中国中药杂志

2020, v.45(21) 5248-5255

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防己黄芪汤对高尿酸血症小鼠降尿酸及肾保护作用机制的研究
Study on mechanism of Fangji Huangqi Decoction on hypouricemic effect and renal protection in hyperuricemia mice

王星;薛宁;李洪雷;陈真;余万;
WANG Xing;XUE Ning;LI Hong-lei;CHEN Zhen;YU Wan;Department of Pharmacology, School of Pharmacy, China Pharmaceutical University;Department of Acupuncture, Jurong Hospital Affiliated to Jiangsu University;Department of Pharmacy, Kangda College of Nanjing Medical University;Department of Neurosurgery, Affiliated Hospital of Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine;

摘要(Abstract):

以氧嗪酸钾盐诱导的高尿酸血症(hyperuricemia,HUA)小鼠为模型,研究防己黄芪汤(Fangji Huangqi Decoction,FHT)降尿酸及改善肾功能的具体机制,为临床防治HUA药物的研发及中药现代化研究提供理论基础。将60只昆明种雄性小鼠随机分组,每组10只,即正常组,模型组(250 mg·kg~(-1)氧嗪酸钾盐),FHT高、中、低给药组(10 920,5 460,2 730 mg·kg~(-1)),阳性药别嘌呤醇组(5 mg·kg~(-1)),每天灌胃给药1次,连续7 d。第6天时,小鼠转至代谢笼中,收集其24 h尿液,用于测定尿液尿酸、肌酐和β2-微球蛋白(β2-microglobulin,β2-MG)水平;7 d后处死动物,测定血清尿酸、肌酐、β2-MG和白细胞介素-1β(interleukin-1β,IL-1β)水平,同时分离肝脏和肾脏组织,肝脏用于后续黄嘌呤氧化酶(xanthine oxidase,XOD)活性测定,肾脏用于后续IL-1β水平、病理学检测及相关蛋白质免疫印迹(Western blot)试验。并在细胞转染试验中,将细胞分成空白组、模型组(4.8 mmol·L~(-1)尿酸处理)、FHT给药组(4.8 mmol·L~(-1)尿酸+200μg·mL~(-1) FHT)、富亮氨酸重复激酶1(leucine-rich repeat kinase 1,LRRK1)-小干扰RNA(small interfering RNA,siRNA)组(4.8 mmol·L~(-1)尿酸+LRRK1-siRNA转染)和LRRK1-siRNA+FHT组(4.8 mmol·L~(-1)尿酸+LRRK1-siRNA转染+200μg·mL~(-1) FHT),孵育24 h后,测定细胞上清液IL-1β水平,并提取细胞蛋白,用于测定LRRK1、表皮生长因子受体(epidermal growth factor receptor,EGFR)、PDZ蛋白激酶1(PDZ kinase 1,PDZK1)和核转录因子(nuclear factor-kappa B,NF-κB)蛋白表达水平。结果显示,FHT可以显著降低HUA小鼠血清尿酸、肌酐、β2-MG和尿液β2-MG水平,升高尿液尿酸和肌酐水平,同时改善HUA小鼠肾脏病理结果,但对肝脏XOD活性没有影响;同时发现HUA小鼠血清及肾脏IL-1β表达水平显著上升,肾脏NF-κB,LRRK1和EGFR蛋白水平显著升高,而肾脏PDZK1蛋白表达水平显著下降,FHT可以显著改善上述异常表达的蛋白,且FHT升高HUA小鼠肾脏有机阴离子转运体1(organic anion transporter 1,OAT1)、OAT3和三磷酸腺苷结合转运蛋白G2(ATP binding transporter G 2,ABCG2)的蛋白表达水平,但是FHT对尿酸转运蛋白1(urate transporter 1,URAT1)的表达没有影响。在细胞转染试验中,LRRK1-siRNA转染后,上清液IL-1β水平、EGFR和NF-κB蛋白表达水平显著降低,PDZK1蛋白表达水平显著上升,再加入FHT后,与LRRK1-siRNA组相比,上清液IL-1β水平及EGFR,PDZK1和NF-κB蛋白表达水平没有显著变化。该研究表明FHT可能通过LRRK1基因调控肾脏尿酸转运系统,提高尿酸排泄能力,从而降低血清尿酸水平;另外,FHT除具有直接的肾保护作用,还可通过降低尿酸水平达到间接的肾保护的作用。
The aim of this paper was to study the specific mechanism of Fangji Huangqi Decoction(FHT) in decreasing uric acid and improving renal function in mice with hyperuricemia(HUA) induced by potassium oxonate, so as to provide theoretical basis for the research and development of drugs for clinical prevention and treatment of HUA and the modernization of traditional Chinese medicine. Sixty Kunming male mice were randomly divided into 6 groups, with 10 mice in each group, namely normal group, model group(250 mg·kg~(-1) potassium oxonate), FHT high, medium and low-dose groups(10 920, 5 460, and 2 730 mg·kg~(-1)) and positive drug allopurinol group(5 mg·kg~(-1)). Drug administration was given once a day for 7 days. On the 6 th day, mice of each group were kept in metabolic cages, and their urine was collected for 24 hours for determination of uric acid, creatinine, and β2-microglobulin(β2-MG) levels. After 7 days, the animals were sacrificed to determine serum uric acid, creatinine β2-MG and interleukin-1β(IL-1β) levels, and their liver and kidney tissues were collected. The liver tissues were used for subsequent determination of xanthine oxidase(XOD) activity, and the kidney tissues were used for subsequent determination of IL-1β levels, pathological tests and related Western blot experiments. In the cell transfection experiment, the cells were divided into blank group, model group(4.8 mmol·L~(-1) uric acid treatment), FHT administration group(4.8 mmol·L~(-1) uric acid+200 μg·mL~(-1) FHT), leucine-rich repeat kinase 1(LRRK1)-small interfering RNA(siRNA) group(4.8 mmol·L~(-1) uric acid+LRRK1-siRNA transfection) and LRRK1-siRNA+FHT group(4.8 mmol·L~(-1) uric acid+LRRK1-siRNA transfection+200 μg·mL~(-1) FHT). After 24 h incubation, the level of IL-1β in the cell supernatant was detected, and the cellular proteins were extracted and used to determine LRRK1, epidermal growth factor receptor(EGFR), PDZ kinase 1(PDZK1) and nuclear factor-kappa B(NF-κB) protein expression levels. The results showed that, FHT could significantly reduce the uric acid, creatinine and β2-MG levels in serum and β2-MG levels in urine, increase the uric acid and creatinine levels in urine, and improve the renal pathological results of the HUA mice, but showed no effect on liver XOD activity; at the same time, we found that the expression level of IL-1β in serum and kidney, NF-κB, LRRK1 and EGFR protein levels in kidney of HUA mice were significantly increased, and the expression level of PDZK1 protein was significantly decreased, while FHT could significantly improve the abnormal expression of these proteins, and FHT increased protein expression of renal organic anion transporter 1(OAT1), OAT3 and ATP bin-ding transporter G2(ABCG2) in HUA mice, but FHT had no effect on the expression of urate transporter 1(URAT1). In the cell transfection experiment, after transfection of LRRK1-siRNA, the levels of IL-1β, EGFR and NF-κB in supernatant were significantly reduced, and the expression of PDZK1 protein was significantly increased. As compared with the LRRK1-siRNA group, the levels of IL-1β, EGFR, PDZK1 and NF-κB did not change significantly with the additional FHT. This study showed that FHT may regulate the renal uric acid transport system through LRRK1 gene, improve the capacity of uric acid excretion, so as to reduce the level of serum uric acid. At the same time, FHT can not only protect the kidney directly, but also in an indirect manner by reducing the level of uric acid.

关键词(KeyWords): 防己黄芪汤;高尿酸血症;尿酸转运系统;LRRK1
Fangji Huangqi Decoction;hyperuricemia;uric acid transport system;LRRK1

Abstract:

Keywords:

基金项目(Foundation): 江苏省自然科学基金项目(BK20180574);; 江苏省中医药局科技项目(YB201820);; 江苏省卫生计生委2018年度医学科研课题(Z2018002);; 江苏省高等学校自然科学研究面上项目(18KJB350005)

作者(Author): 王星;薛宁;李洪雷;陈真;余万;
WANG Xing;XUE Ning;LI Hong-lei;CHEN Zhen;YU Wan;Department of Pharmacology, School of Pharmacy, China Pharmaceutical University;Department of Acupuncture, Jurong Hospital Affiliated to Jiangsu University;Department of Pharmacy, Kangda College of Nanjing Medical University;Department of Neurosurgery, Affiliated Hospital of Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine;

Email:

DOI: 10.19540/j.cnki.cjcmm.20200630.401

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