The Study Of Human Retinal Pigment Epithelium Cells Damage Induced By Blue Light And Mitochondria-participated Mechanism In Vitro

Background and ObjectiveMacular diseases especially age related macular degeneration is the leading course of vision loss or blindness.An increasing number of studies show that if people prolonged exposure to the high density blue light,it could cause retinal photodamage,particularly injuried the macular,and have a big impact on retinal pigment epithelium(RPE)cell.However,the mechanism of photodamage especially retinal photochemical damage is unknown when RPE cell exposes to blue light.Several researches has demonstrated mitochondria played a crucial role in RPE cell damage induced by blue light.But it is not clear what is the mitochondria-participated mechanism in human RPE cell damage,and the relationship between mitochondria-participated damage in RPE cells and blue light irradiation time.To study the mitochondria-participated mechanism of RPE cell damage and cell apoptosis induced by blue light,and the relationship between mitochondria-participated damage in RPE cells and blue light irradiation time,through establishing blue light damage model of human RPE cell in vitro.Part One:The blue light damage model was establishedMethods1.RPE cells blue light damage model was established:Humidified 5%CO2 incubator was converted into blue light incubator box,adjusted the blue light indensity by FL-1D blue light irradiation meter.2.Experiment groups:RPE cells were resurrected and cultured in vitro.RPE cells were used for experiment at passage 4 in logarithmic phase.In pre-experiment,the RPE cells were grouped into 0mW/cm2(control gruop)、1.5mW/cm2、3mW/cm2、4.5mW/cm2 and 6.5mW/cm2 light density group.In order to verify the blue light damage model,the RPE cells were divided into 0-hour(control gruop),1-hour,2-hour,3-hour 4-hour,5-hour,6-hour,12-hour and 24-hour light exposure group,and cell viabilities were measured;cells were grouped into 0-hour,3-hour,6-hour,12-hour and 24-hour light exposure group,and cells features and ultra-structures were observed under the inverted microscope and the transmission electron microscope(TEM);the RPE cells were grouped into 0-hour,1-hour,2-hour,3-hour 4-hour,5-hour,6-hour and 12-hour light exposure group,and cells total apoptotic rates were evaluated.3.In order to select the appropriate blue light indensity,RPE cells were exposed to 6h blue light at these differnet light density,then the cellurlar viability was compared to normal group.Then RPE cell damages were observed under a specific light density in different blue light irrdiation time.4.Blue light damage model was evaluated by these experiment content designs.①Human RPE cell growth curve was drew by Methyl thiazolyl tetrazolium(MTT)method.②The cellular viability in different blue light exposure time was detected by MTT method.③RPE cells total apoptotic rates were evaluated by flow cytometry stained with Annexin-V-PE/7-AAD.④ The morphological characteristics and the ultra-structures of subcellular organelles in RPE cells were examined under the inverted microscope and the TEM.Results1.After 6h blue light illumination,RPE cell was damaged by blue light at density of 3mW/cm2、4.5mW/cm2and 6.5mW/cm2,and the severity of cell damage increased as blue light density raised.The cellular viability was(92.87±6.17)%at 1.5mW/cm2 light density,and it made no significant different compared to cell viability(100.00 ± 5.00)%in control group(P>0.05);while at light density of 3mW/cm2、4.5mW/cm2and 6.5mW/cm2,the cellular viability were(75.67±8.84)%、(65.40±2.74)%and(48.64±5.55)%,it made significant fiffereces compared to the normal control group(at P<0.05),and it made significant differences when the cellurlar viability of these differenet light density groups cpmpared to each other with a P<0.05。Although we can choose 3mW/cm2、4.5mW/cm2and 6.5mW/cm2 light density,as considering the cell viability was easily effected by blue ligt illumination time and systematic error cannot be avoided,so we set up(4 ± 0.5)mW/cm2 was selected as blue light model density for experiment.2.RPE cellurlar viability and cell numbers were changed:cellular viability decreased while cell total apoptotic rates raised,accompanied with the increasing light illumination time.The percentages of cellular viability were(95.73±0.89)%and(94.67±2.56)%when light exposure for 1-hour and 2-hour gruops,and it made no differences compared to control group(100.00±5.00)%(atP>0.05).While the percentages of cellular viability were(84.23±0.16)%,(77.9±2.33)%,(75.43±2.18)%,(66.13± 1.42)%,(53.43± 1.91)%and(47.97+1.36)%,when light exposure for 3-hour,4-hour,5-hour,6-hour,12-hour and 24-hour groups,respectively,showing a significant difference among the groups,and the percentages of light exposure of these groups were significantly lower than the normal control group(all at P<0.05).Besides,the total apoptotic rates of various light irradiation groups were assayed by flow cytometry,it showed that the apoptotic rate increased when light exposure time was more than 4h.and the cell total apoptotic rates were(9.6± 1.1)%、(11.45±2.60)%、(14.90±1.60)%and(23.90±1.20)%after 4-hour、5-hour、6-hour and 12-hour light exposure,compared to normal group apoptotic rate(5.3± 0.64)%,and it made significant differences(all at P<0.05).3.RPE cell morphological features and ultra-structures were changed after blue light irradiation:the cell growth characterictic was inhibited,and there existed slightly changes in cell morphological structure after 3h light illumination;but after 6h illumination,the changes became obvious.Under the inverted microscope,the number of RPE cells were sparse,accompanied with swollen cells and even some floating cells.Under the TEM,the vacuoles-like degeneration,the decreased cells’ number,the mitochondrial bacame swollen and decreased microvilli were found in RPE cells.These changes would become much more obviously with the increaseing light irradiation time.Conclusion4±0.5mW/cm2 blue light density was selected and applied for experiment in preexperiment by converting humidified incubator into blue light incubator box.After more than 3h light irradiation,there exsited changes in the morphological features and ultra-structurces in RPE cells,meanwhile the cellurlar viability decreased,and the cell total apoptotic rates raised significangtly;these changes showed the successful establishment of blue light damage model.Part Two:Mitochondria-participated mechanism of the RPE damage model was induced by blue lightMethods1.Blue light damage model was established at light density of(4±0.5)mW/cm2,then the RPE cells damages were observed at differnet irradiation time.2.Experiment groups:RPE cells were grouped into 0-hour,1/2-hour,1-hour,2-hour,3-hour,4-hour,5-hour and 6-hour light exposure group,and the productions of ROS were measured;cells were grouped into 0-hour,1-hour,2-hour,3-hour,4-hour,5-hour,6-hour and 12-hour light exposure group,the expressions of apoptotic related protein and the expressions of nicotinamide adenine dinucleotidephosphate(NADPH)mRNA and cytochrome C oxidase 1 gene(CO1)mRNA were quanlitified.3.Mitochondria damage and its function were assessed.①The contents of reactive oxygen species(ROS)were assayed by flow cytometry for the assessment of oxidative stress reaction in each gruop.②The expressions of Bcl-2、Bax and Caspase-3 were quantified by Western blot analysis.③ The relative expressions of NADPH mRNA and CO1 mRNA were detected by real time fluorescence quantitative Polymerase chain reaction(RT-PCR).Results1.ROS production in each group:the ROS contents increased as the illumination time of blue light rasised in each light exposure group.The relative ROS contents were(19.04±1.02)%、(22.81 ±3.20)%、(28.75±2.15)%、(33.06±0.96)%、(40.64±2.11)%、(48.25±2.50)%、(60.44±2.68)%in 0.5-hour,1-hour,2-hour,3-hour,4-hour,5-hour and 6-hour light exposure groups,and it showed that after more than 1h blue light irradiation,the relative ROS contents increased significant in RPE cells compared to control group ROS contents(14.75 ± 2.49)%,and it made differnces with a P value<0.05.After 4h light irradiation,it made significant differences when the light exposure group compared to each other with a P value<0.05.2.The relative expression levels of NADPH mRNA and CO1 mRNA:The relative expression levels of NADPH mRNA in the cells were gradually elevated 3 hours after light exposure with the increase of time;while the relative expression levels of CO1mRNA were higher from 2-hour to 5-hour light exposure compared to the normal control group,and the CO1 mRNA levels gradually declined to the normal level.The expression levels of NADPH mRNA and CO1 mRNA were set up as 1 in control group.The relative expression levels of NADPH mRNA were(0.9271±0.0731)and(0.9423±0.1204)in 1-hour and 2-hour light exposure group,and it made no differences compared to control group with a P value>0.05.While the expression of NADPH mRNA were(1.8161±0.0331)、(1.7513±0.1364)、(2.5391±0.1847)、(2.9022±0.3920)and(5.7654±0.7031)in 3-hour,4-hour,5-hour,6-hour and 12-hour light exposure groups,and it made significant differnces compared to control group with a P value＜0.05.The relative expression levels of CO1 mRNA were(1.3217±0.1544)、(1.6626±0.0699).(1.5059±0.0344)and(1.3742±0.1011)in 2-hour,3-hour,4-hour and 5-hour light enxposure groups,and it made significant differnces compared to control group with a P value<0.05.And the relative expression levels of CO1 mRNA were(1.0217±0.0521)、(1.0679±0.0591)and(0.9578±0.0409)in 1-hour,6-hour,12-hour light exposure groups,and it made no differences compared to control group with a P value>0.05.3.The expressions of anti-apoptotic protein Bcl-2、apoptotic protein Bax and Caspase-3:the expression of Bcl-2 decreased obviously after more than 3h blue light irradiatioin;the expression of Bax increased slightly after 3h light irradiatioin,and the expression went up obviously after 5h light exposure,and the expression of Caspase-3 was similary with Bax.Those apoptotic protein by quantitative analysis method indicated:The ratios of Bcl-2 and β-actin were(1.320±0.149)、(1.331 ±0.093)、(0.979±0.155)、(0.743±0.087)、(0.431±0.064)、(0.423±0.044)、(0.263±0.071)in 1-hour,2-hour,3-hour,4-hour,5-hour,6-hour,12-hour light exposure groups,and the expression of Bcl-2 decreased obviously after more than 3h light illumination compared the ratio(1.771 + 0.149)in control group,and the comparisons made significant differences(at P<0.05)，While the ratios of Bax and β-actin were(0.011±0.001)、(0.008±0.001)、(0.069±0.005)、(0.115±0.015)、(0.170±0.024)、(0.245 ±0.032)、(0.484 ± 0.064)in each light exposure group,and the expression of Bax increased slightly after 3h light exposre,and became obvious after 5h light exposure,compared to the ratio(0.016±0.004)in control group,and it made significant differences(at P<0.05).Identically,the ratios of Caspase-3and β-actin were(0.065±0.011)、(0.256±0.053)、(0.310±0.045)、(0.665±0.088)、(0.689±0.096)、(0.943±0.054)、(1.167±0.131)in each light exposure group,and compared to the ratio(0.009±0.002)in control group.We found the similarity protein expression between Bax and Caspase-3,that is the expression of Caspase-3 increased slightly after 3h light iiradiation,and became obvious after 5h light irradiation,and the comparison made significant differences(at P<0.05).Conclusion1.After 1h blue light irradiation,the levels of ROS increased significantly,and the phenomenon caused oxidative stress and cells dysfunction in RPE cell;the epression of CO1 mRNA and NADPH mRNA increased after light irradiation for 2h and 3h,and it indicated the extremely active expression of different motichondrial genome,and caused mitochondria dysfunction after long time illumination.2.The expression of Bcl-2 decreased,accompanied with the expression of Bax and Caspase-3 increased after 3h light irradiation,which indicated the mitochondria mediated apopotosis system was activated after RPE cell encoutering oxidative stress,therefore it cuaesd RPE cell apoptosis.In conclusion,after blue light damage model was established successfully,and we found that it caused oxidative stress and RPE cell dysfunction after RPE cell were illuminated for 1h;then induced mitochondrial genome dysfunction when illumination time reached 2h to 3h;subsequently,the mitochondria mediated apopotosis system was activated,finally blue light induced dammage and changes in the morphological features and ultra-structurces in RPE cells,as well as cell apoptosis and cell necrosis.