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.

Pigment Epithelium-derived Factor in Cataractous Aqueous Humor and Lens Epithelial Cells

Purpose: To study the characteristics of PEDF in cataractous aqueous humor and its expression in human lens epithelium. Methods: The PEDF concentration in the aqueous humor was measured by enzyme -linked immunosorbent assay in senile (130cases) and congenital (18cases) cataract patients who underwent cataract phacoemulsification extraction surgery. Anterior lens capsular specimens were obtained from these patients to count lens epithelial cells (LEC) density. The Lens Opacities Classification System Ⅲ was used to classify the senile cataracts as cortical, nuclear, posterior subcapsular and mixed types of opacity, and quantitative analysis of the nuclear opacities was performed by Pentacam Scheimpflug imaging system. Anterior lens capsular specimens from another senile (10cases) and congenital (10cases) cataract were collected for immunofluorescence with polyclonal antibodies specific to human pigment epithelium -derived factor (PEDF). Results:The mean aqueous level of PEDF was(178. 9±87. 5)ng/ml, and there was negative linear correlation of PEDF level and age (r=0. 811, P<0. 001). In senile cases, the aqueous PEDF concentration decreased with increasing nuclear opacities (r=0. 447, P < 0.01) , and the mean PEDF level in nuclear cataract was significantly lower than that in posterior subcapsular opacity (P < 0.01) . PEDF immunostaining was detected in LEC of all capsular specimens. Conclusion : The PEDF level in human aqueous humor is related to age, types of cataracts and lens opacity. PEDF also express in human LEC. The study results suggest PEDF may regulate and/or protect LEC by paracrine and autocrine, and lack of PEDF may play a role in cataractogenesis.

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.

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.

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。

脂联素对高糖中hRPE细胞活性及T-cad表达的影响

目的观察脂联素对高糖环境下人视网膜色素上皮(hRPE)细胞活性及T-钙黏蛋白(T-cad)表达的影响,以阐明脂联素和T-cad对糖尿病视网膜病变(DR)的影响。方法①将hRPE细胞随机分为正常对照组(5.5mmol/L葡萄糖)、高糖组(33mmol/L葡萄糖)、脂联素组(33mmol/L+2.5μg/ml脂联素),运用MTT法测定不同情况下48h后对细胞活性的影响;②再将上述各组细胞分别培养24h、48h、72h后,运用荧光定量PCR测定T-cad的表达水平。结果①高糖组细胞活性降低,加入脂联素后细胞活力比高糖组升高;②随时间的延长,高糖组T-cad表达下降,脂联素组T-cad表达增加。结论脂联素可通过Tcad保护hRPE细胞的功能。

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.

滋阴补肾片对H_(2)O_(2)诱导人视网膜色素上皮细胞氧化损伤的保护作用

目的探讨滋阴补肾片(ZYBS)对H_(2)O_(2)诱导的人视网膜色素上皮(RPE)细胞ARPE-19氧化应激损伤的保护作用。方法将ARPE-19细胞分为对照组(CG)、模型组(MG)、低剂量ZYBS组(LZYBS)、中剂量ZYBS组(MZYBS)、高剂量ZYBS组(HZYBS),CG组不作任何处理,MG组加入200 mM H_(2)O_(2)处理24 h,LZYBS、MZYBS、HZYBS组分别加入0.5%、1.0%、2.0%的ZYBS含药血清后,再加入200 mM H_(2)O_(2)处理24 h。细胞计数试剂-8(CCK-8)测定各组细胞活性,原位末端转移酶标记技术(TUNEL)细胞原位凋亡试剂盒测定细胞凋亡,生化试剂盒检测细胞上清液中活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化酶(GSH-Px)的表达水平。结果(1)细胞活性:与CG组比较,MG组ARPE-19细胞活性降低(t=27.844,P=0.000);与MG组比较,LZYBS、MZYBS、HZYBS组ARPE-19细胞活性升高(t_(LZYBS)=3.550,P=0.003;t_(MZYBS)=7.402,t_(HZYBS)=10.930,均P=0.000),差异均有统计学意义,且细胞活性随浓度的升高呈剂量依赖性。(2)细胞凋亡:与CG组比较,MG组ARPE-19细胞凋亡率升高(t=25.611,P=0.000);与MG组比较,MZYBS、HZYBS组ARPE-19细胞凋亡率降低(t_(MZYBS)=4.376,P=0.001;t_(HZYBS)=8.166,P=0.000),差异均有统计学意义,且细胞凋亡率随浓度的升高呈剂量依赖性。(3)氧化应激:与CG组比较,MG组ARPE-19细胞ROS、MDA水平升高(t_(ROS)=27.333,t_(MDA)=23.922,均P=0.000),SOD、GSH-Px水平降低(t_(SOD)=11.083,t_(GSH-Px)=14.192,均P=0.000);与MG组比较,LZYBS、MZYBS、HZYBS组ARPE-19细胞ROS(t_(LZYBS)=6.410,t_(MZYBS)=10.417,t_(HZYBS)=10.116,均P=0.000)、MDA(t_(LZYBS)=4.998,t_(MZYBS)=12.319,t_(HZYBS)=19.922,均P=0.000)水平降低,SOD(t_(LZYBS)=6.268,t_(MZYBS)=7.889,t_(HZYBS)=14.894,均P=0.000)、GSH-Px(GSH-Px:t_(LZYBS)=4.453,P=0.001;t_(MZYBS)=6.162,t_(HZYBS)=11.501,均P=0.000)水平升高,差异均有统计学意义,且呈剂量依赖性。结论ZYBS可降低H_(2)O_(2)诱导的RPE细胞的氧化应激损伤,抑制凋亡并减轻氧化应激水平。

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。

枸杞多糖对高糖诱导的人视网膜色素上皮细胞中NLRP3炎症小体/细胞焦亡信号通路的影响

目的 观察枸杞多糖(LBP)对高糖诱导的人视网膜色素上皮细胞(ARPE-19)中NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体/细胞焦亡信号通路的影响。方法 将体外培养的ARPE-19细胞,随机分为5组,即正常组(5.5 mmol·L^(-1)葡萄糖)、高糖组(55.5 mmol·L^(-1)葡萄糖)、LBP低浓度组(55.5 mmol·L^(-1)葡萄糖+100μg·mL^(-1)LBP)、LBP中浓度组(55.5 mmol·L^(-1)葡萄糖+200μg·mL^(-1)LBP)和LBP高浓度组(55.5 mmol·L^(-1)葡萄糖+400μg·mL^(-1)LBP)。采用免疫荧光观察各组细胞中NLRP3蛋白的表达,Western blot检测炎症小体NLRP3、半胱氨酸蛋白酶-1(Caspase-1)、凋亡相关斑点样蛋白(ASC)和其下游细胞焦亡蛋白消皮素D(GSDMD)的相对表达量,ELISA检测细胞上清液中白细胞介素(interleukm,IL)-1β和IL-18的表达水平。结果 免疫荧光结果显示,NLRP3蛋白主要在视网膜色素上皮细胞胞质中表达,且与正常组相比,高糖组NLRP3蛋白的表达水平升高(P<0.05);而与高糖组相比,LBP中、高浓度组NLRP3蛋白的表达水平降低(P<0.01)。Western blot结果显示,与正常组相比,高糖组细胞中NLRP3、Caspase-1、ASC、GSDMD蛋白的相对表达量升高(P均<0.05);与高糖组相比,LBP中、高浓度组中NLRP3、Caspase-1、ASC、GSDMD蛋白的相对表达量降低(P均<0.05)。ELISA结果显示,与正常组相比,高糖组细胞中IL-1β和IL-18表达水平均升高(P均<0.05);与高糖组相比,LBP中、高浓度组中IL-1β、IL-18表达水平均降低(P均<0.05)。结论 LBP可通过抑制NLRP3炎症小体/细胞焦亡信号通路的激活,从而对高糖环境下的人视网膜色素上皮细胞产生保护作用。

基于VEGF/MAPKs通路探讨益肾养肝明目方对RPE细胞凋亡作用的影响

目的探讨益肾养肝明目方(YYM)对过氧化氢(H_(2)O_(2))诱导的人视网膜色素上皮(RPE)细胞凋亡的保护作用及药理机制。方法常规培养ARPE-19细胞,筛选H_(2)O_(2)、康柏西普和YYM的最佳浓度,之后将细胞分为空白组(BG)、模型组(MG)、益肾养肝明目方组(YYM)、阳性对照药康柏西普组(CB)、益肾养肝明目方+康柏西普组(YYM+CB)、益肾养肝明目方对照组(YYM-CG),以400μM H_(2)O_(2)、2μg/mL康柏西普和40μg/mL YYM的最佳浓度处理。使用相差显微镜观察各组ARPE-19细胞形态,流式细胞仪检测细胞凋亡率,化学荧光法检测细胞活性氧(ROS)表达,Western Blot检测血管内皮生长因子/丝裂原活化蛋白激酶(VEGF/MAPKs)通路相关蛋白外源性调节蛋白激酶1/2(eERK1/2)、B细胞淋巴瘤蛋白-2(Bcl-2)、Bcl-2相关蛋白X(Bax)、细胞色素c(Cyc)表达。结果(1)细胞形态:MG组细胞出现皱缩,部分脱落;而YYM、CB组的细胞损伤明显减少,凋亡细胞数量下降。(2)细胞凋亡:与BG组比较,MG组细胞凋亡率较高(t=12.408,P=0.000);与MG组比较,YYM、CB、YYM+CB组细胞凋亡率均降低(t_(YYM)=6.023,t_(CB)=5.967,t_(YYM+CB)=9.804,均P=0.000)。(3)ROS:与BG组比较,MG组ROS表达较高(t=14.388,P=0.000);与MG组比较,YYM、CB、YYM+CB组ROS表达较低(t_(YYM)=8.621,t_(CB)=8.774,t_(YYM+CB)=12.620,均P=0.000)。(4)VEGF/MAPKs:与BG组比较,MG组VEGF、p-p38 MAPKs、p-SAPK/JNK、p-ERK1/2、Bax、Bcl-2、Cyc蛋白表达均较高(t_(VEGF)=18.569,t_(p-p38 MAPK)=22.097,t_(p-SAPK/JNK)=17.548,t_(p-ERK1/2)=18.567,t_(Bax)=20.575,t_(Bcl-2)=18.879,t_(Cyc)=28.196,均P=0.000);与MG组比较,YYM、CB、YYM+CB组VEGF、p-p38 MAPK、p-SAPK/JNK、p-ERK1/2、Bax、Bcl-2、Cyc蛋白表达均较低(YYM组:t_(VEGF)=9.493,t_(p-p38 MAPK)=8.942,t_(p-SAPK/JNK)=8.683,t_(p-ERK1/2)=8.929,t_(Bax)=8.849,t_(Bcl-2)=4.863,t_(Cyc)=11.832,均P=0.000;CB组:t_(VEGF)=16.065,t_(p-p38 MAPK)=16.033,t_(p-SAPK/JNK)=15.558,t_(p-ERK1/2)=13.890,t_(Bax)=16.043,t_(Bcl-2)=6.579,t_(Cyc)=22.028,均P=0.000;YYM+CB组:t_(VEGF)=18.569,t_(p-p38 MAPK)=21.378,t_(p-SAPK/JNK)=17.005,t_(p-ERK1/2)=16.583,t_(Bax)=20.071,t_(Bcl-2)=11.442,t_(Cyc)=26.560,均P=0.000),差异均有统计学意义。结论YYM能通过调控VEGF/MAPKs通路相关蛋白的表达,降低H_(2)O_(2)诱导RPE细胞凋亡率和ROS表达,减轻氧化应激损伤,且联合康柏西普使用效果更佳。

姜黄素在抑制紫外线B诱导的人视网膜色素上皮细胞DNA氧化损伤和凋亡中的作用

目的 探讨姜黄素在紫外线B(UVB)诱导的人视网膜色素上皮(RPE)细胞DNA氧化损伤和凋亡中的作用机制。方法 将人视网膜色素上皮细胞株(ARPE-19)随机分为空白对照组、二甲基亚砜(DMSO)组、姜黄素组、UVB照射组、UVB+DMSO组和UVB+姜黄素组。CCK-8实验检测姜黄素对UVB照射前后ARPE-19细胞活力的影响;免疫荧光检测DNA氧化损伤标志蛋白磷酸化组蛋白H2AX(γH2AX)的定位;Western blot检测γH2AX、凋亡相关蛋白[B淋巴细胞瘤-2基因(BCL-2)和BCL-2相关X蛋白(BAX)]的表达变化;使用X-rosamine衍生物(Mito-Tracker Red CMXRos)和异硫氰酸荧光素(FITC)标记的钙离子依赖的磷脂结核蛋白Annexin V(Annexin V-FITC)来检测UVB诱导的ARPE-19细胞线粒体膜电位变化和细胞凋亡情况。结果 与空白对照组相比,DMSO组DMSO处理24 h后的ARPE-19细胞活力无明显改变(P>0.05)。与DMSO组相比,姜黄素组采用15μmol·L^(-1)姜黄素处理24 h后ARPE-19细胞活力轻度升高(P<0.05),表明15μmol·L^(-1)姜黄素对ARPE-19细胞无毒性作用,后续实验采用该浓度处理细胞。与空白对照组相比,UVB照射组ARPE-19细胞活力降低,γH2AX和BAX蛋白表达水平升高,BCL-2蛋白表达水平降低,线粒体膜电位降低,细胞凋亡增加(均为P<0.05)。与UVB照射组相比,UVB+DMSO组ARPE-19细胞活力,γH2AX、BAX以及BCL-2蛋白表达水平均无明显变化(均为P>0.05),线粒体膜电位和细胞凋亡均无明显改变(均为P>0.05)。与UVB+DMSO组相比,UVB+姜黄素组ARPE-19细胞活力升高,γH2AX和BAX蛋白表达水平降低,BCL-2蛋白表达水平升高,线粒体膜电位升高,细胞凋亡减少(均为P<0.05)。结论 姜黄素能减轻UVB诱导的RPE细胞DNA氧化损伤和凋亡,有望为姜黄素在年龄相关性黄斑变性中的作用机制研究提供新的思路。