摘要
目的探讨高浓度尿酸盐对肠道细胞HT-29尿酸转运功能的影响,明确高浓度尿酸盐干预下三磷酸腺苷结合盒转运蛋白G2(ABCG2)表达及功能的改变并探讨可能的机制。方法实验组分别使用2.5、5.0、7.5、10.0、15.0、20.0 mg/dl浓度梯度的尿酸对HT-29细胞进行干预,对照组使用0 mg/dl干预,通过细胞计数试剂盒(CCK8法)检测细胞活性,流式细胞术检测ABCG2转运功能,Western blot法和实时荧光定量聚合酶链反应(RT-qPCR)分别检测β-catenin/ABCG2蛋白及mRNA表达水平,免疫共沉淀(Co-IP)检测ABCG2与β-catenin的结合水平,激光共聚焦显微镜观察细胞ABCG2及β-catenin的蛋白定位。结果与对照组相比,2.5、5.0 mg/dl浓度尿酸处理24 h后,HT-29细胞的增殖活性无明显改变,而10.0、15.0、20.0 mg/dl浓度尿酸处理24 h后,细胞活性受损较为显著,细胞活性分别下降至(80.67±1.59)%、(77.85±2.41)%、(69.49±1.45)%,差异有统计学意义(均P<0.05)。与对照组比较,低浓度尿酸(2.5、5.0 ml/dl)干预下,β-catenin/ABCG2在蛋白水平和mRNA水平表达差异均无统计学意义(均P>0.05),高浓度尿酸(7.5、10.0、15.0、20.0 mg/dl)干预下β-catenin/ABCG2在蛋白水平和mRNA水平表达均显著下降(均P<0.05)。选择对照(0 mg/dl)和高浓度(15.0 mg/dl)尿酸进行进一步相关实验,研究发现高浓度尿酸显著下调了HT-29细胞的转运功能(P<0.05)。免疫共沉淀证实ABCG2和β-catenin蛋白间存在相互结合作用,与对照组相比,高浓度尿酸干预组中ABCG2和β-catenin蛋白结合呈下降趋势但无统计学意义(均P>0.05)。激光共聚焦显微镜下观察显示,与对照组相比,高浓度尿酸组中ABCG2与β-catenin共定位减少,且定位于胞膜的ABCG2数量减少(P<0.05)。结论高浓度尿酸盐可下调肠道细胞β-catenin/ABCG2的表达及功能,进而影响细胞的转运功能,且高浓度尿酸作用下破坏了β-catenin/ABCG2的共定位。
Objective To investigate the effect of high concentration uric acid(UA)on uric acid transport function in intestinal HT-29 cells,to clarify the changes of adenosine triphosphate binding box transporter G2(ABCG2)expression and function under high concentration of urate,and to explore the possible mechanism.Methods HT-29 cells were treated with UA of gradient concentrations(2.5,5.0,7.5,10.0,15.0,and 20.0 mg/dl).Cells nontreated with UA were used as controls.Cell activity was detected by cell counting kit-8(CCK8)assay.ABCG2 transport function was detected by flow cytometry.The mRNA and protein expression ofβ-catenin and ABCG2 was detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot,respectively.The interactions of ABCG2 andβ-catenin was detected by co-immunoprecipitation(Co-IP)assay.Laser confocal fluorescence microscopy was used to observe the distribution and co-localization ofβ-catenin/ABCG2 protein in HT-29 cells.Results The proliferative activity of cells in the high concentration UA(10.0,15.0,and 20.0 mg/dl)groups was significantly decreased to about 80.67%±1.59%,77.85%±2.41%,69.49%±1.45%,respectively,when compared with that in the control group(P<0.05).In contrast,after treatment with 2.5 and 5.0 mg/dl UA for 24 h,the proliferation activity of HT-29 cells was not significantly changed.There was no significant difference inβ-catenin/ABCG2 protein and mRNA expression between the low concentration UA(2.5 and 5.0 ml/dl)groups and the control group.Compared with control cells,the protein and mRNA expression ofβ-catenin/ABCG2 was significantly decreased in the high concentration uric acid(7.5,10.0,15.0,and 20.0 mg/dl)groups(P<0.05).Thus,15.0 mg/dl UA(HUA)was used for subsequent experiments.Compared with control cells,the intestinal drug efflux functions of HT-29 cells in the HUA group were compromised(P<0.05),as revealed by flow cytometry.Co-IP assay confirmed that there was interaction betweenβ-catenin and ABCG2 in control cells.The interaction betweenβ-catenin and ABCG2
作者
杨帆
丁雪丽
李晓宇
宋玉君
田字彬
郭英杰
Yang Fan;Ding Xueli;Li Xiaoyu;Song Yujun;Tian Zibin;Guo Yingjie(Department of Gastroenterology,the Affiliated Hospital of Qingdao University,Qingdao 266071,China)
出处
《中华临床医师杂志（电子版）》
CAS
2024年第1期57-63,共7页
Chinese Journal of Clinicians(Electronic Edition)
基金
国家自然科学基金(82100581)。
