saRNA激活ABCG2表达增强肾和肠细胞排泄尿酸的研究  

Enhanced Expression of ABCG2 by saRNA increases Uric Acid Excretion of Renal and Intestinal Cells in Mice

作　　者：贾少平 葛科立[2] 杨柳[1] 张志霞 张金玉[1] 葛银林[1] JIA Shaoping;GE Keli;YANG Liu;ZHANG Zhixia;ZHANG Jinyu;GE Yinlin(Department of Biochemistry and Molecular Biology,School of Basic Medicine,Qingdao University,Qingdao 266071,China;Department of Immunology,School of Basic Medicine,Qingdao University,Qingdao 266011,China;Shandong Institute of Commerce and Technology,Jinan 250103,China)

机构地区：[1]青岛大学基础医学院生物化学与分子生物学系,山东青岛266071 [2]青岛大学基础医学院免疫学系,山东青岛266011 [3]山东商业职业技术学院,济南250103

出　　处：《中国药学杂志》2024年第8期713-723,共11页Chinese Pharmaceutical Journal

基　　金：山东省科技计划资助(2014GGH218023);山东省重点研发项目资助(2017GSF218084);青岛市应用基础研究项目资助(19-6-2-31-cg)。

摘　　要：目的筛选、研究小激活RNA(small activating RNA,saRNA)增强ABCG2基因表达对肾、肠细胞排泄尿酸(uric acid,UA)的影响,探索促进UA排泄的全新一类治疗机制药物。方法设计靶向小鼠和人ABCG2基因的saRNA,转染小鼠肾小管上皮细胞(TCMK-1)和人肾小管上皮细胞(HK-2)细胞,逆转录-聚合酶链反应(RT-qPCR)和Western Blot筛选强激活效果的saRNA;检测强激活saRNA增强TCMK-1和HK-2细胞排泄UA的作用。筛选的saRNA通过尾静脉注射高尿酸血症(hyperuricemia,HUA)模型小鼠,检测血尿酸(serum uric acid,SUA)、尿尿酸(urine uric acid,UUA)、肠道UA、血尿素氮(blood urea nitrogen,BUN)和血清肌酐(serum creatinine,SCr)含量,以及肝脏黄嘌呤氧化酶(xanthine oxidase,XOD)活性,验证所选saRNA促进UA排泄的作用。苏木精-伊红染色法(H&E)染色观察小鼠肾脏和小肠病理改变。RT-qPCR,Western blot和免疫组化分析肾脏和小肠ABCG2基因表达水平。结果筛选到小鼠和人ABCG2 saRNA各2条,分别命名为saRNA-1/2和hsaRNA-1/2,能够提高TCMK-1和HK-2细胞ABCG2基因的表达(P<0.05),促进胞内UA排泄(P<0.05)。saRNA-1/2能够提高HUA模型小鼠UUA和肠道UA含量(P<0.05),降低SUA,BUN和SCr的含量(P<0.05),改善HUA对肾脏和小肠的损伤。saRNA-1/2促进了小鼠肾脏和小肠中ABCG2基因的表达,但对小鼠肝脏中XOD的活性没有明显影响(P>0.05)。结论所筛选的saRNA能够提高ABCG2基因的表达,并促进细胞内和小鼠体内UA的排泄,降低SUA水平。OBJECTIVE To screen and study the effects of small activating RNA(saRNA)enhancing the expression of ABCG2 gene on renal and intestinal cell excretion of uric acid(UA)to explore a new type of therapeutic mechanism drugs to promote UA excretion.METHODS saRNAs targeting mice and human ABCG2 genes were designed and screened by real-time quantitative reverse transcription PCR(RT-qPCR)and Western blot in mice TCMK-1 and human HK-2 cells.The effect of the selected saRNA on the excretion of UA was examined in TCMK-1 and HK-2 cells.The mice saRNA was injected into the tail vein of hyperuricemia(HUA)model mice.The levels of serum uric acid(SUA),urinary uric acid(UUA),intestinal uric acid,blood urea nitrogen(BUN)and serum creatinine(SCr),and the activity of liver xanthine oxidase(XOD)in the mice were detected to verify the ability of selected saRNA to promote UA excretion.The pathological changes of kidney and small intestine were analyzed through H&E.The expression of ABCG2 in kidney and small intestine was analyzed by RT-qPCR,Western blot and immunohistochemistry.RESULTS Four saRNAs targeting ABCG2 were selected,2 for mice and 2 for human,named saRNA-1/2 and hsaRNA-1/2 respectively,which increased the expression of ABCG2 in TCMK-1 and HK-2 cells(P<0.05),and promoted the extracellular excretion of UA(P<0.05).In HUA model mice,saRNA-1/2 increased the levels of UUA and intestinal UA(P<0.05),reduced the levels of SUA,BUN,and SCr(P<0.05),decreased the damage of HUA to kidney and small intestine,and enhanced the expression of ABCG2 in kidney and small intestine,but did not show significant effect on the activity of liver XOD(P>0.05).CONCLUSION The selected saRNAs could enhance the expression of ABCG2 and promote the excretion of UA in cells and mice,reducing SUA level.

关 键 词：高尿酸血症 尿酸 小激活RNA ABCG2 基因表达 

分 类 号：R34[医药卫生—基础医学] R966