Network pharmacology and experimental validation reveal mechanisms of Ilicis Cornutae Folium water extract in treating hyperuricemia

Abstract

Ethnopharmacological relevance: Ilicis Cornutae Folium, the dried leaves of Ilex cornuta, has been traditionally used in Chinese medicine for heat-clearing, detoxification, and liver-kidney tonification.

Aim of the study: To investigate the therapeutic mechanisms of Ilicis Cornutae Folium water extract (ICFWE) on hyperuricemia (HUA) through integrated computational and pharmacological approaches.

Materials and methods: We established hyperuricemic mouse models using an adenine/potassium oxonate combination to evaluate ICFWE's therapeutic effects. First, serum uric acid (UA) levels were measured along with renal/hepatic function markers and hepatic xanthine oxidase (XOD) activity. Next, renal histopathology was assessed through HE and Masson staining, while urate transporter expression was examined by immunohistochemistry (IHC) and real-time PCR. For chemical characterization, we employed UPLC-Q-TOF/MS analysis, while network pharmacology combined with molecular docking helped predict and validate potential targets. Finally, we analyzed apoptotic markers (BAX/Bcl-2/Caspase-3) and NF-κB pathway-related targets (pp65/p65/TNF-α/IL-1β) by Western blot and real-time PCR.

Results: ICFWE (240-960 mg/kg/d) significantly reduced serum UA levels in hyperuricemic mice, improved renal function indicators, and inhibited hepatic XOD activity. Histopathological analysis showed that it decreased renal fibrosis area. At the molecular level, ICFWE upregulated the expression of renal ABCG2, OAT1, and OAT3 while downregulating GLUT9 expression. UPLC-Q-TOF/MS identified 35 chemical components, and network pharmacology screening revealed seven core active components primarily targeting the TNF, Caspase-3, Bcl-2, and NF-κB pathways. Molecular docking demonstrated that pedunculoside, isorhamnetin, and quercetin had strong binding affinity with Caspase-3, TNF, Bcl-2, and ABCG2. Mechanistic studies showed that ICFWE significantly downregulated BAX and cleaved caspase-3 protein expression, upregulated Bcl-2 expression, and suppressed NF-κB phosphorylation along with the production of downstream inflammatory factors TNF-α and IL-1β. These effects were further validated in an LPS/UA-stimulated HK-2 cell model.

Conclusion: ICFWE alleviates HUA by dual regulation of UA metabolism by inhibiting XOD production and promoting excretion and renal protection through anti-inflammatory/anti-apoptotic pathways.

Keywords: Apoptosis; Hyperuricemia; Ilicis Cornutae Folium; Inflammatory; Network pharmacology.