Standardized Extract of Flavonoids from Smilax glabra Alleviates Gouty Arthritis by Multi-Target Inhibition of Neutrophil Extracellular Traps via the Raf/ERK and Histone Citrullination Pathways

Chenxi Wu,1,* Xinru Xu,1,* Yueyue Shi,1,* Cantao Li,1,* Fenfen Li,1 Ziye Xu,1 Liangxin Chen,1 Wenjing Xu,1 Yihuan Wang,1 Xiaoxi Zhang,2 Daozong Xia1 1School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, People’s Republic of China; 2Academy of Chinese Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, People’s Republic of China*These authors contributed equally to this workCorrespondence: Daozong Xia, School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, People’s Republic of China, Email [email protected]: Neutrophil extracellular traps (NETs) are involved in the pathogenesis of gouty arthritis (GA). This study aimed to investigate whether standardized extract of flavonoids from Smilax glabra Roxb. (SFSG) alleviates GA by targeting NETs formation and to elucidate its underlying mechanisms involving neutrophil-driven inflammation and signaling pathways.Methods: SFSG’s effects were evaluated in a monosodium urate (MSU)-induced GA model. Neutrophil depletion (via mAb 1A8 antibody) validated their pathogenic role. SFSG was administered for 7 days to assess joint swelling, histopathology (HE staining), neutrophil infiltration (immunohistochemistry), and inflammatory cytokines (IL-1β and IL-8, measured by ELISA). In MSU and PMA-stimulated neutrophils, SFSG (0– 200 μg/mL, non-toxic by CCK8) was tested for NETs formation (immunofluorescence, transmission electron microscopy), oxidative stress (ROS), lactate dehydrogenase (LDH) activity, Raf/ERK signaling, and citrullinated histone H3 (CitH3) (Western blotting).Results: Neutrophil depletion significantly alleviated MSU-induced joint inflammation, confirming neutrophils as key drivers of GA pathology. SFSG treatment dose-dependently suppressed MSU-induced joint inflammation in vivo. Notably, the high dose inhibited ankle swelling by ~66% (reducing it from 58.36% to 19.75%), matching the efficacy of colchicine. Concomitant reductions in neutrophil infiltration and IL-1β/IL-8 levels were also observed. Additionally, SFSG attenuated PADI4 and NOX2 gene expression. In vitro, SFSG inhibited NETs formation, ROS production, and LDH release while downregulating Raf/ERK signaling and CitH3 expression.Conclusion: SFSG alleviates GA progression by targeting neutrophil-driven inflammation and NETs formation through suppression of Raf/ERK signaling and CitH3 expression, thereby demonstrating its potential as a phytotherapeutic agent for neutrophil-associated pathologies. Keywords: standardized extract of flavonoids from Smilax glabra, gouty arthritis, neutrophil extracellular traps, neutrophil depletion, Raf/ERK signaling pathway