Objective To investigate the effect of ZnO nanoparticles on the expressions of plasma membrane calcium ATPasel (PMCA1) of human lens epithelial cell B-3 (HLEB-3) at both mRNA and protein levels in the presence and absence of ultraviolet B (UVB) irradiation.Methods HLEB-3 was cultured in RPMI 1640 medium,and the cytotoxic effect of different concentrations of ZnO (0 μg · mL-1,2.5 μg · mL-1,5.0μg · mL-1,10.0 μg · mL-1) on HLEB-3 was investigated in the presence and absence of UVB irradiation.DAPI staining was used to monitor the effect of ZnO on HLECB-3 nuclei,and cell apoptosis was evaluated using annexin V-FITC/PI staining in the presence and absence of UVB irradiation.In addition,the intracellular calcium ion (Ca2 +)levels were assayed using Fluo-3/AM staining,and the expression levels of both PMCA1 mRNA and protein within HLEB-3 were detected by real-time PCR and Western blot,respectively.Results DAPI staining showed that the ZnO-treated HLEB-3 displayed a concentration-dependent apoptosis,and UVB irradiation could further aggravate the cytotoxic effect of ZnO on HLEB-3.In addition,in the presence of UVB irradiation,concentration gradient of ZnO (2.5 μg · mL-1,5.0 μg · mL-1,10.0 μg · mL-1) increased the intracellular calcium ion levels [from (156.34 ±4.59) nmol · L-1 to (173.88 ±7.17)umol · L-1,(289.02 ± 9.09) nmol · L-1,(488.36 ± 48.16) nmol · L 1,respectively] and upregulated HLEB-3 apoptosis,with statistical difference (all P ＜ 0.05).Moreover,the expression level of PMCA1 in the 2.5 μg · mL-1,5.0 μg · mL-1,10.0 μg · mL-1 ZnO-treated epithelial cells was accordingly 0.75,0.57 and 0.41 as much as that in the 0μg · mL-1 ZnO-treated cells in the absence of UVB irradiation (all P ＜ 0.05),and was accordingly 0.64,0.24 and 0.09 in the present of UVB irradiation,with significant difference (all P ＜ 0.05).Conclusion Both ZnO nanoparticle and UVB irradiation can exert cosuppression effect on HLEB-3 via calcium-mediated signaling pathway,indicating it has great potential for the treatment of posterior capsular opacification with UVB irradiation.%目的 研究纳米材料氧化锌在有/无紫外线B(ultraviolet B,UVB)照射下对人晶状体上皮细胞系B-3(human lens epithelial cell B-3,HLEB-3)细胞质膜Ca2+-ATP酶1(plasma membrane calcium ATPase1,PMCA1)表达的影响.方法 HLEB-3细胞进行培养和传代,在有/无UVB照射情况下,加入不同浓度的氧化锌,利用DAPI染色细胞核;Annexin V/PI染色检测细胞凋亡;Fluo-3/AM染色检测细胞内Ca2浓度的变化;实时荧光定量PCR检测PMCA1 mRNA表达水平;Western blot方法检测PMCA1蛋白表达水平.结果 DAPI细胞核染色结果显示,氧化锌以浓度依赖方式使HLEB-3细胞产生典型的细胞凋亡反应;UVB照射可增加氧化锌对HLEB-3细胞的毒性效应;在UVB照射下,经氧化锌(2.5 μg· mL-1、5.0 μg·mL-1、10.0 μg· mL-1)处理的HLEB-3细胞凋亡及细胞内Ca2浓度增加(均为P＜0.05),细胞内Ca2水平分别从(156.34±4.59) nmol·L-增加到(173.88±7.17)nmol· L-1、(289.02±9.09)nmol·L-1、(488.36±48.16) nmol·L-,差异均有统计学意义(均为P＜0.05);在无UVB照射下,2.5 μg· mL-1、5.0 μg· mL-1、10.0μg·mL-1氧化锌组细胞PMCA1 mRNA表达分别是0μg·mL-1氧化锌组的0.75倍、0.57倍和0.41倍,差异均有统计学意义(均为P ＜0.05);而在有UVB照射下,各浓度氧化锌组细胞PMCA1 mRNA表达分别是0μg·mL-1氧化锌组的0.64倍、0.24倍和0.09倍,差异均有统计学意义(均为P＜0.05).结论 氧化锌和UVB照射通过Ca2信号转导通路对HLEB-3细胞有协同抑制作用,提示氧化锌在有UVB照射的情况下对后发性白内障的治疗有巨大潜在作用.
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