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@#AIM: To investigate the effects of blue light and white light on the proliferation of human retinal pigment epithelial cell line(ARPE-19)at different times, and lay a foundation for further detecting the changes of related factors during photodamage and further studying the signal transduction mechanism during light damage. <p>METHODS:Well-grown ARPE-19 cells were collected for experimentation. The standard curve of CCK-8 was made to determine the proper cell density of ARPE-19 cells and the reaction time of CCK-8 reagent. The cells were divided into dark group, blue light group and white light group, which were irradiated for 3, 6 and 9h respectively. After 12h of light-repellent treatment, CCK-8 method was used to examine the effects of different light sources on the proliferation rate of ARPE-19 cells at different times. <p>RESULTS: The number of cells per well was selected by CCK-8 standard curve to be 20 000, and the corresponding absorbance value was measured after 4h of the action of CCK-8 solution. The CCK-8 test results showed that the cell proliferation rates of the three groups were significantly different(<i>P</i><0.01). The cell proliferation rate of the blue light group was significantly different(<i>P</i><0.001)at 3, 6 and 9h, and the cell proliferation rate decreased gradually with the extension of the illumination time. The cell proliferation rate of the white light group was significantly different(<i>P</i><0.05)at 3, 6 and 9h; there was a statistically significant difference in the rate of cell proliferation at 3h and 6h in white light(<i>P</i><0.05), however, there was no significant difference in the rate of cell proliferation at 9h illumination compared with 3h and 6h illumination respectively(<i>P</i>=0.253, 0.120). The proliferation rate of cells under white light for 3-6h showed a downward trend, while that of cells under light for 6-9h showed an upward trend. At the same illumination time, the proliferation rate of the cells in the blue and white groups was lower than that in the dark group, and the cell proliferation rate in the blue group was lower than that in the white group. The difference was statistically significant(<i>P</i><0.05). <p>CONCLUSION: The proliferation of ARPE-19 cells was inhibited by light irradiation. The proliferation rate of cells in blue light group was significantly lower than that of white light group. With the increase of light time, the cell proliferation rate decreased.
ABSTRACT
Background Retinal pigment epithelial (RPE) cell transplantation is a novel approach to the treatment of hereditary retinal diseases, however, human-derived RPE cell line occurs de-differentiation during in vitro cell culture.Studies showed that early abnormal activation of Akt/mammalian target of rapamycin (mTOR) signal pathway is the primary cause of RPE cell line de-differentiation, therefore, the inhibition of mTOR pathway will be helpful for the retard of de-differentiation of RPE cells.Objective This study aimed to investigate whether rapamycin can suppress the activation of mTOR pathway and promote differentiation of ARPE19 cells.Methods ARPE-19 cells were incubated in 12-well plate and divided into control group and rapamycin-treated group.DMSO or rapamycin with the final concentration of 400 nmol/L was added in the medium of the control group and the rapamycin-treated group, respectively.The cells of each group were collected 24 hours and 48 hours after cultured.The expression of zonula occludens-1 (ZO-1) in the cells was examined by immunofluorescence.The relative expression levels of RPE cell specific genes and proteins were assayed by using real-time quantitative PCR and Western blot, respectively.The detected results were compared between the two groups.Results ZO-1 was expressed in both group,but the fluorescence intensity was evidently enhanced in the rapamycin-treated group.The relative expression levels of RPE65, MERKT and LRAT mRNA in the cells increased by 25.97％ , 29.71％ and 13.00％ in the rapamycin treated group compared with the control group 24 hours after cultured (P=0.04,0.04,0.04) , and the expression levels of RPE65, LRAT, rLBP1, BEST1 , keratin18 and MERKT mRNA elevated by 174.00％ , 88.00％ , 56.18％ ,193.81％ ,10.83％ and 35.02％ in the rapamycin-treated group in comparison with the control group 48 hours after cultured (P =0.00,0.04,0.01,0.04,0.04,0.03).In addition, the expressions of p-mTOR, p-P70S6 and p-S6 protein were weaker in the rapamyein-treated group than those in the control group both 24 hours and 48 hours after cultured.Twenty four hours after cultured,the expression level of ZO-1 protein raised by 40％ in the rapamycin-treated group compared with the control group (P =0.01);while 48 hours after cultured,the expression levels of ZO-1 ,MERKT, catenin and LRAT proteins elevated by 36.00％ ,57.37％, 13.68％ and 41.07％ in the rapamycintreated group in comparison with the control group (P=0.01,0.00,0.04,0.04).Conclusions Rapamycin can suppress the activation of mTOR signaling pathway and up-regulate the expressions of RPE specific genes in ARPE19 cells.Inhibition of mTOR pathway might be an effective way for culturing RPE cells in vitro.
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Purpose: Our goal was to identify the cellular and molecular effects of 2-ethylpyridine (2-EP, a component of cigarette smoke) on human retinal pigment epithelial cells (ARPE-19) in vitro. Materials and Methods: ARPE-19 cells were exposed to varying concentrations of 2-EP. Cell viability (CV) was measured by a trypan blue dye exclusion assay. Caspase-3/7 and caspase-9 activities were measured by fl uorochrome assays. The production of reactive oxygen/nitrogen species (ROS/RNS) was detected with a 2’,7’-dichlorodihydrofl uorescein diacetate dye assay. The JC-1 assay was used to measure mitochondrial membrane potential (m). Mitochondrial redox potential was measured using a RedoxSensor Red kit and mitochondria were evaluated with Mitotracker dye. Results: Aft er 2-EP exposure, ARPE-19 cells showed signifi cantly decreased CV, increased caspase-3/7 and caspase-9 activities, elevated ROS/RNS levels, decreased m value and decreased redox fl uorescence when compared with control samples. Conclusions: These results show that 2-EP treatment induced cell death by caspase-dependent apoptosis associated with an oxidative stress and mitochondrial dysfunction. These data represent a possible mechanism by which smoking contributes to age-related macular degeneration and other retinal diseases and identify mitochondria as a target for future therapeutic interventions.
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AIM:To investigate the effects of resveratrol ( Res) on the proliferation of ARPE-19 cells and to ex-plore the possible mechanisms.METHODS:After ARPE-19 cells were treated with Res at concentrations of 0, 50, 100, 150, 200 and 300 μmol/L for 24 h, 48 h and 72 h, the effects of Res on the proliferation of the cells were tested by CCK-8 assay.The ARPE-19 cells were treated with Res at concentrations of 0, 100, 150 and 200 μmol/L for 48 h.The effects of Res on the cell cycle and apoptosis were detected by flow cytometry with Annexin V-FITC/PI staining.The protein expression of proliferating cell nuclear antigen (PCNA) was detected by immunofluorescent assay.The mRNA expression of PCNA, P21 and P27 was determined by real-time PCR.RESULTS:The results of CCK-8 assay showed that Res inhibited the prolifera-tion of ARPE-19 cells in a time-and dose-dependent manner.The treatment with Res for 48 h resulted in an arrest of cell cycle at S phase without increasing cell apoptosis.Res inhibited the protein expression of PCNA in ARPE-19 cells.The re-sults of real-time PCR showed that Res increased the mRNA expression of P21 and P27, and decreased the mRNA expres-sion of PCNA.CONCLUSION: Res inhibits the proliferation of ARPE-19 cells and induces the cell cycle arrest at S phase.The mechanism may be related to up-regulation of P21 and P27, and down-regulation of PCNA.
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AIM: To evaluate the antioxidant activity of naringenin in human retinal pigment epithelium (ARPE-19) cells andhuman umbilical vein endothelial cells (HUVEC).·METHODS: MTT assay was used to measure theviability and proliferation of ARPE-19 cells and HUVEC.·RESULTS: Three and 10mg/L naringenin significantlyincreased the proliferation of ARPE-19 cells by 10.8% and11.4%, respectively. Ten mg/L naringenin increasedhypoxia-, 0.3mmol/L NAN3-, and 200μmol/L H2O2- induced damage of ARPE-19 cells by 55.2%, 69.2%, and50.3%, respectively. One mg/L naringenin increased theviability of 50μmol/L t-BHP-, and 30mg/L NalO3-treatedARPE-19 cells by 20.2% and 30.4%, respectively. Thirtymg/L naringenin also increased the proliferation of50μmol/L t-BHP-treated ARPE-19 cells by 32.2%, and1mg/L naringenin increased the proliferation of 30, 100and 300 mg/L NalO3-treated ARPE-19 cells by 30.3%,10.3% and 18.5%, respectively. The reduction of HUVECwas 23.9%, 70.4% and 77.9% in the 3, 10 and 30mg/Lnaringenin-treated groups, respectively. Furthermore, 1and 3mg/L naringenin increased hypoxia-induced damagein HUVEC by 10.7% and 13.1%, and 300mg/L NalO3- induced damage in HUVEC by 41.2% and 37.7%. Threemg/L naringenin increased 200 and 400μmol/L H2O2-in-jured HUVEC by 20.1% and 21.5%, respectively.·CONCLUSION: Naringenin increases the proliferation ofARPE-19 cells and inhibites the growth of HUVEC, and haspotent antioxidant activity in ARPE-19 cells and HUVEC.