氧化锌抑制紫外线B诱导的人晶状体上皮细胞Ca^2＋-ATP酶1表达

Zinc oxide nanoparticles inhibits Ca^(2+)-ATPase1 expression induced by UVB irradiation in human lens epithelial cells

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 ma 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(2.5μg·m L^(-1)1、5.0μg·m L^(-1)、10.0μg·m L^(-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^(-1)增加到(173.88±7.17)nmol·L^(-1)、(289.02±9.09)nmol·L^(-1)、(488.36±48.16)nmol·L^(-1),差异均有统计学意义(均为P<0.05);在无UVB照射下,2.5μg·m L^(-1)、5.0μg·m L^(-1)、10.0μg·m L^(-1)氧化锌组细胞PMCA1 mRNA表达分别是0μg·m L^(-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·m L^(-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Ca^(2+)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。

Objective To investigate the effect of ZnO nanoparticles on the expressions of plasma membrane calcium ATPase1 （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^-1,5.0 μg·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） nmol·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 0 μg·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.