基于复方配伍环境探究番红花去柱头花部治疗高尿酸血症合并痛风性关节炎的药效与作用机制

Exploring Efficacy and Mechanism of Saffron Floral Bio-residues for Treatment of Hyperuricemia Combined with Gouty Arthritis Based on Compound Compatibility Environment

摘要:

目的 在复方配伍环境下，探究番红花去柱头花部（SFB）治疗高尿酸血症（HUA）合并痛风性关节炎（GA）的药效与作用机制。方法 基于数据分析和网络靶点距离计算，筛选复方及SFB的对照中药。80只SD大鼠适应性喂养7 d后，随机取10只大鼠作为空白组，其余70只大鼠腹腔注射3%氧嗪酸钾并灌胃1%腺嘌呤，连续14 d，第13天于大鼠右踝关节腔注射2.5%尿酸钠溶液，诱导HUA合并GA模型。将成模大鼠随机分为模型组、苯溴马隆组（0.02 g·kg^-1）、痛风舒片组（9 g·kg^-1）、痛风舒颗粒组（9 g·kg^-1）、SFB颗粒组（3.6 g·kg^-1）、车前子颗粒组（3.6 g·kg^-1）和新方组（SFB替代痛风舒颗粒中的车前子，9 g·kg^-1），每组10只。各给药组按剂量灌胃相应药物，空白组和模型组灌胃等体积蒸馏水，1次/d，连续14 d，造模期间同步给药。给药结束后，检查大鼠步态、踝关节肿胀度和机械痛阈值变化，并检测血清尿酸、肌酐、尿素氮、黄嘌呤氧化酶（XOD）；酶联免疫吸附测定法（ELSIA）检测大鼠血清肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β和IL-6水平；苏木素-伊红（HE）染色观察大鼠肝、肾和踝关节病理变化；蛋白免疫印迹法（Western blot）检测大鼠肾脏尿酸转运体1（URAT1）、葡萄糖转运体9（GLUT9）、有机阴离子转运体1（OAT1）、三磷酸腺苷（ATP）结合盒转运蛋白G2（ABCG2）及肝脏XOD蛋白的表达水平。结果 通过网络药理学分析，确定车前子作为候选对照药，痛风舒片作为复方配伍环境，用于探究SFB的降血酸和治疗GA疗效。动物实验表明，与空白组比较，模型组的步态评分和关节肿胀度均显著升高，机械痛阈显著降低（P<0.01）；与模型组比较，各给药组大鼠步态评分、关节肿胀度及机械痛阈均有不同程度改善。生化指标检测结果显示，与空白组比较，模型组大鼠的血清尿酸、肌酐、尿素氮、XOD水平均显著上升（P<0.01）；与模型组比较，各给药组大鼠血清尿酸、XOD水平显著下降（P<0.01）。ELISA结果显示，与空白组比较，模型组大鼠血清TNF-α、IL-1β、IL-6水平显著升高（P<0.01）；与模型组比较，痛风舒片组、痛风舒颗粒组与新方组大鼠的血清TNF-α、IL-1β和IL-6水平明显降低（P<0.05，P<0.01）。Western blot结果显示，与空白组比较，模型组大鼠肾组织URAT1、GLUT9蛋白表达显著升高，OAT1、ABCG2蛋白表达显著降低，肝组织XOD蛋白表达显著升高（P<0.01）；与模型组比较，SFB组、痛风舒颗粒组及新方组大鼠肾组织URAT1、GLUT9蛋白表达明显降低，OAT1、ABCG2蛋白表达明显增加，肝组织XOD蛋白表达明显降低（P<0.05，P<0.01）。病理学分析显示，模型组大鼠肝组织中可见中性粒细胞灶性浸润、细胞坏死和胞核碎裂，肾组织出现尿酸钠沉积结晶和肾小管扩张，踝关节滑膜增生和炎细胞浸润，各给药组大鼠的肝、肾脏和踝关节组织异常程度减轻。结论 SFB替代车前子后的通风舒颗粒新方在治疗HUA合并GA具有同等效果。该文提出了一种在复方配伍环境下进行中药新资源药效探究的新策略，可为中药新资源的研究与开发提供新示范。

Abstract:

Objective To investigate the efficacy and mechanism of saffron floral bio-residues（SFB） in the treatment of hyperuricemia（HUA） combined with gouty arthritis（GA） in a compound compatibility setting.Method Screening candidate control Chinese medicines for compound and SFB based on network target distance calculation and data analysis. After adaptive feeding of 80 SD rats for 7 days， 10 rats were randomly selected as the blank group， while the remaining 70 rats were intraperitoneally injected with 3% potassium oxonate and orally administered with 1% adenine for 14 consecutive days. On the 13^th day， rats were injected with 2.5% sodium urate solution into the right ankle joint cavity to induce swelling of the joint capsule on the opposite side， inducing a HUA combined with GA model. At the same time， the modeling rats were randomly divided into 7 groups， including the model group， benzbromarone group（positive drug， 0.02 g·kg^-1）， Tongfengshu tablets group（9 g·kg^-1）， Tongfengshu granules group（9 g·kg^-1）， SFB granules group（3.6 g·kg^-1）， Plantaginis Semen granules group（3.6 g·kg^-1）， and new formula group（SFB replacing Plantaginis Semen in Tongfengshu granules， 9 g·kg^-1）， with 10 rats in each group. Each treatment group was orally administered with the corresponding drugs according to body weight， while the control and model groups were given equal volume of distilled water by gavage once a day for 14 consecutive days. After 14 days of synchronous administration and modeling， changes in gait， ankle joint swelling and mechanical pain threshold in rats were observed， and serum uric acid， creatinine， urea nitrogen and xanthine oxidase（XOD） were measured. Enzyme-linked immunosorbent assay（ELSIA） was used to detect the levels of tumor necrosis factor（TNF）-α， interleukin（IL）-1β and IL-6 in rat serum， hematoxylin-eosin（HE） staining was used to observe the pathological changes in the liver， kidney and ankle joints of rats， Western blot was used to detect the expression levels of uric acid transporter 1（URAT1）， glucose transporter 9 （GLUT9）， organic anion transporter 1（OAT1）， adenosine triphosphate（ATP） binding cassette transporter G2（ABCG2）， and liver XOD proteins.Result Through network pharmacology analysis， Plantaginis Semen was selected as a candidate control herb， and Tongfengshu tablets was used as a compound compatibility environment to explore the efficacy of SFB in reducing blood uric acid levels and treating GA. Animal experiments showed that compared with the blank group， the gait score and joint swelling degree of the model group were significantly increased， and the mechanical pain threshold was significantly decreased（P<0.01）. Compared with the model group， the gait score， joint swelling degree and mechanical pain threshold of rats in each medication group were improved to varying degrees. Biochemical indicators showed that compared with the blank group， the serum uric acid， creatinine， urea nitrogen and XOD levels of the model group were significantly increased（P<0.01）. Compared with the model group， the serum uric acid and XOD levels of rats in each treatment group were significantly decreased（P<0.01）. ELISA results showed that compared with the blank group， the levels of serum TNF-α， IL-1β and IL-6 in the model group were significantly increased（P<0.01）. Compared with the model group， the levels of TNF-α， IL-1β and IL-6 in the benzbromarone group， Tongfengshu tablets group， Tongfengshu granules group and new formula group were significantly reduced（P<0.05，P<0.01）. Western blot results showed that compared with the blank group， the expression levels of URAT1 and GLUT9 proteins in renal tissue and OXD protein in liver tissue of the model group were significantly increased， while the expression levels of renal OAT1 and ABCG2 were significantly decreased（P<0.01）. Compared with the model group， the expression levels of renal URAT1 and GLUT9 in the SFB granules group， Tongfengshu granules group and new formula group were significantly decreased， while the expression levels of renal OAT1 and ABCG2 were significantly increased， and the expression of XOD protein in liver tissue was significantly decreased（P<0.05， P<0.01）. Pathological analysis showed that focal infiltration of neutrophils， cell necrosis and nuclear fragmentation were observed in the liver tissue of the model group， sodium urate deposition crystals and tubular dilation appeared in renal tissue， synovial hyperplasia and inflammatory cell infiltration appeared in ankle joint. Compared with the model group， the abnormal degrees of liver， kidney and ankle joint tissue of rats in each treatment group were alleviated.Conclusion The new formula of SFB replacing Plantaginis Semen has the same effect in the treatment of HUA combined with GA. This study proposes a new strategy to investigate the efficacy of new resources of Chinese medicine in a compound compatibility environment， which can provide a new demonstration for the research and development of new resources of Chinese medicine.