摘要
目的 研究硫酸镍(NiSO4)对人支气管上皮细胞(Beas-2B)的毒作用及损伤机制,并探讨Wnt/β-catenin通路在损伤中的作用.方法 选择不同浓度硫酸镍(0、50、100、200 μmol/L)处理Beas-2B细胞24 h,CCK-8法检测硫酸镍的细胞毒性;使用丙二醛和总铁离子检测试剂盒检测细胞内丙二醛、总铁离子含量;使用免疫印迹法(Westernblot)检测Wnt/β-catenin信号通路蛋白Wnt3a、β-catenin和铁死亡相关蛋白谷胱甘肽过氧化物酶4(GPX4)、胱氨酸/谷氨酸反转运蛋白(xCT)以及凋亡相关蛋白B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2关联X蛋白(Bax)表达水平;通路抑制剂XAV-939处理Beas-2B细胞后100 μmol/L硫酸镍染毒24 h,检测GPX4、xCT表达水平.结果 Beas-2B细胞存活率随着硫酸镍浓度的升高而降低(F=12.530,P＜0.05);丙二醛和总铁离子含量下降,Wnt3a、β-catenin表达逐渐增加(F=9.209、3.936、7.639、23.350,P＜0.05),GPX4、xCT表达增加,Bax表达逐渐增加和Bcl-2表达逐渐降低(F=6.903、17.76、52.930、14.460,P＜0.05);与 100μmol/L染毒组比较,XAV-939 处理组 Beas-2B 细胞中 Wnt3a、β-catenin、GPX4和xCT表达均降低(t=8.055、8.049、9.659、11.700,P＜0.05).结论 体外实验硫酸镍可诱导Beas-2B细胞凋亡的同时可能通过激活Wnt/β-catenin通路抑制细胞铁死亡.
Abstract
Objective To study the toxic effect and injury mechanism on human bronchial epithelial cells(Beas-2B)by NiSO4,and to explore the role of the Wnt/β-catenin pathway in it.Methods Different concentrations of NiSO4(0,50,100,200 μmol/L)were selected to treat Beas-2B cells for 24 hours,the cytotoxicity of NiSO4 was detected by CCK-8 assay,and the contents of malondialdehyde and total iron ions in cells were detected by lipid peroxidation mda assay kit and total iron content colorimetric assay kit.Western blot was used to detect the expression of Wnt3a,β-catenin,Ferroptosis related proteins GPX4,xCT,Apoptosis related proteins Bcl-2,Bax.Beas-2B cells were treated with XAV-939,a pathway inhibitor,and then treated with 100 μmol/L NiSO4 for 24 h.The expression levels of GPX4 and xCT were detected.Results With the increase of NiSO4 concentration,Beas-2B cell survival rate significantly reduced(F=12.530,P＜0.05),the contents of malondialdehyde and total iron ion decreased,the expression of Wnt3a,β-catenin gradually increased(F=9.209,3.936,7.639,23.350,P＜0.05),the expression of GPX4,xCT increased,the expression of Bax gradually increased and the expression of Bcl-2 decreased gradually(F=6.903,17.760,52.930,14.460,P＜0.05).Compared with 100 μmol/L group,the expression of Wnt3a,β-catenin,GPX4 and xCT in Beas-2B cells treated with XAV-939 decreased(t=8.055,8.049,9.659,11.70,P＜0.05).Conclusion In vitro experiments showed that NiSO4 can induce apoptosis in Beas-2B cells while possibly activating Wnt/β-catenin pathway inhibits cell ferroptosis.
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