| Objective:Retinal pigmented epithelial cell (RPE) oxidative stress is known to have a vital role in the etiology of age-related macular degeneration (AMD). The protection of retinal pigment epithelial cells (RPEs) is promising treatments for AMD. The present study aimed to investigate whether allicin, a natural product with antioxidant activity, was able to protect RPEs (ARPE-19) from hydrogen peroxide (H2O2)-induced damage, and to determine the underlying mechanisms.Methods:1. ARPE-19 cells were grown to 80% confluence, and were treated with H2O2 (250 or 500 ?M) for 12 or 24 h following supplementation with allicin (5,10,20 or 40?g/ml; 98% purity) for 4 h. Cells were harvested after the appropriate treatments. Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyl tetrazolium bromide (MTT) assay.2. An Intracellular ROS Assay kit was used to detect the intracellular ROS levels. Briefly,100 ?l 2',7'-dichlorodihydro fluorescein diacetate (DCFH-DA) was added to the culture media of the ARPE-19 cells for 30 min at 37?. Subsequently, the cells were washed twice with phosphate-buffered saline (PBS). DCFH fluorescence of the cell lysate was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using FLUO star optima and was quantified using Image J version 1.41 software. The average fluorescence intensity was analyzed from five fields for each treatment. The relative fluorescence intensity was expressed as a percentage increase with reference to the intensity of the normal control cells. The glutathione (GSH)/glutathione disulfide (GSSG) ratio and malondialdehyde (MDA) concentrations were assessed using the GSH/GSSG Assay kit and the MDA Assay kit, according to the manufacturer's protocols. The superoxide dismutase (SOD) activity was measured using the SOD Assay kit, according to the manufacturer's protocols.3. Effects of allicin on reactive oxygen species-associated enzymes in ARPE-19 retinal pigmented epithelial cells following treatment with hydrogen peroxide (H2O2). Relative mRNA expression levels of NOX4 and NQO1 were measured by reverse transcription-quantitative polymerase chain reaction. Protein expression levels of NOX4 and NQO1 were detected by western blotting.The NOX4 protein expression levels of ARPE-19 cells treated with H2O2 (control group) were denoted as 100%. All experiments were performed in triplicate. Activity of superoxide dismutase (SOD) was measured using a SOD assay kit. Normal group, untreated cells; control group, cells treated with 250 ?M H2O2.4. Nuclear factor erythroid 2-related factor 2 (Nrf2) is associated with allicin-mediated protection of retinal pigmented epithelial cells (RPEs) against hydrogen peroxide (H2O2)-induced injury. Relative mRNA expression levels of Nrf2 were measured by reverse transcription-quantitative polymerase chain reaction. ARPE-19 cells exposed to H2O2 (control group) were denoted as 100%. Expression levels of cytosolic Nrf2 protein were determined by western blotting. Expression levels of nuclear Nrf2 protein were determined by western blotting. Efficiency of Nrf2 knockdown was evaluated by western blotting. Differences in cell viability were compared between the groups. Normal group, untreated cells; control group, cells treated with 250 uM H2O2; 20 ?g/ml+knockout (KO) group, cells with small interfering RNA (siRNA)-mediated Nrf2 knockdown, treated with 20 ?g/ml allicin and 250 ?M H2O2 20 ?g/ml+KO control group, cells transfected with non-target siRNA, treated with 20 ?g/ml allicin and 250?M H2O2.Results:1. Treatment with H2O2 resulted in a significant loss of cell viability after 12 or 24 h exposure. Notably, treatment with allicin reversed the effects of H2O2 on RPEs. The percentage of viable cells was increased in response to 10-40 ?g/ml allicin in a dose-dependent manner (P<0.05).2. Oxidative stress is characterized by a pathological state of excessive ROS production or abnormal ROS homeostasis. Allicin (10 and 20 ?g/ml) markedly reduced H2O2-induced ARPE-19cells intracellular ROS levels (P=0.047 and P=0.033), as compared with in the cells not treated with allicin. The GSH/GSSG ratio reflects the extent of oxidative stress in cells. In the present study, RPEs preincubated with allicin (10 ?g/ml, P=0.037; 20 ?g/ml, P=0.007) were better at combating H2O2 injury, as compared with cells in the H2O2 group. Treatment with H2O2resulted in a significant increase in MDA levels, which was downregulated following allicin preincubation in a dose dependent manner, especially when administered at 20 ?g/ml (P=0.027).3. Allicin-mediated protection of RPEs is associated with regulation of ROS-associated enzymes, SOD, NOX4 and NQO1. NOX4,which is the key enzyme of the ROS generation system, SOD, which is an important biomarker of oxidative stress, and NQO1, which is an important antioxidant enzyme. Treatment with allicin (20 ?g/ml) significantly downregulated the mRNA and protein expression levels of NOX4 (mRNA, P=0.031; protein, P=0.003). H2O2 significantly inhibited the levels of SOD, and pretreatment with allicin significantly attenuated this inhibition when administered at a concentration of 20 ug/ml (P=0.009). In addition, allicin was able to markedly increase the protein expression levels of NQO1 when administered at a concentration of 10 ?g/ml or 20 ?g/ml (P<0.001).4. Nrf2, which has an essential role eliminating oxidants by reinforcing cellular antioxidant capacity. The relative mRNA expression levels of Nrf2 were enhanced by allicin in a dose-dependent manner (P<0.05), as compared with the H2O2-treated control cells. The cytosolic protein expression levels of Nrf2 were significantly increased following treatment with 20?g/ml allicin (P=0.008; Fig.4B and C). The nuclear protein expression levels of Nrf2 were significantly elevated following treatment with allicin at both 10 ?g/ml (P=0.025) and 20 ?g/ml (P=0.008), as compared with the control cells. The Nrf2-siRNA effectively silenced Nrf2 gene expression in the RPEs. Nrf2 knockdown significantly attenuated the protection of allicin against H2O2-mediated loss of viability (P=0.032). However, Nrf2 knockdown did not completely suppress the protective effects of allicin (Control group 53.34±5.01% vs.20 ?g/ml+knockdown group 63.06±7.85%).Conclusions:1. Exogenous H2O2 induces ARPE-19 oxidative stress and cell injury. Allicin was able to protect ARPE-19 cells from H2O2-induced damage in a dose-dependent manner.2. Allicin due adjusting the intracellular ROS level, GSH/GSSG, MDA and SOD level to realize the protective effect of RPEs.3. Allicin-mediated protection of RPEs is associated with regulation of ROS-associated enzymes, SOD, NOX4,NQO1 and Nrf2. |
PDF Full Text Request