Lutein-stevioside nanoparticle attenuates H_(2)O_(2)-induced oxidative damage in ARPE cells

In order to improve the bioavailability of lutein(LUT),a novel lutein-stevio side nanoparticle(LUT-STE)were prepared previously,but the information about LUT-STE on protecting of eye health was limited.This study investigated the effect of LUT-STE on antioxidant activity of H_(2)O_(2)-induced human retinal pigment epithelial(ARPE)cells.LUT and LUT-STE(final concentration of 5μg/mL)significantly enhanced cell viability from(74.84±5.10)%to(81.92±10.01)%(LUT)and(89.33±4.34)%(LUT-STE),and inhibited the cell apoptosis(P<0.05).After pretreatment with LUT-STE in ARPE cells,the levels of superoxide dismutase(SOD),catalase(CAT)and glutathion peroxidase(GSH-Px)in ARPE cells were significantly increased(P<0.05),the contents of reactive oxygen species(ROS)and malondialdehyde(MDA)were decreased.In addition,the vascular endothelial growth factor(VEGF)levels were inhibited by 13.61%and 17.39%,respectively,pretreatment with LUT and LUT-STE.Western blotting results showed that the pretreatment with LUT-STE inhibited the expression of caspase-9 and caspase-3 and up-regulated Bcl-2/Bax pathway to inhibit H_(2)O_(2)-induced apoptosis.In summary,the novel delivery LUT-STE had more pronounced inhibitory effect on H_(2)O_(2)-induced damage in human ARPE cells.

LIN28A attenuates high glucose-induced retinal pigmented epithelium injury through activating SIRT1-dependent autophagy

AIM:To evaluate the effects of LIN28A(human)on high glucose-induced retinal pigmented epithelium(RPE)cell injury and its possible mechanism.METHODS:Diabetic retinopathy model was generated following 48h of exposure to 30 mmol/L high glucose(HG)in ARPE-19 cells.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot tested the expression of the corresponding genes and proteins.Cell viability as well as apoptosis was determined through cell counting kit-8(CCK-8)and flow cytometry assays.Immunofluorescence assay was adopted to evaluate autophagy activity.Caspase 3 activity,oxidative stress markers,and cytokines were appraised adopting their commercial kits,respectively.Finally,ARPE-19 cells were preincubated with EX527,a Sirtuin 1(SIRT1)inhibitor,prior to HG stimulation to validate the regulatory mechanism.RESULTS:LIN28A was downregulated in HG-challenged ARPE-19 cells.LIN28A overexpression greatly inhibited HGinduced ARPE-19 cell viability loss,apoptosis,oxidative damage as well as inflammatory response.Meanwhile,the repressed autophagy and SIRT1 in ARPE-19 cells challenged with HG were elevated after LIN28A overexpression.In addition,treatment of EX527 greatly inhibited the activated autophagy following LIN28A overexpression and partly abolished the protective role of LIN28A against HG-elicited apoptosis,oxidative damage as well as inflammation in ARPE-19 cells.CONCLUSION:LIN28A exerts a protective role against HG-elicited RPE oxidative damage,inflammation,as well as apoptosis via regulating SIRT1/autophagy.

Comparison of vegetable oils on the uptake of lutein and zeaxanthin by ARPE-19 cells

AIM:To compare the effect of vegetable oils on the uptake of lutein and zeaxanthin by adult retinal pigment epithelial(ARPE)-19 cells in vitro.METHODS:ARPE-19 cells were cultured in Dulbecco’s Modified Eagle Medium-F-12 supplemented with 10%foetal bovine serum and 1%penicillin–streptomycin in a humidified 5%CO_(2) incubator maintained at 37℃.Cells were treated with 247μmol/L lutein,49μmol/L zeaxanthin and 1%(v/v)of either coconut oil,corn oil,peanut oil,olive oil,sunflower oil,soybean oil,castor oil,or linseed oil for 48h.Lutein and zeaxanthin concentration in the cells were quantified by high performance liquid chromatography.RESULTS:Among the oils tested,the highest lutein and zeaxanthin uptake was observed with coconut oil while the lowest was observed with linseed oil.CONCLUSION:ARPE-19 uptake of lutein and zeaxanthin are found to be dependent on the type of oils.

Morroniside ameliorates lipopolysaccharide-induced inflammatory damage in iris pigment epithelial cells through inhibition of TLR4/JAK2/STAT3 pathway

AIM:To investigate the effect of morroniside(Mor)on lipopolysaccharide(LPS)-treated iris pigment epithelial cells(IPE).METHODS:IPE cells were induced by LPS and treated with Mor.Cell proliferation was detected by cell counting kit(CCK)-8,apoptosis was detected by flow cytometry,the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,and IL-8 were measured by enzyme-linked immunosorbent assay(ELISA)kits,and the protein expression of TLR4,JAK2,p-JAK2,STAT3,and p-STAT3 was analyzed by Western blotting.In addition,overexpression of TLR4 and Mor treatment of LPS-stimulated IPE cells were also tested for the above indices.RESULTS:Mor effectively promoted the proliferation and inhibited the apoptosis of LPS-treated IPE cells.In addition,Mor significantly reduced the levels of TNF-α,IL-6,and IL-8 and significantly inhibited the expression of TLR4,p-JAK2,and p-STAT3 in LPS-treated IPE cells.The effect of Mor on LPS-treated IPE cells was markedly attenuated after overexpression of TLR4.CONCLUSION:These findings suggest that Mor may ameliorate LPS-induced inflammatory damage and apoptosis in IPE through inhibition of TLR4/JAK2/STAT3 pathway.

Protective Effect and Autophagy Mechanism of Lycium barbarum Polysaccharides on Retinal Pigment Epithelial Cells Under High-Glucose Conditions

Objective:To study the effects of Lycium barbarum polysaccharide(LBP)on the proliferation,apoptosis,and autophagy of retinal pigment epithelial(RPE)cells cultured under high-glucose conditions.Methods:The ARPE-19 cell line was randomly divided into a control group(normally cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12[DMEM/F-12]medium),a high-glucose group(HG;50 mmol/L glucose added to DMEM/F-12 medium),and a HG+LBP group(incubated in DMEM/F-12 medium containing 1 mg/mL LBP for 24 h,and then treated with 50 mmol/L glucose for 24 h).Following Ad-mCherry-GFP-LC3B infection,cell proliferation,apoptosis,mammalian target of rapamy-cin(mTOR)expression,and autophagic flux were determined by Cell Counting Kit-8(CCK-8),AnnexinV-APC/7-AAD Apoptosis Detection Kit,Western blot,and laser confocal microscopy,respectively.Results:The proliferation rate of ARPE-19 cells in the HG group was significantly lower than that in the control group(P<0.05),while the proliferation rate of ARPE-19 cells in the HG+LBP group was significantly higher than that in the HG group(P<0.05).The apoptosis rate of ARPE-19 cells in the HG group was significantly higher than that in the control group(P<0.05),while the apoptosis rate of ARPE-19 cells in the HG+LBP group was significantly lower than that in the HG group(P<0.05).The relative expression of phosphorylated mTOR(p-mTOR)of ARPE-19 cells in the HG group was significantly lower than that in the control group(P<0.05),with enhanced autophagic flux;when compared with the HG group,the HG+LBP group had significantly higher expression of p-mTOR(P<0.05),with diminished autophagic flux.Conclusion:LBP has a protective effect on RPE cells with high glucose-induced injury,and its mechanism may be related to LBP inhibition of high glucose-induced abnormal autophagy.

Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

AIM:To explore the effect of epidermal growth factor receptor(EGFR)inhibition by erlotinib and EGFR siRNA on epidermal growth factor(EGF)-induced activation of retinal pigment epithelium(RPE)cells.METHODS:Human RPE cell line(ARPE-19 cells)was activated by 100 ng/mL EGF.Erlotinib and EGFR siRNA were used to intervene EGF treatment.Cellular viability,proliferation,and migration were detected by methyl thiazolyl tetrazolium(MTT)assay,bromodeoxyuridine(BrdU)staining assay and wound healing assay,respectively.EGFR/protein kinase B(AKT)pathway proteins and N-cadherin,α-smooth muscle actin(α-SMA),and vimentin were tested by Western blot assay.EGFR was also determined by immunofluorescence staining.RESULTS:EGF treatment for 24h induced a significant increase of ARPE-19 cells’viability,proliferation and migration,phosphorylation of EGFR/AKT proteins,and decreased total EGFR expression.Erlotinib suppressed ARPE-19 cells’viability,proliferation and migration through down regulating total EGFR and AKT protein expressions.Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin,α-SMA,and vimentin proteins.Similarly,EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation,viability,and migration,phosphorylation of EGFR/AKT proteins,and up-regulation of N-cadherin,α-SMA,and vimentin proteins.CONCLUSION:Erlotinib and EGFR-knockdown suppress EGF-induced cell viability,proliferation,and migration via EGFR/AKT pathway in RPE cells.EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy(PVR).

Hepatocyte growth factor promotes retinal pigment epithelium cell activity through MET/AKT signaling pathway

AIM:To explore the effects of hepatocyte growth factor(HGF)on retinal pigment epithelium(RPE)cell behaviors.METHODS:The human adult retinal pigment epithelial cell line-19(ARPE-19)were treated by HGF or mesenchymalepithelial transition factor(MET)inhibitor SU11274 in vitro.Cell viability was detected by a Cell Counting Kit-8 assay.Cell proliferation and motility was detected by a bromodeoxyuridine incorporation assay and a wound healing assay,respectively.The expression levels of MET,phosphorylated MET,protein kinase B(AKT),and phosphorylated AKT proteins were determined by Western blot assay.The MET and phosphorylated MET proteins were also determined by immunofluorescence assay.RESULTS:HGF increased ARPE-19 cells’viability,proliferation and migration,and induced an increase of phosphorylated MET and phosphorylated AKT proteins.SU11274 significantly reduced cell viability,proliferation,and migration and decreased the expression of MET and AKT proteins.SU11274 suppressed HGF-induced increase of viability,proliferation,and migration in ARPE-19 cells.Additionally,SU11274 also blocked HGF-induced phosphorylation of MET and AKT proteins.CONCLUSION:HGF enhances cellular viability,proliferation,and migration in RPE cells through the MET/AKT signaling pathway,whereas this enhancement is suppressed by the MET inhibitor SU11274.HGF-induced MET/AKT signaling might be a vital contributor of RPE cells survival.

Bone morphogenetic protein-6 suppresses TGF-β_(2)-induced epithelial-mesenchymal transition in retinal pigment epithelium

AIM:To evaluate the effect of bone morphogenetic protein-6(BMP-6)on transforming growth factor(TGF)-β_(2)-induced epithelial-mesenchymal transition(EMT)in retinal pigment epithelium(RPE).METHODS:Adult retinal pigment epithelial cell line(ARPE-19)were randomly divided into control,TGF-β_(2)(5μg/L),and BMP-6 small interfering RNA(siRNA)group.The cell morphology was observed by microscopy,and the cell migration ability were detected by Transwell chamber.The EMT-related indexes and BMP-6 protein levels were detected by Western blotting.Furthermore,a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group,TGF-β_(2)+empty plasmid group,BMP-6 overexpression group,and TGF-β_(2)+BMP-6 overexpression group.The EMT-related indexes and extracellular regulated protein kinases(ERK)protein levels were detected.RESULTS:Compared with the control group,the migration of RPE cells in the TGF-β_(2) group was significantly enhanced.TGF-β_(2) increased the protein expression levels ofα-smooth muscle actin(α-SMA),fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6(P<0.05)in RPE.Similarly,the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced.BMP-6 siRNA increased the protein expression levels ofα-SMA,fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin(P<0.05).Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-β_(2) and prevented TGF-β_(2) from affecting EMT-related biomarkers(P<0.05).CONCLUSION:BMP-6 prevents the EMT in RPE cells induced by TGF-β_(2),which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

Diabetes and high-glucose could upregulate the expression of receptor for activated C kinase 1 in retina

BACKGROUND Diabetic retinopathy(DR)is a major ocular complication of diabetes mellitus,leading to visual impairment.Retinal pigment epithelium(RPE)injury is a key component of the outer blood retinal barrier,and its damage is an important indicator of DR.Receptor for activated C kinase 1(RACK1)activates protein kinase C-ε(PKC-ε)to promote the generation of reactive oxygen species(ROS)in RPE cells,leading to apoptosis.Therefore,we hypothesize that the activation of RACK1 under hypoxic/high-glucose conditions may promote RPE cell apoptosis by modulating PKC-ε/ROS,thereby disrupting the barrier effect of the outer blood retinal barrier and contributing to the progression of DR.AIM To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR.METHODS In this study,Sprague-Dawley rats and adult RPE cell line-19(ARPE-19)cells were used as in vivo and in vitro models,respectively,to explore the role of RACK1 in mediating PKC-εin early DR.Furthermore,the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model.RESULTS Streptozotocin-induced diabetic rats showed increased apoptosis and upregulated expression of RACK1 and PKC-εproteins in RPE cells following a prolonged modeling.Similarly,ARPE-19 cells exposed to high glucose and hypoxia displayed elevated mRNA and protein levels of RACK1 and PKC-ε,accompanied by an increases in ROS production,apoptosis rate,and monolayer permeability.However,silencing RACK1 significantly downregulated the expression of PKC-εand ROS,reduced cell apoptosis and permeability,and protected barrier function.CONCLUSION RACK1 plays a significant role in the development of early DR and might serve as a potential therapeutic target for DR by regulating RPE apoptosis and barrier function.

PI3K/AKT/mTOR signaling pathway inhibitors in proliferation of retinal pigment epithelial cells

AIM: To determine whether the PI3K/AKT/mTOR pathway is activated in proliferative vitreoretinopathy (PVR) in homo-sapiens. METHODS: The retina of controls and patients with PVR were collected and their levels of PI3K, phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP-1 were determined by Western blot. The cultured human retinal pigment epithelial cell line D407 was treated with a specific mTOR inhibitor, rapamycin (RAPA) or a PI3K inhibitor, LY294002, of various concentrations and durations. Cell morphology was observed by phase contrast microscopy and the proliferation and apoptosis of treated cells were determined by MTT assay and flow cytometry. RESULTS: Levels of PI3K, phospho-AKT, phospho-mTOR, phospho-P70S6K and phospho-4EBP1 was increased in the retina in PVR (P <0.05). In D407 cells, both RAPA and LY294002 significantly inhibited cell proliferation and cell cycle progression, and promoted apoptosis (P <0.05); morphologically, the cells became smaller. Both RAPA and LY294002 reduced levels of phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP1 expression (P <0.05). RAPA, but not LY294002, had no significant effect on PI3K expression. CONCLUSION: PI3K/AKT/mTOR signaling pathway is highly activated in the retinal pigment epithelial cells of PVR. The inhibitors of PI3K/AKT/mTOR signaling pathway, RAPA and LY294002, could inhibited the PI3K/AKT/mTOR signaling pathway by reducing the levels of phosphorylation of mTOR pathway components.

Up-regulation of HIF-1α and VEGF Expression by Elevated Glucose Concentration and Hypoxia in Cultured Human Retinal Pigment Epithelial Cells

Summary： In order to explore the effect of high glucose concentration and high glucose concentration with hypoxia on the production of hypoxia-inducible factor-1α （HIF-1α） and vascular endothelial growth factor （VEGF）, human RPE cells were cultured in 5,56 mmol/L glucose （control group）, 5.56 mmol/L glucose with 150 !a mol/L COCl2 （hypoxic group）, 25 mmol/L glucose （high glucose group） and 25 mmol/L glucose with 150 μmol/L COCl2 （combination group）. RT-PCR was used to detect the expression of HIF-1α and VEGF mRNAs. Western blot analysis was used to measure the levels of HIF-1α and VEGF proteins. Although the small amount of HIF-1α protein was able to be detected in high glucose group but not in control group, there was no significant difference between the expression of HIF-1α mRNA of RPE cells in high glucose group and that of RPE cells in control group. As compared with RPE cells in control group, the mRNA expression and the protein synthesis of VEGF in high glucose group were up-regulated. As compared with RPE cells in hypoxic group, the expression of HIF-1α mRNA of RPE cells in combination group was not different, but the protein synthesis of HIF-1α, the mRNA expression and the protein synthesis of VEGF were more obviously up-regulated. In conclusion, high concentration glucose mainly influence the protein synthesis of HIF-1α of RPE cell, and HIF-1α protein is able to be accumulated in high concentration glucose. Under hypoxia, the HIF-1α protein induced by high concentration glucose is more stable, and the expression of VEGF is obviously increased. It is suggested that high concentration glucose may play a role in retinal neovascularization, especially at ischemia stage of diabetic retinopathy.

Effects of Lycium barbarum polysaccharide on the photoinduced autophagy of retinal pigment epithelium cells

AIM:To investigate the relationship between autophagy and apoptosis in photoinduced injuries in retinal pigment epithelium(RPE)cells and how Lycium barbarum polysaccharide(LBP)contributes to the increased of RPE cells to photoinduced autophagy.METHODS:In vitro cultures of human RPE strains(ARPE-19)were prepared and randomly divided into the blank control,model,low-dose LBP,middle-dose LBP,high-dose LBP,and 3-methyladenine(3MA)groups.The viability of the RPE cells and apoptosis levels in each group were tested through cell counting kit-8(CCK8)method with a flow cytometer(Annexin V/PI double staining technique).The expression levels of LC3II,LC3I,and P62 proteins were detected with the immunofluorescence method.The expression levels of beclin1,LC3,P62,PI3K,P-mTOR,mTOR,P-Akt,and Akt proteins were tested through Western blot.RESULTS:LBP considerably strengthens cell viability and inhibits the apoptosis of RPE cells after photoinduction.The PI3K/Akt/mTOR signal pathway is activated because of the upregulation of the phosphorylation levels of Akt and mTOR proteins,and thus autophagy is inhibited.CONCLUSION:LBP can inhibit the excessive autophagy in RPE cells by activating the PI3K/Akt/mTOR signaling pathways and thereby protect RPE cells from photoinduced injuries.

Distribution and ultrastructure of pigment cells in the skins of normal and albino adult turbot, Scophthalmus Maximus

The distribution and ultrastructure of pigment cells in skins of normal and albino adult turbots were examined with transmission electron microscopy （TEM）. Three types of pigment cells of melanophore, iridophore and xanthophore have been recognized in adult turbot skins. The skin color depends mainly on the amount and distribution of melanophore and iridophore, as xanthophore is quite rare. No pigment cells can be found in the epidermis of the skins. In the pigmented ocular skin of the turbot, melanophore and iridophore are usually co-localized in the dermis. This is quite different from the distribution in larvae skin. In albino and white blind skins of adult turbots, however, only iridophore monolayer still exists, while the melanophore monolayer disappears. This cytological evidence explains why the albino adult turbot, unlike its larvae, could never resume its body color no matter what enviroumental and nutritional conditions were provided. Endocytosis is quite active in the cellular membrane of the iridophore. This might be related to the formation of reflective platelet and stability of the iridophore.

Inhibition of PCNA Antisense Oligonucleotides Mediated by Liposome on mRNA Expression and Proliferation of h-RPE Cells

Summary： The proliferating cell nuclear antigen （PCNA） gene expression was blocked and retinal pigment epithelium （RPE） proliferation was inhibited by using antisense oligonucleotides （AS-ODN） mediated by liposome, to find a new genetic therapy of proliferative vitreoretinopathy （PVR）. RPE cells cultured in vitro were transfected with synthetic fluorescence labled AS-ODN mediated by liposome-Lipofectamine, and the intracellular distribution and persistence time of AS-ODN were dynamically observed. AS-ODN （0.07, 0.28 and 1.12 μ mol/L and sense oligonucleotides （S-ODN with the same concentrations as AS-ODN） mediated by liposome were delivered to the RPE cells cultured in vitro, and CPM values were measured by ^3H-TdR incorporation assay and analyzed statistically by variance by comparison with blank control group. Expression ofPCNA mRNA in RPE cells was detected by in situ hybridization after the treatment of different concentrations of PCNA AS-ODN and S-ODN, and the average optic density （AOD） was measured by image analysis system and was subjected to q-test and correlation analysis with CPM. Our results showed that AS-ODN mediated by liposome could quickly aggregate in cellular plasma and nuclei in 30 min and 6 h, and stayed for as long as 6 days. AS-ODN （0.28 and 1.12 μmol/L） markedly suppressed proliferation of RPE cells in a dose-dependent manner with the difference being statistically significant （P〈0.05 and P〈0.01, repectively） as compared with blank control group. AOD was well correlated with CPM （r=0.975）. It is concluded that liposome could increase transfection efficiency of AS-ODN in RPE cells, and AS-ODN could sequence-specifically suppress PCNA mRNA expression and proliferation of human RPE cells.

Peroxynitrite-induced expression of inducible nitric oxide synthase and activated apoptosis via nuclear factor-kappa B pathway in retinal pigment epithelial cells and antagonism of cholecystokinin octapeptide-8 in vitro

AIM: To explore that if peroxynitrite induced the expression of inducible nitric oxide synthase (iNOS)via nuclear factor-kappa B (NF-kappa B)pathway in retinal pigment epithelial (RPE) cells and the antagonism of cholecystokinin octapeptide-8 (Melatonin, CCK-8) in vitro. METHODS: RPE cells were obtained from eyes of C57BL/6 mouse and divided into control, peroxynitrite and CCK-8 groups. Control group was treated with saline, peroxynitrite group was treated with peroxynitrite, and CCK-8 group was treated with CCK-8 after added with peroxynitrite. All changes were observered at 6, 12 and 24 hours after treatment. Gene array analysis, Reverse Transcription Polymerase Chain Reaction (RT-PCR) were used to determine the expression of inducible nitric oxide synthase ( iNOS)mRNA in RPE cells. Western blotting was used to test the apoptosis of RPE cells. Immunofluorescent staining was used to determine the NF-kappa B pathway signal transduction. RESULTS: Compared to the control group, the expression of iNOS mRNA was up-regulated in peroxynitrite group and down-regulated in CCK-8 group with gene array analysis. Apoptosis was increased in peroxynitrite group and decreased in CCK-8 group with western blotting. The NF-kappa B pathway signal transduction was more and more stronger in the peroxynitrite group. But in CCK-8 group, little stronger could be observed at 12 hours, then weak at 24 hours with immunofluorescent staining (P<0.001). CONCLUSION: This study suggested that apoptosis of RPE cells was partly induced by peroxynitrite, which may be the new way of oxidative damage to the RPE cells. The NF-kappa B signal transduction may affect and reinforce apoptosis mediated by peroxynitrite. CCK-8 decreased apoptosis of RPE cells induced by peroxynitrite and is a potential agent for therapy of retinopathy. The mechanism of CCK-8 dealing with RPE cells may be related to its direct inhibition of the formation of iNOS to produce peroxynitrite and antagnism of damage of peroxynitrite to the RPE cells.

Acetylcholinesterase function in apoptotic retina pigment epithelial cells induced by H_2O_2

AIM:To investigate the acetylcholinesterase（AChE）expression involved in retina pigment epithelial（RPE）apoptosis induced by higher concentrations H2O2.METHODS:The human retinal pigment epithelium cell line ARPE-19 was from ATCC（Rockville,MD）.Cultured ARPE-19 cells were treated with H2O2 at 0,250,500,1000,2 000μmol/L and cell viability was measured with MTT assay.AChE expression and DNA fragments were analyzed by immunocytochemistry,TUNEL and PARP-1Western blotting.RESULTS:Immunofluorescence detected AChE exist in the normal human retinal tissue.When H2O2】500μmol/L,AChE expression showed an increase after 2h,and this concentration was selected for the present study.RPE cell was induced with 1 000μmoI/L H2O2 for 2h,compared to the control group,cell activity decline detected by MTT,AChE and PARP-1 protein expression was significantly increased detected by Western blotting.AChE immunofluorescence staining was positive in RPE cell after HO2 incubate 2h.In addition,pretreatment with100|jmol/L epigallocatechin gallate（EGCG）,cell viability increased from 31.20%±3.90%to 70.23%±12.96%.CONCLUSION:AChE is weakly expressed in normal human RPE cells.Stimulation with H2O2 caused the stable increase of AChE expression in RPE cells,which may indicate that AChE may be an important role in AMD.

Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells

Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspase-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite.

Mesenchymal stem cells-derived exosomes ameliorate blue light stimulation in retinal pigment epithelium cells and retinal laser injury by VEGF-dependent mechanism

AIM： To observe the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells（h UCMSCs） on the expression of vascular endothelial growth factor-A（VEGF-A） in blue light injured human retinal pigment epithelial（RPE） cells and laser-induced choroidal neovascularization（CNV） in rats.METHODS： Exosomes were isolated from h UCMSCs and characterized by transmission electron microscope and Western blot. MSCs-derived exosomes were cultured with RPE cells exposed to blue light. The m RNA and protein expression of VEGF-A were determined by real time-polymerase chain reaction（PCR） and Western blot, respectively. Immunofluorescence assay was used for the detection of the expression level of VEGF-A. We injected different doses of MSCs-derived exosomes intravitreally to observe and compare their effects in a mouse model of laserinduced retinal injury. The histological structure of CNV in rats was inspected by hematoxylin-eosin（HE） staining and fundus fluorescein angiography. The expression of VEGF-A was detected by immunohistochemistry.RESULTS： Exosomes exhibited the typical characteristic morphology（cup-shaped） and size（diameter between 50 and 150 nm）. The exosomes marker, CD63, and h UCMSCs marker, CD90, showed a robust presence. In vitro, MSCsderived exosomes downregulated the m RNA（Exo-L： t=6.485, 7.959, 9.286; Exo-M： t=7.517, 10.170, 13.413; Exo-H： t=10.317, 12.234, 14.592, P〈0.05） and protein（Exo-L： t=2.945, 4.477, 6.657; Exo-M： t=4.713, 6.421, 8.836; Exo-H：t=6.539, 12.194, 12.783; P〈0.05） expression of VEGF-A in RPE cells after blue light stimulation. In vivo, we found that the MSCs-derived exosomes reduced damage, distinctly downregulated VEGF-A（Exo-H： t=0.957, 1.382; P〈0.05）, and gradually improved the histological structures of CNV for a better visual function（Exo-L： 0.346, Exo-M： 3.382, Exo-H： 8.571; P〈0.05）. CONCLUSION： MSCs-derived exosomes ameliorate blue light stimulation in RPE cells and laser-induced retinal injury via downregulation of VEGF-A.

Cross-talk between microRNA-let7c and transforming growth factor-β2 during epithelial-to-mesenchymal transition of retinal pigment epithelial cells

AIM: To explore the roles of microRNA-let7 c(miR-let7 c) and transforming growth factor-β2(TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition(EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial(ARPE-19) cells were cultured with no serum for 12 h, and then with recombinant human TGF-β2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7 c mimcs(miR-let7 cM), miR-let7 c mimcs negative control(miR-let7cMNC) and miR-let7 c inhibitor(miR-let7 cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B(NF-κB) signaling pathway was activated after induction by TGF-β2(P<0.05). In turn, overexpressed miR-let7 c significantly inhibited TGF-β2-induced EMT(P<0.05). However, miR-let7 c was unable to inhibit TGF-β2-induced EMT when the NF-κB signaling pathway was inhibited by BAY11-7082(P<0.01). CONCLUSION: The miR-let7 c regulates TGF-β2-induced EMT through the NF-κB signaling pathway in ARPE-19 cells.

Gac fruit extracts ameliorate proliferation and modulate angiogenic markers of human retinal pigment epithelial cells under high glucose conditions

Objective: To investigate the impact of the extracts of Gac fruit parts(peel, pulp, seed, and aril) on the cell viability and angiogenesis markers of human retinal pigment epithelial(ARPE-19) cells under high glucose conditions. Methods: The effect of the extracts of Gac fruit peel, pulp, seed and aril on the ARPE-19 cells was determined using MTT viability assay, Trypan blue dye and morphological changes were observed using light microscopy. Enzyme-linked immunosorbent-based assay was performed to evaluate the effect of Gac fruit parts on the reactive oxygen species(ROS), vascular endothelial growth factor(VEGF) and pigmented epithelium-derived factor(PEDF) secretions. Results: High glucose(HG) at 30 mmol/L increased ARPE-19 cell viability and ROS and VEGF secretions. While, the exposure of ARPE-19 cells in high glucose condition to Gac fruit extracts led to inhibition of cell viability, induced morphological changes, decreased ROS and VEGF secretions, and increased PEDF level. Gac pulp, seed, and aril at 1 000 μg/mL showed significant inhibition activities [(7.5 ± 5.1)%,(2.7 ± 0.5)%,(3.2 ± 1.1)%, respectively] against HG-induced ARPE-19 cell viability. The findings also demonstrated that Gac aril at 250 μg/mL significantly decreased ROS and VEGF levels [(40.6 ± 3.3) pg/mL,(107.4 ± 48.3) pg/mL, respectively] compared to ROS [(71.7 ± 2.9) pg/mL ] and VEGF [(606.9 ± 81.1) pg/mL] in HG untreated cells. Moreover, 250 μg/mL of Gac peel dramatically increased PEDF level [(18.2 ± 0.3) ng/mL] compared to that in HG untreated cells [(0.48 ± 0.39) ng/mL]. Conclusions: This study indicates that the extracts of Gac peel, pulp, seed and aril reduced cell viability, minimized ROS generations and showed angiogenic activities. Therefore, our findings open new insights into the potentiality of Gac fruit against HG-related diabetic retinopathy disease.