Effect of acetyl L-carnitine on human retinal pigment epithelium-19 cells in hypoxic conditions

AIM:To investigate the effect of acetyl-L-carnitine(ALCAR)on cell viability,morphological integrity,and vascular endothelial growth factor(VEGF)expression in human retinal pigment epithelium(ARPE-19)cells using a hypoxic model.METHODS:In the first set of experiments,the optimal CoCl_(2) dose was determined by exposing ARPE-19 cell cultures to different concentrations.To evaluate the effect of ALCAR on cell viability,five groups of ARPE-19 cell culture were established that included a control group,a sham group(200μM CoCl_(2)),and groups that received 1,10 and 100 mM doses of ALCAR combined with 200μM CoCl_(2),respectively.The cell viability was measured by MTT assay.The morphological characteristics of cells were observed by an inverted phase contrast microscope.The levels of VEGF and HIF-1α secretion by ARPE-19 cells were detected by enzyme linked immunosorbent assay(ELISA)assay.RESULTS:ARPE-19 cells were exposed to different doses of CoCl_(2) in order to create a hypoxia model.Nevertheless,when exposed to a concentration of 200μM CoCl_(2),a notable decrease in viability to 83% was noted.ALCAR was found to increase the cell viability at 1 mM and 10 mM concentrations,while the highest concentration(100 mM)did not have an added effect.The cell viability was found to be significantly higher in the groups treated with a concentration of 1 mM and 10 mM ALCAR compared to the Sham group(P=0.041,P=0.019,respectively).The cell viability and morphology remained unaffected by the greatest dose of ALCAR(100 mM).The administration of 10 mM ALCAR demonstrated a statistically significant reduction in the levels of VEGF and HIF-1α compared with the Sham group(P=0.013,P=0.033,respectively).CONCLUSION:The findings from the current study indicate that ALCAR could represent a viable therapeutic option with the potential to open up novel treatment pathways for retinal diseases,particular relevance for age-related macular degeneration(AMD).However,to fully elucidate ALCAR’s application potential in retinal diseases,additional investigation is necessary to clearly define the exact mechanisms involved.

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.

4-HNE Induces Apoptosis of Human Retinal Pigment Epithelial Cells by Modifying HSP70

The role of heat shock protein 70 (HSP70) in apoptosis of human retinal pigment epithelial cells (ARPE-19) induced by 4-hydroxy-2-nonenal (4-HNE) was explored. Different concentrations of 4-HNE were used to stimulate ARPE-19 cells, and apoptosis was measured by flow cytometry. The expression of apoptotic-related proteins, HSP70, X-linked inhibitorof- apoptosis (XIAP), Bcl-2, and Bax were quantified by Western blotting. HSP70 and XIAP overexpression plasmids, or their corresponding siRNAs were transfected into ARPE-19 cells using Lipofectamine. 2000. Co-immunoprecipitation and Western blotting were used to detect the effect of 4-HNE on the expression of HSP70 and the binding level between 4-HNE and HSP70. The results showed that 4-HNE induced late apoptosis in ARPE-19 cells, accompanied by elevated levels of 4-HNE-modified IISP70, but it did not affect HSP70 protein expression. 4-HNE-modified HSP70 down-regulated the expression of the apoptosis inhibitory protein XIAP. Overexpression of HSP70 or XIAP inhibited 4-HNE-induced apoptosis of ARPE-19 cells. It was suggested that 4-HNE could promote XIAP degradation by modification of HSP70 to induce late apoptosis of human retinal pigment epithelial 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.

Influence of IL-1β and TNF-α on Fas Expression of Human Retinal Pigment Epithelial Cells in Vitro

Purpose:To observe Fas expression change of cultured human retinal pigment epithelial (RPE) cells by IL-1β and TNF-α.Methods:With flow cytometry, immunohischemistry, and color imaging system, Fas expressions by exposure to IL-1β and/or TNF-α were measured.Results:The gray degree values of Fas expression were 67.5±6.1 in IL-1β+TNF-α-treated group, 80.1±9.2 in IL-1β-treated group, and 70.4±6.4 in TNF-α-treated group, respectively. There were significant differences (P < 0.005) compared with control group (107.0±10.2). Flow cytometry showed that 15.0% cultured human RPE cells expressed Fas. Fas-positive in IL-1β, TNF-α, and IL-1β+TNF-α-treated groups expressed was 28.1%, 34.5%, and 65.2%, respectively. Conclusion:IL-1β, TNF-α, and combining both of them can up-regulate Fas protein expression, which may contribute to more Fas (+) cells in proliferative vitreoretinopathy (PVR). Minimizing this process by means of inducing apoptosis of Fas (+) proliferative cells of Fas/FasL pathway is a future preventive and therapeutic possibility for PVR. Eye Science 2004;20:39-41.

Low level of activin A secreted by fibroblast feeder cells accelerates early stage differentiation of retinal pigment epithelial cells from human pluripotent stem cells

Human pluripotent stem cells (hPSC) differentiated to retinal pigment epithelial cells (RPE) provide a promising tool for cell replacement therapies of retinal degenerative diseases. The in vitro differentiation of hPSC-RPE is still poorly understood and current differentiation protocols rely on spontaneous differentiation on fibroblast feeder cells or as floating cell aggregates in suspension. The fibroblast feeder cells may have an inductive effect on the hPSC-RPE differentiation, providing variable signals mimicking the extraocular mesenchyme that directs the differentiation in vivo. The effect of the commonly used fibroblast feeder cells on the hPSCRPE differentiation was studied by comparing suspension differentiation in standard RPEbasic (no bFGF) medium to RPEbasic medium conditioned with mouse embryonic (mEF-CM) and human foreskin (hFF-CM) fibroblast feeder cells. The fibroblast secreted factors were found to enhance early hPSC-RPE differentiation. The onset of pigmentation was faster in the conditioned media (CM) compared to RPEbasic for both human embryonic (hESC) and induced pluripotent (iPSC) stem cells, with the first pigments appearing around two weeks of differentiation. After four weeks of differentiation, CM conditions consistently contained higher number of pigmented cell aggregates. The ratio of PAX6 and MITF positive cells was quantified to be clearly higher in the CM conditions, with mEFCM containing most positive cells. The mEF cells were found to secrete low levels of activin A growth factor that is known to regulate eye field differentiation. As RPEbasic was supplemented with corresponding, low level (10 ng/ml) of recombinant human activin A, a clear increase in the hPSC-RPE differentiation was achieved. Thus, inductive effect provided by feeder cells was at least partially driven by activin A and could be substituted with a low level of recombinant growth factor in contrasts to previously reported much higher concentrations.

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.

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.

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.

非接触共培养体系下抑制ARPE-19中CAMKⅡ表达对HUVECs迁移和侵袭及管腔形成的影响

目的:探讨非接触共培养体系下抑制人视网膜色素上皮细胞(ARPE)中Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ(CAMKⅡ)表达对人脐静脉内皮细胞(HUVECs)迁移、侵袭、管腔形成的影响。方法:将过表达CAMKⅡ-δ的ARPE-19样本进行RNA测序,应用生物信息学分析差异基因参与的功能。使用transwell小室构建ARPE-19和HUVECs非接触共培养体系,根据实验干预措施分为:空白组:仅接种未共培养的HUVECs,无ARPE-19细胞;对照组:ARPE-19和HUVECs细胞均使用完全培养基进行共培养;AIP组(CAMKⅡ抑制组):ARPE-19使用含有AIP(160 nmol/L)的完全培养基,HUVECs使用完全培养基,进行共培养。检测HUVECs迁移、侵袭和管腔形成能力的变化,并通过Western blotting检测CAMKⅡ/AMPK/mTOR/VEGFA蛋白表达水平。结果:生信分析发现差异基因参与细胞生长与死亡和细胞运动等生物学过程。划痕和transwell迁移实验均表明AIP组的HUVECs相对迁移率均明显低于对照组(均P<0.05)。而侵袭和小管形成实验表明,AIP组的相对侵袭率和相对管腔形成率较对照组无明显改变(均P>0.05)。Western blotting结果表明AIP组CAMKⅡ、P-mTOR、VEGFA蛋白表达较对照组均明显下调,而P-AMPK蛋白表达较明显上调(均P<0.05)。结论:在非接触共培养体系下抑制ARPE-19细胞中CAMKⅡ表达可以显著降低HUVECs迁移能力,但不能改变侵袭和管腔形成能力,这可能是通过AMPK/mTOR/VEGFA信号通路实现的。

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.

GLS1通过激活Nrf2/HO-1轴抑制过氧化氢诱导的ARPE-19细胞氧化应激、自噬与凋亡

目的探究GLS1对过氧化氢诱导的ARPE-19细胞氧化应激、自噬与凋亡的影响及机制。方法ARPE-19细胞分为对照组、H_(2)O_(2)组、H_(2)O_(2)+Vector组、H_(2)O_(2)+GLS1组,对照组细胞不做任何处理,H_(2)O_(2)组细胞用200μmol/L H_(2)O_(2)诱导48h,H_(2)O_(2)+Vector组和H_(2)O_(2)+GLS1组细胞转染Vector和GLS1质粒后用200μmol/L H_(2)O_(2)诱导48 h,比色法测定各组细胞SOD、GSH和MDA浓度,透射电镜观察各组细胞自噬小体,流式细胞术检测各组细胞凋亡水平,Western blot检测各组细胞Nrf2、HO-1、LC3-Ⅱ、p62的表达。结果与对照组比较,H_(2)O_(2)组ARPE-19细胞SOD、GSH表达显著下降,MDA显著增加,自噬小体数目显著增加,细胞凋亡显著增加,LC3-Ⅱ表达显著上调,P62、抗核因子红系2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)、血红素氧合酶1(heme Oxygenase-1,HO-1)表达显著下调。与H_(2)O_(2)+Vector组比较,H_(2)O_(2)+GLS1组细胞SOD、GSH表达显著增加,MDA显著降低,自噬小体数目显著减少,细胞凋亡显著减少,LC3-Ⅱ表达显著下调,P62、Nrf2、HO-1表达显著上调。结论GLS1可抑制H_(2)O_(2)诱导的ARPE-19细胞氧化应激、自噬与凋亡,其机制为激活Nrf2/HO-1信号通路。

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.

止血化瘀利水方对缺氧诱导ARPE-19细胞VEGF及HIF-1α表达的影响

目的探讨止血化瘀利水方含药血清对CoCl2诱导的人视网膜色素上皮细胞(ARPE-19细胞)血管内皮生长因子(VEGF)、缺氧诱导因子-1α(HIF-1α)蛋白及mRNA表达的影响。方法体外培养ARPE-19细胞,用CoCl2诱导构建缺氧模型,将RPE细胞分为正常组、缺氧模型组、空白对照组、阳性对照组、中药组,以CCK-8法检测细胞增殖活性,ELISA法检测VEGF蛋白浓度,应用荧光定量PCR检测HIF-1α和VEGF的mRNA表达水平。结果 200μmol/LCoCl2对RPE细胞吸光度值(OD值)影响最大,设立为缺氧细胞模型浓度。与正常组比较,缺氧模型组细胞增殖活性增强,VEGF蛋白浓度增高,HIF-1α及VEGF的m RNA表达水平上调。与缺氧模型组比较,中药组、阳性对照组在24 h、48 h、72 h细胞VEGF蛋白均含量减少,差异均有统计学意义(中药组:t24 h=4.242,P24 h<0.001;t48 h=3.430,P48 h<0.001;t72 h=2.501,P72 h=0.006;阳性对照组:t24 h=2.897,P24 h=0.001;t48 h=2.308,P48 h=0.013;t72 h=4.748,P72 h<0.001),与空白对照组比较,差异无统计学意义(P>0.05)。中药组、阳性对照组在48 h细胞HIF-1αmRNA表达下调,与缺氧模型组比较,差异有统计学意义(中药组:t=15.358,P<0.001;阳性对照组:t=14.918,P<0.001),24 h、72 h差异无统计学意义(P>0.05)。中药组、阳性对照组在24 h、48 h细胞VEGF m RNA表达受到抑制,与缺氧模型组比较,差异均有统计学意义(中药组:t24 h=3.084,P24 h=0.003;t48 h=14.837,P48 h<0.001;阳性对照组:t24 h=3.437,P24 h=0.001;t48 h=13.554,P48 h<0.001)。结论缺氧损伤可诱导ARPE-19细胞增殖,VEGF蛋白表达量增加,HIF-1α和VEGF mRNA表达水平上调;止血化瘀利水方对缺氧RPE细胞的增殖及VEGF蛋白、HIF-1α和VEGF mRNA的表达有抑制作用。与对照组复方血栓通胶囊比较,止血化瘀利水方对RPE细胞增殖的抑制作用更持久,在24 h、48 h控制VEGF蛋白表达水平更接近正常组;而二者在抑制VEGF和HIF-1αmRNA表达方面作用相似。

黄芪含药血清对氯化钴诱导ARPE-19细胞缺氧损伤的保护作用

目的:研究黄芪含药血清对氯化钴(CoCl_(2))诱导人视网膜色素上皮(ARPE-19)细胞缺氧损伤的保护作用,从而探讨黄芪是否通过抗氧化应激来改善糖尿病视网膜病变(DR)。方法:建立CoCl_(2)诱导ARPE-19细胞缺氧模型,并分为以下5组:正常组(细胞正常培养,不加任何处理)、缺氧模型组(200μmol/L CoCl_(2))、空白血清组(200μmol/L CoCl_(2)+空白血清)、低剂量含药血清组(200μmol/L CoCl_(2)+10%含药血清)、高剂量含药血清组(200μmol/L CoCl_(2)+20%含药血清)。CCK-8检测细胞活性;试剂盒检测细胞上清液中还原型谷胱甘肽(GSH)、丙二醛(MDA)水平;ELISA检测细胞培养基中缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)的含量;实时荧光定量PCR(qPCR)检测VEGF、HIF-1α和脯氨酰羟化酶-2(PHD-2)的mRNA水平;Western Blot法检测VEGF、HIF-1α和PHD-2的表达情况。结果:采用200μmol/L的CoCl_(2)浓度可成功建立ARPE-19细胞缺氧模型。低剂量和高剂量黄芪含药血清可抑制缺氧诱导的ARPE-19细胞增殖(P<0.05),升高ARPE-19细胞缺氧损伤中GSH水平,降低MDA含量(P<0.05)。低剂量和高剂量黄芪含药血清可抑制ARPE-19细胞缺氧损伤上清液中HIF-1α和VEGF的表达(P<0.05),以及ARPE-19细胞中VEGF、HIF-1α、PHD-2的mRNA表达和蛋白表达(P<0.05)。结论:低剂量和高剂量黄芪含药血清通过抗氧化作用减轻CoCl_(2)诱导ARPE-19细胞缺氧损伤。

京尼平调节Nrf2/HO-1通路抑制ARPE-19细胞氧化损伤

目的研究京尼平(genipin)具有抑制高糖缺氧导致的人视网膜色素上皮(ARPE-19)细胞氧化应激损伤的作用及机制。方法体外培养ARPE-19细胞,建立氧化应激损伤模型。细胞随机分为4组培养,即对照组、模型组、京尼平组和Nrf2抑制剂组。倒置显微镜下观察各组细胞形态;吖啶橙-溴乙锭(AO/EB)染色法分析细胞凋亡;DCFH-DA法检测细胞内ROS含量;试剂盒检测谷胱甘肽过氧化物酶(GSH-Px)活性;Western blot法检测细胞核因子E_(2)相关因子2(Nrf2)和血红素氧化酶-1(HO-1)蛋白相对表达量。结果京尼平明显改善高糖缺氧环境的细胞生长状态,减少细胞凋亡率,降低ROS水平,升高GSH-Px活力值。Western blot法检测Nrf2/HO-1通路相关蛋白,模型组与空白组比较,Nrf2和HO-1蛋白表达水平均降低;京尼平组与模型组比较,上调Nrf2和HO-1蛋白表达量,加入Nrf2抑制剂后则明显降低,差异均有统计学意义(P<0.05)。结论对于高糖缺氧导致氧化应激损伤的ARPE-19细胞,京尼平具有抑制作用,其机制与调节Nrf2/HO-1通路有关。

信号转导/转录激活因子3抗ARPE-19细胞氧化应激研究

目的本研究旨在观察信号转导/转录激活因子3(signal transduction/activation of transcription factor3,STAT3)对视网膜色素上皮细胞(retinal pigment epithelium cells,RPE)氧化应激损伤的保护作用,并探讨STAT3在年龄相关性黄斑变性(age-related macular degeneration,AMD)发病机制中的意义。方法体外培养ARPE-19细胞系,氧化低密度脂蛋白及H2O2干预培养细胞诱导氧化应激损伤,通过对细胞增殖、凋亡、活性氧族(reactive oxygen species,ROS)水平及细胞衰老分析研究,评估氧化应激损伤对RPE的影响;实时荧光定量技术分析氧化应激过程中STAT3-mRNA表达状况;STAT3过表达载体转染ARPE-19细胞,烟酰胺预处理细胞再经H2O2及氧化低密度脂蛋白干预后通过对增殖、凋亡、ROS及细胞衰老状态的分析,了解STAT3抗RPE氧化应激效果。结果与对照组相比,H2O2和氧化低密度脂蛋白能显著增加ROS水平,促使细胞衰老增加,细胞的增殖显著下降而细胞凋亡显著上升(均为P<0.05)。氧化应激状况下STAT3上游产物表达上升,氧化低密度脂蛋白及H2O2组荧光表达强度分别是3.3±1.2及3.5±1.1,与对照组相比差异均有统计学意义(均为P<0.05);STAT3能保护ARPE-19细胞抗氧化应激损伤,使细胞增殖增加、凋亡减少及ROS累积,但不造成细胞衰老状况加剧,说明ARPE-19细胞衰老不受STAT3调节。结论STAT3在细胞氧化损伤中能独立地发挥抗氧化应激作用,提示了STAT3在AMD治疗过程中的应用前景。

白藜芦醇对脂多糖诱导人视网膜色素上皮ARPE-19细胞损伤的保护机制研究

目的观察白藜芦醇(RES)对脂多糖(LPS)诱导人视网膜色素上皮(hRPE)ARPE-19细胞损伤的保护作用,及其对Aldolase/AMPK信号通路的影响。方法将细胞分为5组:(1)对照组(NC组),采用正常完全培养液培养;(2)LPS组,采用含100 ng/mL的LPS培养液培养;(3)LPS+RES组(LR组),采用100 ng/mL的LPS和40μM的RES共同处理;(4)LPS+二氢脱氧吗啡(CC)组(LC组),采用100 ng/mL的LPS和10μM的CC共同处理;(5)LPS+CC+RES组(LCR组),采用100 ng/mL的LPS和10μM的CC和40μM的RES共同处理。5组均处理24 h后,通过CCK8法和Hoechst染色法检测ARPE-19细胞增殖活性,流式细胞术检测ARPE-19细胞凋亡率和线粒体膜电压(MMP),ATP测定试剂盒分析ARPE-19细胞中ATP含量,Westernblot分析ARPE-19细胞中Aldolase和p-AMPK蛋白的表达水平。结果(1)ARPE-19细胞增殖:NC组、LPS组和LR组之间比较,差异具有统计学意义(F=108.412,P=0.000)。组间两两比较均有统计学意义(P<0.05)。(2)ARPE-19细胞凋亡:NC组、LPS组和LR组之间比较,差异具有统计学意义(F=43.437,P=0.000)。组间两两比较均有统计学意义(P<0.05)。(3)ARPE-19细胞ATP水平:NC组、LPS组和LR组之间比较,差异具有统计学意义(F=46.803,P=0.000)。组间两两比较均有统计学意义(P<0.05)。(4)ARPE-19细胞的MMP:NC组、LPS组和LR组之间比较,差异具有统计学意义(F=57.815,P=0.000)。与NC组比较,LPS组明显降低(P<0.05),LR组降低不明显(P>0.05);与LPS组比较,LR组明显升高(P<0.05)。(5)p-AMPK蛋白表达水平:5组比较,差异有统计学意义(F=19.804,P=0.000)。与NC组比较,LPS组、LR组和LC组升高(P<0.05),LCR组升高不明显(P>0.05)。与LPS组比较,各组均有统计学意义(P<0.05)。与LR组比较,各组均有统计学意义(P<0.05)。与LC组比较,LCR组升高不明显(P>0.05)。(6)细胞增殖活力:5组比较,差异有统计学意义(F=31.209,P=0.000)。与NC组比较,各组均有统计学意义(P<0.05)。与LPS组比较,LR组和LC组有统计学意义(P<0.05),LCR组升高不明显(P>0.05)。与LR组比较,各组均有统计学意义(P<0.05)。与LC组比较,LCR组升高(P<0.05)。(7)ARPE-19细胞Aldolase表达:NC组、LPS组和LR组之间比较,差异具有统计学意义(F=51.024,P=0.000)。与NC组比较,各组均有统计学意义(P<0.05);与LPS组比较,LR组升高(P<0.05)。结论RES可通过调节Aldolase/AMPK信号通路活性改善LPS诱导的hRPE细胞损伤。

DRP1调控线粒体稳态对人视网膜色素上皮细胞上皮-间充质转化的影响

目的探讨线粒体动力相关蛋白(DRP1)对人视网膜色素上皮(ARPE-19)细胞上皮-间充质转化(EMT)进程的影响。方法构建H_(2)O_(2)干预ARPE-19细胞模型,将ARPE-19细胞分为3组,NG组:采用含体积分数10%胎牛血清的DMEM/F12培养基培养细胞6 h;H_(2)O_(2)组:先采用650μmol·L^(-1)H_(2)O_(2)干预细胞,此后培养方式及时间与NG组相同;H_(2)O_(2)+Mdivi-1组:先采用10μmol·L^(-1)Mdivi-1处理ARPE-19细胞2 h,再给予650μmol·L^(-1)H_(2)O_(2)干预,此后培养方式及时间与NG组相同。Western blot检测各组细胞p-DRP-1/DRP1、E-钙黏蛋白、N-钙黏蛋白、α-平滑肌肌动蛋白(α-SMA)、波型蛋白(Vimintin)及紧密连接蛋白(ZO-1)表达水平;线粒体红色荧光探针检测各组细胞线粒体形态;线粒体超氧化物红色荧光探针检测各组细胞线粒体活性氧(ROS)水平;JC-1染色试剂盒检测各组细胞线粒体膜电位;免疫荧光检测各组细胞中ZO-1表达水平。结果H_(2)O_(2)组细胞p-DRP-1/DRP1蛋白表达比值高于NG组,H_(2)O_(2)+Mdivi-1组细胞p-DRP-1/DRP1蛋白表达比值低于H_(2)O_(2)组,差异均有统计学意义(均为P<0.05)。H_(2)O_(2)+Mdivi-1组细胞较H_(2)O_(2)组线粒体碎片化程度得到改善。H_(2)O_(2)组细胞线粒体ROS水平(4.42±0.29)与NG组(1.00±0.17)及H_(2)O_(2)+Mdivi-1组(2.15±0.18)比较,差异均有统计学意义(均为P<0.05)。H_(2)O_(2)组细胞红/绿荧光强度比值(0.16±0.12)与NG组(1.00±0.09)及H_(2)O_(2)+Mdivi-1组(0.42±0.05)比较,差异均有统计学意义(均为P<0.05)。H_(2)O_(2)组细胞上皮样标志物表达下降,间质样标志物表达上升,H_(2)O_(2)+Mdivi-1组细胞上皮样标志物表达上升,间质样标志物表达下降。各组细胞α-SMA、N-钙黏蛋白、E-钙黏蛋白、Vimintin及ZO-1相对表达量比较,H_(2)O_(2)组与NG组及H_(2)O_(2)+Mdivi-1组比较,差异均有统计学意义(均为P<0.05)。ZO-1免疫荧光染色实验显示,H_(2)O_(2)+Mdivi-1组的细胞连接紧密程度优于H_(2)O_(2)组。结论DRP1可调控线粒体动态平衡,靶向DRP1可改善线粒体功能并抑制EMT进展,从而减轻H_(2)O_(2)诱导的RPE细胞功能障碍。