有机阴离子转运蛋白1对大鼠滑膜细胞吸收甲氨蝶呤、增殖及迁移的影响

Effects of organic anion transporter 1 on absorption,proliferation and migration of methotrexate by rat synovial cells

目的探讨有机阴离子转运蛋白1(OAT1)对胶原诱导性关节炎(CIA)大鼠成纤维样滑膜细胞(FLS)吸收甲氨蝶呤(MTX)以及对其增殖和迁移的影响。方法对CIA大鼠FLS使用转染技术上调OAT1的表达,并用Western blot、RT-qPCR方法验证转染效率。将FLS分为阴性对照组(NC组)、Overexpress组,并用肿瘤坏死因子-α(TNF-α)(20 ng/ml)刺激。另取正常大鼠FLS为对照组(Control),给予同浓度(100 ng/ml)MTX和OAT1特异性底物对氨基马尿酸(PAH),采用UPLC-MS/MS方法评估FLS对MTX及PAH吸收功能。给予MTX(100 ng/ml)后使用CCK-8、Transwell方法检测FLS增殖效应及迁移能力。结果与NC组相比,Overexpress组的OAT1及Slc22a6表达均上升;与NC组比较,OAT1过表达组FLS对MTX和PAH的吸收在15、30、45、60、120 min时均增加;与NC组比较,Overexpress组FLS的增殖效应在12 h时下降,并趋近于Control组水平;与NC组比较,12 h时OAT1过表达组的迁移能力下降,并趋近于Control组水平。结论OAT1表达的升高能增加炎性FLS对MTX的吸收,并可抑制其异常的增殖效应和迁移能力。

Objective To investigate the effect of organic anion transporter1(OAT1)on the absorption of methotrexate(MTX)and its proliferation and migration in fibroblast-like synovial cells(FLS)of rats with collagen induced arthritis(CIA).Methods The CIA model was established in rats and the FLS was isolated and cultured.The transfection technique was used to up-regulate the expression of OAT1 in FLS,and Western blot and RT-qPCR were used to verify the transfection efficiency.FLS were divided into negative control(NC)and OAT1 overexpression groups and stimulated with tumor necrosis factor-α(TNF-α)(20μg/L).In addition,normal rats FLS were taken as the Control group.The MTX and p-aminohippuric acid(the specific substrate of OAT1,PAH)were given at the same concentration(100 ng/ml).And the absorption function of FLS for MTX and PAH was evaluated by UPLC-MS/MS/MS.At the same time,after MTX(100 ng/ml)was administered,the proliferation effect and migration ability of FLS were detected by CCK-8 and Transwell.Results Compared with the NC group,the expressions of OAT1 and Slc22a6 in the overexpression group significantly increased.Compared with the NC group,the absorption of MTX and PAH by FLS in the OAT1 overexpression group significantly increased at 15,30,45,60 and 120 min.Compared with the NC group,the proliferation of FLS in the OAT1 overexpression group significantly decreased at 12 h and approached the level of the Control group.Compared with the NC group,the migration ability of OAT1 overexpression group decreased significantly after 12 h and approached the level of the Control group.Conclusion The increased expression of OAT1 can significantly increase the absorption of MTX by inflammatory FLS and inhibit its abnormal proliferation effect and migration ability.