| The retinal pigment epithelium (RPE), a monolayer of cells which differentiatedfrom neuroderm, is located between neural retina and choriocapillaris. The normalRPE layer keeps the visual cycle going on wheels, and also plays a crucial role inmaintenance of photoreceptor cells structure and the biological function. The RPEdegeneration could induce a series of retinal diseases. The mechanism of RPEdegeneration isn’t elucidated well so far, nevertheless, these retinal diseases occurwith RPE oxidative stress. So that could be the point of penetration for themechanism research of retinal degenerative-related diseases. The known factors thatcould evoke RPE oxidative stress response are superoxide anion free radicals, bluelight exposure, toxicants exposure, smoking, obesity, etc. Studies have found that theheavy metal lead could induce some somatic cells or tissues to produce free radicalswhich result in cell oxidative stress response and break the redox homeostasis ofcellular milieu interieur seriously. However, the previous reports indicate that leadand cadmium deposition occurred in eyeground, especially in RPE layer. Accordingly,we speculated whether chronic lead exposure should be a potential pathogeneticfactor that induce RPE oxidative stress and cause its degeneration afterwards.Concerning our research, two RPE cell lines that are fetal RPE-derived fRPE-13established in our lab and the prevalent one adult RPE-derived ARPE-19wereinvestigated. To confirm whether heavy metals lead-induced oxidative damage ofretinal pigment epithelium could be a latent pathologic factor, RPE cells wereexposured to various lead doses. After that, we checked the changes of cellmorphology and the expression variation of moleculars related to oxidative stresspathways in RPE cells. In addition, the lead-induced impact on RPE physiologicalfunctions also been determinated. The results presented as follows:1. The RPE cell viability was measured by CCK-8assay after acute lead-treatedculturing. The results indicate that both fetal-derived fRPE-13and adult-derivedARPE-19presented excitatory effect of low dose (≤20μM) lead, while RPE cell viability decreased significantly when doses above50μM; what’smore, RPE hashigher tolerance level against to lead toxicity compared with other cell types studiedpreviously. Hence, we determined the investigated lead doses of follow-upexperiments:0μM,10μM,50μM,250μM.2. The cell morphology changed significantly after RPE treated with chroniclead-exposure at intervals of12hours for3days,5days and8days respectively. Withtreatment for3days,0μM,10μM group cells were eugenic and plump withisland-like, whereas50μM and250μM dose group showed slow-growing and fewcell islands relatively. After5days, the dish peripheral cells of fRPE-1310μM groupstarted shrinked, and50μM group of ARPE-19as narrow hook-shaped, while250μM group has been completely collapsed. Cell shrinkage came out largely in10μMgroup of fRPE-13at day8. And simultaneously, the cell morphology was irregular in10μM group of ARPE-19, even cobblestone-like epithelial appeared partially. Thecells were collapsed completely by higher dose-treatment in both cell lines.3. The epithelial was carried out scratch assay when cells reached100%confluence and subsequently lead-exposured for3days. The migration extent wasrecorded by light microscope at a certain time interval. The cell-free area pixels werecalculated for statistical analysis. The migration capacity of both lines seems to beinhibited almost when lead concentration up to50μM, even low concentrations oflead (10μM) also reduced the ability of healing. Our results showed that thescratch-migration inhibiton of lead submited dose-dependant effect.4. Cells were collected after culturing by lead chronic-exposure for5days andreseeded on96-well plate. The cell viability was measured by CCK-8assay atintervals of24hours, and then the growth curve was made. The rightward shift ofgrowth curve indicated that lead reduced the proliferation activity of RPE.5. Cells were fixed and stained when cells achieved logarithmic growth phaseafter culturing for3.5days, according to the features of RPE cell growth curve. Theresult of RPE cell cycle was analysed by flow cytometric system. The cells in phaseof S or G2M were increased markedly in low dose-treated group, which comparedwith lead-free cultured group. That result confirmed the excitatory effect of low doselead exposure further. However, the cells that turned into mitotic phase began decreased when lead dose level above to50μM.6. Total RNA was extracted from RPE cells which have been exposureed for5days, and subsequently reverse transcribed into cDNA, then qPCR was performed.The members of RPE oxidative stress response that are antioxidant enzyme or proteinand molecular chaperones (such as prohibitin, HSP70) were upregulated distinctly inboth RPE cell lines, which treated with chronic lead-exposure. In addition, the keytranscription factor of oxidative stress response, Nrf2, also had a significantupregulation.7. After lead-exposured culturing, the adherent or non-adherent cells werecollected for apoptotic analysis. The result indicated that the apoptosis rate presenteddose-dependant effect.In conclusion, the results from research of RPE cell morphology, physiologicalfunctions or transcriptional level indicated that chronic lead-exposure induced RPEoxidative stress, further resulted in cell damage and triggered cell apoptosis ornecrosis ultimately. Accordingly, we could draw a conclusion that heavy metal leadprobably a important pathogenetic factor that induce RPE oxidative stress and causethe oxidative injury, which provides more references, evidences and supports for theetiological mechanism research of retinopathy-related diseases. |
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