背景:巴马汀是黄柏的主要生物活性生物碱,在中国,这是治疗高尿酸血症(HUA)的常用处方。巴马汀的代谢产物对其突出的生物活性至关重要。9-羟基-8-氧代棕榈素(9-OPAL)是一种新型的肝脏介导的次生代谢产物。
目的:目前的研究是评估9-OPAL的疗效,一种来自黄柏的新的肝脏介导的次生代谢产物,并在实验HUA小鼠模型中进一步探讨其潜在机制。
方法:使用肝脏样品进行OPAL的体外代谢实验。我们分离并鉴定了一种新的肝脏代谢产物,并研究了其在小鼠体内的抗HUA作用。每天用氧酸钾和次黄嘌呤诱导HUA小鼠一周。建模1小时后,小鼠口服给予不同剂量的9-OPAL(5、10和20mg/kg)。使用苏木精-伊红染色(H&E)评估肾脏的病理变化。评估了9-OPAL的急性毒性。9-OPAL对血清尿酸(UA)水平的影响腺苷脱氨酶(ADA),黄嘌呤氧化酶(XOD),肌酐(CRE),采用酶联免疫吸附试验(ELISA)或生化法测定血尿素氮(BUN)和炎性细胞因子。此外,蛋白质印迹,采用实时定量PCR(qRT-PCR)和分子对接技术研究9-OPAL对HUA小鼠肾尿酸转运体表达和NLRP3信号通路的影响。
结果:9-OPAL首次被发现是一种新的肝脏介导的黄花素代谢产物。用9-OPAL治疗显着降低了UA,CRE以及BUN水平,并且还有效地减轻了肾脏组织病理学异常恶化,具有良好的安全性。此外,9-OPAL显著降低XOD和ADA的血清和肝脏活性,显著抑制UA转运蛋白1(URAT1)和葡萄糖转运蛋白9(GLUT9)的上调,并逆转了有机阴离子转运蛋白1(OAT1)的下调。此外,9-OPAL有效缓解肾脏炎症标志物(TNF-α,IL-1β,IL-6和IL-18),并下调肾Nod样受体家族pyrin结构域包含3(NLRP3)的转录和翻译表达,HUA小鼠的caspase-1,凋亡相关斑点样(ASC)和IL-1β。分子对接结果显示9-OPAL与XOD牢固结合,OAT1、GLUT9、URAT1、NLRP3、caspase-1、ASC和IL-1β。
结论:发现9-OPAL是一种新的肝脏介导的次级代谢产物,具有良好的安全性。9-OPAL具有突出的抗高尿酸血症和肾脏保护作用,机制可能与抑制XOD活动密切相关,调节肾尿酸转运蛋白表达并抑制NLRP3炎性体激活。我们的研究也可能为黄柏在HUA治疗中的传统应用提供进一步的实验证据。
BACKGROUND: Palmatine is a main bioactive alkaloid of Cortex Phellodendri, which has been commonly prescribed for the treatment of hyperuricemia (HUA) in China. The metabolites of palmatine were crucial to its prominent biological activity. 9-Hydroxy-8-oxypalmatine (9-OPAL) is a novel liver-mediated secondary oxymetabolite of palmatine.
OBJECTIVE: The current study was to assess the efficacy of 9-OPAL, a novel liver-mediated secondary oxymetabolite of palmatine derived from Cortex Phellodendri, in experimental HUA mouse model and further explore its underlying mechanism.
METHODS: An in vitro metabolic experiment with oxypalmatine was carried out using liver samples. We separated and identified a novel liver metabolite, and investigated its anti-HUA effect in mice. HUA mice were induced by potassium oxonate and hypoxanthine daily for one week. After 1 h of modeling, mice were orally administered with different doses of 9-OPAL (5, 10 and 20 mg/kg). The pathological changes of the kidneys were evaluated using hematoxylin-eosin staining (H&E). The acute toxicity of 9-OPAL was assessed. The effects of 9-OPAL on serum levels of uric acid (UA), adenosine deaminase (ADA), xanthine oxidase (XOD), creatinine (CRE), blood urea nitrogen (BUN) and inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA) or biochemical method. Furthermore, Western blot, quantitative real-time PCR (qRT-PCR) and molecular docking were used to investigate the effect of 9-OPAL on the expression of renal urate transporters and NLRP3 signaling pathway in HUA mice.
RESULTS: 9-OPAL had been discovered to be a novel liver-mediated oxymetabolite of palmatine for the first time. Treatment with 9-OPAL significantly reduced the UA, CRE as well as BUN levels, and also effectively attenuated abnormal renal histopathological deterioration with favorable safety profile. Besides, 9-OPAL significantly decreased the serum and hepatic activities of XOD and ADA, dramatically inhibited the up-regulation of UA transporter protein 1 (URAT1) and glucose transporter protein 9 (GLUT9), and reversed the down-regulation of organic anion transporter protein 1 (OAT1). Additionally, 9-OPAL effectively mitigated the renal inflammatory markers (TNF-α, IL-1β, IL-6 and IL-18), and downregulated the transcriptional and translational expressions of renal Nod-like receptor family pyrin domain containing 3 (NLRP3), caspase-1, apoptosis-associated speck-like (ASC) and IL-1β in HUA mice. Molecular docking results revealed 9-OPAL bound firmly with XOD, OAT1, GLUT9, URAT1, NLRP3, caspase-1, ASC and IL-1β.
CONCLUSIONS: 9-OPAL was found to be a novel liver-mediated secondary metabolite of palmatine with favorable safety profile. 9-OPAL had eminent anti-hyperuricemic and renal-protective effects, and the mechanisms might be intimately associated with repressing XOD activities, modulating renal urate transporter expression and suppressing the NLRP3 inflammasome activation. Our investigation might also provide further experimental evidence for the traditional application of Cortex Phellodendri in the treatment of HUA.