5-氟尿嘧啶通过下调Wnt/β-catenin信号通路抑制骨髓基质细胞增殖

5-Fluorouracil Inhibits Proliferation of Bone Marrow Stromal Cells via Down-Regulating Wnt/β-catenin Signaling Pathway

该研究探讨了5-氟尿嘧啶(5-fluorouracil, 5-FU)抑制人骨髓基质细胞HS-5增殖的可能机制,寻找改善化疗药物对骨髓基质细胞损伤的治疗靶点。实验分3组,对照组:常规培养;5-FU组:常规培养基础上加入25μg/mL 5-FU;氯化锂(LiCl)+5-FU组:10 mmol/L LiCl预处理细胞, 6 h后加入25μg/mL 5-FU,各组培养48 h。EdU检测HS-5细胞增殖,流式细胞术检测细胞周期, Western blot检测β-catenin、Cyclin D1、C-myc蛋白表达, DCFH-DA荧光法检测细胞内活性氧(reactive oxygen species, ROS)水平, Western blot检测缝隙连接蛋白Cx43表达。与对照组相比, 5-FU组HS-5细胞增殖能力下降,细胞阻滞在G0/G1期,胞内ROS水平显著升高,β-catenin、 Cyclin D1、C-myc、Cx43蛋白表达下调。与5-FU组相比, LiCl+5-FU组HS-5细胞增殖能力回升,细胞G1期阻滞减轻,胞内ROS水平降低,β-catenin、Cyclin D1、C-myc、Cx43蛋白表达上调。5-FU可通过下调Wnt/β-catenin信号通路抑制HS-5细胞增殖,其作用机制可能与5-FU诱导细胞发生氧化应激,下调细胞间隙连接蛋白Cx43表达有关。

This article was to explore the possible mechanism of 5-fluorouracil(5-FU)inhibiting the proliferation of human bone marrow stromal cells(HS-5),and to find a therapeutic target for improving the hematopoietic damage of chemotherapy drugs.HS-5 cells were divided into three groups.The control group was routinely cultured;the 5-FU group was treated by 5-FU on the concentration of 25μg/mL;the LiCl+5-FU group was pretreated with LiCl on the concentration of 10 mmol/L,and 25μg/mL 5-FU was added after 6 h,each group was cultured for 48 h.The proliferation of HS-5 cells was detected by EdU.The cell cycle was analyzed by flow cytometry.The expressions ofβ-catenin,CyclinD1,and C-myc proteins were measured by Western blot.The levels of reactive oxygen species(ROS)in cells were detected by DCFH-DA fluorescence.The expression of Cx43 protein was measured by Western blot.Compared with the control group,the proliferative capacity of HS-5 cells decreased,the cell cycle was blocked,intracellular ROS level was significantly increased,and the expressions ofβ-catenin,CyclinD1,C-myc,and Cx43 proteins were down-regulated in the 5-FU group.Compared with the 5-FU group,the proliferation of HS-5 cells in the LiCl+5-FU group was increased,the cell cycle arrest was attenuated,intracellular ROS level was decreased,and the expressions ofβ-catenin,CyclinD1,C-myc and Cx43 proteins were up-regulated.5-FU can inhibit the proliferation of HS-5 cells by the mechanism of down-regulating Wnt/β-catenin signaling pathway,which may lead to 5-FU-induced oxidative stress and down-regulation of Cx43 expression.