Effect of miR-27b-3p and Nrf2 in human retinal pigment epithelial cell induced by high-glucose

AIM:To determine whether the microRNA-27b-3p(miR-27b-3p)/NF-E2-related factor 2(Nrf2)pathway plays a role in human retinal pigment epithelial(hRPE)cell response to high glucose,how miR-27b-3p and Nrf2 expression are regulated,and whether this pathway could be specifically targeted.METHODS:hRPE cells were cultured in normal glucose or high glucose for 1,3,or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species(ROS)levels using a dihydroethidium kit.miR-27b-3p,Nrf2,NAD(P)H quinone oxidoreductase 1(NQO1)and heme oxygenase-1(HO-1)mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction(RT-qPCR)and immunocytofluorescence(ICF),respectively.Western blot analyses were performed to determine nuclear and total Nrf2 protein levels.Nrf2,NQO1,and HO-1 expression levels by RT-qPCR,ICF,or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection.Finally,the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine.RESULTS:Persistent exposure to high glucose gradually suppressed hRPE Nrf2,NQO1,and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels.High glucose also promoted ROS release and inhibited cellular proliferation.Nrf2,NQO1,and HO-1 mRNA levels decreased after miR-27b-3p overexpression and,conversely,both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor.After treating hRPE cells exposed to high glucose with pyridoxamine,ROS levels tended to decreased,proliferation rate increased,Nrf2,NQO1,and HO-1 mRNA and protein levels were upregulated,and miR-27b-3p mRNA levels were suppressed.CONCLUSION:Nrf2 is a downstream target of miR-27b-3p.Furthermore,the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.

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.

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.

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.

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.

Modulation of Matrix Metalloproteinase and TIMP-1 Expression by TGF-β_1 in Cultured Human RPE Cells

In order to investigate the effects of TGF-β1 on the expression of MMP-2, -9 and TIMP- 1 in human retinal pigment epithelial （RPE） cells, the third-sixth passage cultured RPE cells were treated with TGF-β1 at different concentrations （0.01, 0. 1, 1.0, 10 ng/mL）, the expression of MMP-2, -9 and TIMP-1 mRNA was detected by semi-qudntitative RT-PCR assays. MMP-2, -9 and TIMP-1 mRNA were expressed in the cultured RPE cells. The values of MMP-2/β-actin in the cells treated with 0.1, 1.0, 10 ng/mL TGF-β1 were 1.04±0.04, 1.07±0.02 and 1.11±0.03, respectively, significantly higher than in the control group （0.96±0.03, P〈0. 05-0.01）. The expression of MMP-2 mRNA could be up-regulated by TGF-β, , in a dose-dependent manner. The expression of MMP-9 mRNA in the cultured RPE cells was slightly up-regulated by various TGF-β1 concentrations treatment. The values of TIMP-1/β-actin in the cells treated with 0.01 and 0.1 ng/ mL TGF-β1 were 0.85 ±0.01 and 0.97 ± 0.02 respectively, significantly lower than in the control group （1.07±0.04, P〈0.01）, indicating that the expression of TIMP-1 mRNA was down-regulated by TGF-β1 at low concentrations. But along with the increase of TGF-β1 concentrations （1.0 and 10 ng/mL）, the expression of TIMP-1 mRNA was slightly up-regulated, not significantly different from that in the control group （P〉0.05）. It was concluded that TGF-β1 might play an important role in the up-regulation of the expression of MMP-2 in RPE cells and result in a directional shift in the balance between MMP and TIMP. This may be facilitated for RPE cells to migrate in the pathogenesis of vitreoretinopathy.

Effect of bevacizumab on the expression of fibrosis-related inflammatory mediators in ARPE-19 cells

AIM：To investigate the effect of anti-vascular epithelial growth factor（VEGF）agents on the expression of fibrosisrelated inflammatory mediators under normoxic and hypoxic conditions,and to further clarify the mechanism underlying fibrosis after anti-VEGF therapy. METHODS：Human retinal pigment epithelial（RPE）cells were incubated under normoxic and hypoxic conditions.For hypoxia treatment,CoCl_2 at 200μmol/L was added to the media. ARPE-19 cells were treated as following：1）control group：no treatment; 2）bevacizumab group：bevacizumab at 0.25 mg/mL was added to the media; 3）hypoxia group：CoCl_2 at 200 μmol/L was added to the media; 4）hypoxia＋bevacizumab group：CoCl_2 at 200 μmol/L and bevacizumab at 0.25 mg/mL were added to the media.The expression of interleukin（IL）-1β,IL-6,IL-8 and tumor necrosis factor（TNF）-α were evaluated using real-time polymerase chain reaction（RT-PCR）and enzyme-linked immunosorbent assay（ELISA）at 6,12,24 and 48 h. RESULTS：Both m RNA and protein levels of IL-1β,IL-6 and IL-8 were statistically significantly higher in the bevacizumab group than in the control group at each time point,and TNF-α gene and protein expression was only significantly higher only at 24 and 48h（P〈0.05）. Under hypoxic conditions,bevacizumab significantly increased the expression of IL-1β,IL-6,IL-8 and TNF-α at 6,12,24 and 48h（P〈0.05）. IL-1β,IL-8 and TNF-α peaked at 24 h and IL-6 peaked at 12 h after the bevacizumab treatment under both normoxic and hypoxic conditions. CONCLUSION：Treatment of ARPE-19 cells with bevacizumab can significantly increase the expression of fibrosis-related inflammatory mediators under bothnormoxic and hypoxic conditions. Inflammatory factors might be involved in the process of fibrosis after antiVEGF therapy,and the up-regulation of inflammatory factors induced by anti-VEGF drugs might promote the fibrosis process.