterol complex.

Fig.5 Effects of JWQLY on body weight and arthritis in rats with antigen-induced arthritis (AIA) (Mean±SD, n=8). A: Representative images of paw swelling in rats from each group. B: Changes in body weight of the rats in different groups. C: Arthritis index scores of the rats in different groups. #P<0.05 vs Control group; *P<0.05 vs Model group.

Fig.6 Effects of JWQLY on synovial tissue of AIA rats (Mean±SD, n=8). A: HE staining of synovial tissues from rats in different groups (Original magnification: ×200). B: Histopathological scores of synovial tissues of the rats in AIA model and treatment groups. Red arrows indicate fibroblast proliferation, blue arrows indicate inflammatory cell infiltration, and yellow arrows indicate pannus formation. *P<0.05 vs Model group.

Fig.7 TUNEL staining of synovial cell apoptosis in AIA rats treated with JWQLY (Mean±SD, n=3).

Fig.8 Results of ELISA for detecting serum levels of IL-1β (A) and VEGF (B) in rats from each group (Mean±SD, n=6). #P<0.05 vs Control group; *P<0.05 vs Model group.

Fig.9 Effects of JWQLY and inhibitor on body weight and arthritis in the rats (Mean±SD, n=8). A: Representative images of paw swelling in rats from each group. B: Changes in body weight of the rats in different groups. C: Arthritis index scores of the rats in different groups. #P<0.05 vs Control group; *P<0.05 vs Model group.

Fig.10 Effect of JWQLY on the protein expressions of BAX and BCL-2 in each group detected by immunohistochemistry. Red arrows indicate the positive expression areas (cytoplasmic brown-yellow staining).

Fig.11 Effect of JWQLY on protein expression of Ihh, Smo, Gli1, cleaved caspase-3 and cleaved caspase-9 in each group (Mean±SD, n=3). #P<0.05 vs Control group; *P<0.05 vs Model group.

Fig.12 Effect of JWQLY and Hedgehog inhibitor on proteins expression of Ihh, Smo, Gli1, cleaved caspase-3 and cleaved caspase-9 in each group (Mean±SD, n=3). #P<0.05 vs Control group; *P<0.05 vs Model group.

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