The Effects Of Different Concentrations Of Zinc Chloride Against UVB Radiation-induced Damage In Cultured Human Lens Epithelial Cells And Their Mechanisms

Objective: To investigate the protective effect of appropriate concentrations of zinc ions on human lens epithelial B-3(HLE B-3) cells upon ultraviolet B(UVB) radiation in vitro, and the alterations in the levels of intracellular calcium ions(Ca2+) and relevant Ca2+-ATPase expression. Methods: HLE B-3 cells were cultured and pretreated with different concentrations of zinc chloride(ZnCl2) for 2 h, then exposed to UVB radiation(40 mJ/cm2) and further cultured different time prior to the following experiments: a real-time cell electronic sensing system was used to explore the effects of ZnCl2 in the presence and absence of UVB irradiation on cell proliferation; integrity of mitochondrial membrane potential(ΔΨm) was assessed using JC-1 staining; meanwhile, DCFH-DA staining was used to investigate the change in the levels of intracellular reactive oxygen species(ROS); Annexin V-FITC/PI staining was used to measure the rate of cell apoptosis; Fluo-3/AM staining was used to measure the intracellular Ca2+ level. Moreover, real-time PCR was used to assay the abundance of plasma membrane calcium ATPase 1(PMCA1), plasma membrane calcium ATPase 4(PMCA4), sarcoplasmic endoplasmic reticulum Ca2+-ATPase 2b(SERCA2b) and sarcoplasmic endoplasmic reticulum Ca2+-ATPase 3(SERCA3) mRNA in HLE B-3 cells; Enzyme-Linked Immuno Sorbent Assay(ELISA) was used to determine the levels of PMCA1, PMCA4, SERCA2 b and SERCA3 proteins. Results: ZnCl2 could markedly reduce UVB radiation-induced HLE B-3 cell apoptosis, and this inhibitory effect requires appropriate zinc ion concentrations. The pretreatment of appropriate concentrations of ZnCl2 could efficiently prevent HLE B-3 cells from the loss of Δψm, maintain intracellular calcium homeostasis, reduce the generation of UVB radiation-induced ROS and decrease HLE B-3 cell apoptosis. Meanwhile, ZnCl2-pretreatment could also decrease the UVB-induced expression of SERCA2 b and SERCA3 and causes an upregulation of PMCA1 and PMCA4. Conclusions: UVB-induced apoptosis in HLE B-3 cells proceeds through the calcium-mediated mitochondrial apoptotic pathway. Pretreatment with appropriate concentrations of ZnCl2 can significantly upregulate the expression of PMCA1 and PMCA4 to restore the intracellular Ca2+ level under UVB radiation, efficiently maintains the mitochondrial membrane potential, inhibits the overproduction of reactive oxygen species, and thus maintains normal physiological functions within cells.