Table2_Integrating network pharmacology and experimental validation to clarify the anti-hyperuricemia mechanism of cortex phellodendri in mice.XLSX

Hyperuricemia (HUA), a common metabolic disease, is treated as the second-largest metabolic disease after diabetes in China. Cortex Phellodendri (CP) is one of the most frequently used herbal medicines for treating gout or HUA. However, the mechanism underlying the anti-HUA effect of CP is still unrevealed. Hence, this study aimed to explore the pharmacological mechanism of CP against HUA using network pharmacology coupled with in vivo experimental validation. Active compounds and potential targets of CP, as well as the potential targets related to HUA, were retrieved from multiple open-source databases. The drug-disease overlapping targets were obtained by Venn diagram analysis and used to construct the herb-component-target (HCT), protein-protein-interaction (PPI), and component-target-pathway (CTP) networks. The functional enrichment analysis was also performed for further study. Furthermore, a HUA mouse model was induced by a combination of intraperitoneal injection of potassium oxonate (PO, 300 mg/kg) and intragastric administration of hypoxanthine (HX, 300 mg/kg) daily for 10 days. Different dosages of CP (200, 400, and 800 mg/kg) were orally given to mice 1 h after modeling. The results showed that 12 bioactive compounds and 122 drug-disease overlapping targets were obtained by matching 415 CP-related targets and 679 HUA-related targets, and berberine was one of the most important compounds with the highest degree value. The core targets of CP for treating HUA were TP53, MAPK8, MAPK3, IL-6, c-Jun, AKT1, xanthine oxidase (XOD), and ATP-binding cassette subfamily G member 2 (ABCG2). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results showed that the anti-HUA effect of CP mainly involved the pathways of inflammation and apoptosis, such as PI3K/Akt, TNF, MAPK, TLR, AMPK, NF-κB, and NLRP3 signaling pathways. In vivo animal experiment further confirmed the hypouricemic effect of CP in a HUA mouse model, as evidenced by significantly restored kidney histological deteriorations, and considerably decreased levels of serum uric acid (sUA), creatinine (Cre), blood urea nitrogen (BUN), and hepatic UA. Furthermore, the hypouricemic action of CP in vivo might be attributed to its suppression of XOD activity in the liver, rather than ABCG2 in the kidney. Real-time qPCR (RT-qPCR) and Western blot analysis also confirmed the key roles of the hub genes in CP against HUA. In conclusion, CP exhibited therapeutic effect against HUA via multi-compounds, multi-targets, and multi-pathways. It possessed anti-HUA and nephroprotective effects via suppressing XOD activity, and reversed the progression of renal injury by exerting anti-inflammatory and anti-apoptotic effects.

高尿酸血症（Hyperuricemia, HUA）是一种常见代谢性疾病，在我国已成为仅次于糖尿病的第二大代谢类疾病。黄柏（Cortex Phellodendri, CP）是治疗痛风或高尿酸血症最常用的中药材之一，但其抗高尿酸血症的作用机制仍未阐明。因此，本研究旨在通过网络药理学结合体内实验验证，阐明黄柏抗高尿酸血症的药理作用机制。从多个开源数据库中检索黄柏的活性成分、潜在靶点以及与高尿酸血症相关的潜在靶点。通过韦恩图分析得到药物-疾病重叠靶点，并用于构建草药-成分-靶点（HCT）、蛋白质相互作用（PPI）以及成分-靶点-通路（CTP）网络，同时开展功能富集分析以进行深入研究。此外，本研究通过每日腹腔注射氧嗪酸钾（potassium oxonate, PO, 300 mg/kg）联合灌胃次黄嘌呤（hypoxanthine, HX, 300 mg/kg），连续造模10天，构建高尿酸血症小鼠模型。造模1小时后，分别以200、400、800 mg/kg三种不同剂量的黄柏对小鼠灌胃给药。研究结果显示，通过匹配415个黄柏相关靶点与679个高尿酸血症相关靶点，共得到12个生物活性成分与122个药物-疾病重叠靶点，其中小檗碱（berberine）是度值最高的关键活性成分之一。黄柏治疗高尿酸血症的核心靶点包括TP53、MAPK8、MAPK3、IL-6、c-Jun、AKT1、黄嘌呤氧化酶（xanthine oxidase, XOD）以及ATP结合盒亚家族G成员2（ATP-binding cassette subfamily G member 2, ABCG2）。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析结果表明，黄柏抗高尿酸血症的作用主要涉及炎症与细胞凋亡相关通路，如PI3K/Akt、TNF、MAPK、TLR、AMPK、NF-κB及NLRP3信号通路。体内动物实验进一步证实了黄柏在高尿酸血症小鼠模型中的降尿酸作用，表现为肾脏组织病理损伤显著恢复，血清尿酸（serum uric acid, sUA）、肌酐（Cre）、血尿素氮（BUN）及肝脏尿酸水平均显著降低。此外，黄柏在体内的降尿酸作用可能与其抑制肝脏黄嘌呤氧化酶活性相关，而非通过调控肾脏ABCG2实现。实时荧光定量PCR（RT-qPCR）与蛋白质印迹实验同样证实了核心靶点在黄柏抗高尿酸血症过程中的关键作用。综上，黄柏通过多成分、多靶点、多通路发挥抗高尿酸血症的治疗作用，其通过抑制黄嘌呤氧化酶活性实现降尿酸与肾脏保护功效，并通过抗炎与抗凋亡作用逆转肾损伤进展。