痛风宁对高尿酸血症模型大鼠肾脏尿酸盐转运体表达的影响

Effect of Tongfengning on Expression of Renal Urate Transporter in Hyperuricemia Rats

目的:通过观察痛风宁对高尿酸血症(HUA)模型大鼠肾脏尿酸盐转运体蛋白及mRNA表达的影响,探讨该方促进尿酸排泄的机制。方法:将80只SD大鼠随机分为空白组20只和造模组60只。造模组行腹腔注射氧嗪酸钾(OAPS)联合盐酸乙胺丁醇(EMB)灌胃造模,于第21天时鉴定模型,确定造模成功后将60只造模大鼠随机分为模型组、痛风宁组和苯溴马隆组,每组20只。痛风宁组和苯溴马隆组分别予15. 3 g·kg^-1·d^-1痛风宁药液及5. 2 mg·kg^-1·d^-1苯溴马隆混悬液灌胃,空白组和模型组予等量生理盐水灌胃,于干预第14,21天分批采集大鼠尿液、血液、肾脏组织。采用酶比色法检测血尿酸(SUA),尿尿酸(UUA)含量,脲酶法检测血尿素氮(BUN)含量,肌氨酸氧化酶法检测血肌酐(CRE)含量;蛋白免疫印迹法(Western blot)及实时荧光定量PCR(Real-time PCR)检测肾脏尿酸盐转运体蛋白和mRNA表达。结果:药物干预第14,21天,与空白组比较,模型组SUA,CRE,BUN升高,UUA显著降低(P<0. 01),尿酸盐转运体1(URAT1),葡萄糖转运体9(GLUT9)蛋白与mRNA表达明显升高(P<0. 05),三磷酸腺苷结合盒转运蛋白G2(ABCG2),有机阴离子1(OAT1),有机阴离子3(OAT3)蛋白与mRNA表达明显下降(P<0. 05);与模型组比较,痛风宁组及苯溴马隆组SUA,CRE,BUN明显降低,UUA明显升高(P<0. 05),URAT1,GLUT9蛋白与mRNA表达明显下降(P<0. 05),ABCG2,OAT1,OAT3蛋白与mRNA表达明显升高(P<0. 05),痛风宁组CRE,BUN明显降低(P<0. 05,P<0. 01),且明显优于苯溴马隆组(P<0. 05);干预第21天痛风宁组,除BUN外,其余指标改善趋势均更明显。结论:腹腔注射OAPS联合EMB灌胃可致HUA,并伴有肾功能异常;痛风宁可促进尿酸排泄,降低血尿酸,并下调肾脏URAT1,GLUT9表达,上调ABCG2,OAT1,OAT3表达,这可能是其促进尿酸排泄而降尿酸的作用机制之一;痛风宁对肾功能具有一定的保护作用。

Objective:Study on the mechanism of Tongfengning in promoting uric acid excretion from the perspective of urate transporter and mRNA in renal of hyperuricemia(HUA)model rats. Method: The 80 sprague-dawley rats were randomly divided into two groups,the blank group with 20 rats and the model group with 60 rats. Rats in model group were established as hyperuricemia(HUA)models by intraperitoneal injection of oxonic acid potassium salt(OAPS)and intragastric administration ethambutol hydrochloride(EMB)once a day for 21 days. After successful modeling,rats in the model group were divided into the model group,Tongfengning group and benzbromarone group,with 20 rats per group. Tongfengning solution(15. 3 g·kg^-1·d^-1)was administered to the Tongfengning group by gavage feeding. Rats in benzbromarone group were administered5. 2 mg·kg^-1·d^-1 benzbromarone suspension,whereas those in the blank group and the model group were administered the equivalent amount of normal saline for 21 days. On days 14 thand 21 stfollowing intervention,urine,blood,and kidney were collected from rats,serum uric acid(SUA)and urinary uric acid(UUA)levels,blood urea nitrogenand(BUN)and creatinine(CRE)levels and the expression of urate transporter proteins and their mRNAs of all rats were detected by enzyme-colorimetric method,urease method,sarcosine oxidase method,Western blot and Real-time quantitative PCR(Real-time PCR),respectively. Result: On days 14 thand21 thfollowing intervention,compared with blank group,SUA,CRE and BUN levels,and urate transporter 1(URAT1),glucose transporter 9(GLUT9)expression increased(P<0. 05,P<0. 01),whereas UUA level,and adenosine triphosphate-binding cassette transporter protein G2(ABCG2),organic anion 1(OAT1),organic anion 3(OAT3)expression decreased in the model group(P<0. 05,P<0. 01). Compared with model group,SUA,CRE and BUN levels,and URAT1,GLUT9 expression decreased in Tongfengning group and the benzbromarone group(P<0. 05),whereas UUA level,and ABCG2,OAT1,OAT3 expression increased(P<0. 05). Creatinine and BUN levels decreased in the Tongfengning group(P<0. 05,P<0. 01),with the trend much better than the benzbromarone group(P<0. 05). On day 21 st,except for the BUN level did not change much compared with day 14 th,all the rest indicators got improved obviously. Conclusion: Intraperitoneal injection of OAPS and intragastric administration of EMB can cause HUA models with renal dysfunction.Tongfengning reduced URAT1,GLUT9 mRNA and protein expression,and upregulated ABCG2,OAT1,OAT3 mRNA and protein expression in the rat kidney,which may be one of the mechanisms of promoting uric acid excretion. Tongfengning has a certain protective effect on renal function.