ABSTRACT
@#AIM: To investigate the potential toxic effects of paclitaxel(PTX)on the proliferation, apoptosis, cell cycle, morphology, and blood-retinal barrier(BRB)of human retinal pigment epithelial cells(ARPE-19). <p>METHODS: ARPE-19 cells were cultured <i>in vitro</i> and divided into two groups: Control group(Control)and drug plus group(PTX). ARPE-19 cells were treated with different concentrations of PTX(0.005, 0.05, 0.5, 5mg/L)for a certain period of time(12, 24, 36, 48, 72h), and CCK8 assay and flow cytometry were used to detect the effects of drug on proliferation and apoptosis of ARPE-19 cells at different concentrations and time points. The same time, the cell cycle was detected by flow cytometry. Morphological changes of cells were observed by immunofluorescence. Expressions of apoptosis-related proteins and barrier function-related proteins were detected by Western blot. The effect of the drug on the cell barrier was measured by measuring the transepithelial resistance of the cells. <p>RESULTS: PTX reduced the proliferation ability of ARPE-19 cells. After 36h of treatment with low concentration of 0.005mg/L paclitaxel, cell proliferation began to be affected. At the same time, PTX accelerated cell apoptosis was dependent on drug concentration and time. Flow cytometry showed that the cells were arrested in the G2-M phase. In addition, PTX causes significant morphological changes in cells, with normal cells fusiform or irregular. In the PTX group, the number of cells decreased and the cell shape tended to be round. PTX affected retinal barrier function, and the transepithelial resistance of cells was significantly decreased after treatment, and the expression of tight junction proteins ZO-1 and Occludin were significantly decreased compared with the control group(<i>P</i><0.05). The expression levels of Cleaved-caspase-3 and Bax were significantly increased compared with the control group, while the expression levels of Bcl-2 were significantly decreased(<i>P</i><0.05)and was dependent on drug concentration and time. <p>CONCLUSION: PTX can affect the proliferation and apoptosis of ARPE-19 cells, and it depends on time and concentration. In addition, PTX affected the cell cycle and morphology of ARPE-19 cell. At the same time PTX can destroy the barrier function of the retina,suggesting that anti-tumor drugs have a potential toxic effect on the retina.
ABSTRACT
@#AIM:To study the effect of vitamin E on the injury of human retinal pigment epithelial(hRPE)cells induced by high-dose blue light, and provide experimental evidence for intercepting blue light damaged hRPE cells. <p>METHODS: The hRPE cell injury model was established with 3000±150Lx blue light. The apoptosis rate and reactive oxygen species(ROS)of the six groups of hRPE cells were detected by flow cytometry at 0, 3, 6, 9, 12 and 24h respectively. Apoptosis and ROS in hRPE cells were detected by cytometry in 0h-irradiation group, 6h-irradiation group, and vitamin E added groups(vitamin E concentration 10, 50, 100μmol/L)before or after 6h-irradiation. The fluorescence intensity of hRPE cells was observed under a fluorescence microscope using Hoechst 33258 staining reagent.<p>RESULTS: Compared with the 0h-irradiation group, the relative amount of reactive oxygen species increased significantly in 3, 6, 9, 12 and 24h groups(all <i>P</i><0.01), the apoptosis rate of hRPE cells increased significantly in 6, 9, 12 and 24h groups(all <i>P</i><0.01), the apoptosis rate of the 3h-irradiation group was not statistically significantly increased(<i>P</i>=0.46). Compared with the 6h-irradiation group, the relative amounts of ROS and apoptotic rate of the six groups of hRPE cells added vitamin E were significantly decreased, and the blue fluorescence of Hoechst 33258 released in the cells gradually decreased, which was concentration dependent(all <i>P</i><0.01),except for apoptosis rate of hRPE cells in the 10 μmol/L vitamin E group before irradiation(<i>P</i>=0.66). Compared with the 0h-irradiation group, the difference in the relative amount of ROS and apoptosis rate of hRPE cells in added groups were statistically significant(all <i>P</i><0.01). At the same concentration of vitamin E, the relative amount of ROS and apoptosis rate of hRPE cells added vitamin E after irradiation were significantly lower than those added vitamin E before irradiation(all <i>P</i><0.01), except for apoptosis rate of hRPE cells in the 10 μmol/L vitamin E group, which had no difference between added before and after irradiation(<i>P</i>=0.08).<p>CONCLUSION: After hRPE cells had been irradiated by blue light, the increase in the relative amount of intracellular ROS was earlier than that of apoptosis. Elimination of intracellular ROS is the idea of intercepting high doses of blue light induced hRPE cell injury. Vitamin E protects RPE cell against damage induced by high doses of blue light, and the effect becomes stronger as the concentration of vitamin E increases, which is better when added after irradiating. However, it doesn't take effect until the concentration reaches a certain level. And the damage can't be completely repaired.
ABSTRACT
@#AIM: To investigate the effect and mechanism of curcumin on inhibiting choroidal neovascularization(CNV)<i>in vitro</i>. METHODS: Human retinal pigment epithelial(ARPE-19)cells chemical hypoxia model was established by cobalt chloride(CoCl2). CCK-8 method was used to detect the effect of curcumin on the activity of ARPE-19 cells induced by CoCl2. RT-qPCR and Western blot were used to detect the expression of AKT, HIF-1α, VEGF mRNA and protein in ARPE-19 cells hypoxia model induced by CoCl2. Cell scratch test, transwell chamber migration test, transwell chamber invasion test and matrigel matrix hose lumen formation test were used to observe the effects of conditioned medium of curcumin in ARPE-19 cells on the proliferation, migration, invasion and lumen formation of human umbilical vein endothelial cells(HUVEC)in non-contact condition. RESULTS:Chemical hypoxia model of ARPE-19 cells can successfully establish by CoCl2 at 100μmol/L. CoCl2 at the final concentration of 100μmol/L can promote the expression of AKT, HIF-1α and VEGF mRNA and p-AKT, HIF-1α and VEGF protein in ARPE-19 cells. Curcumin at the final concentration of 100μmol/L can reduce the expression of AKT, HIF-1α and VEGF mRNA in ARPE-19 hypoxia model. Curcumin at the final concentration of 100μmol/L can reduce the expression of AKT, HIF -1α and VEGF proteins in ARPE-19 hypoxia model. The conditioned medium of low(6.25μmol/L), medium(25μmol/L)and high dose(100μmol/L)curcumin in ARPE-19 cells can significantly inhibit the level migration of HUVEC. The conditioned medium in high dose group can significantly inhibit the vertical migration and cell invasion of HUVEC. The conditioned medium of middle and high dose curcumin in ARPE-19 cells can inhibit the lumen formation of HUVEC. CONCLUSION:Curcumin at 100μmol/L can protect ARPE-19 cells from hypoxia induced by CoCl2. Curcumin can inhibit the formation of blood vessels at the cellular level.
ABSTRACT
@#AIM: To study the protective effect of astragalus-containing serum on cobalt chloride(CoCl2)-induced hypoxia injury of human retinal pigment epithelial cells(ARPE-19), so as to explore whether astragalus can improve diabetic retinopathy(DR)by anti-oxidative stress.<p>METHODS: The ARPE-19 hypoxia model induced by CoCl2 was established and divided into the following 5 groups: normal group(cells were cultured normally without any treatment), hypoxia model group(200μmol/L CoCl2), blank serum group(200μmol/L CoCl2+blank serum), low-dose drug-containing serum group(200μmol/L CoCl2+10% medicated serum)and high-dose drug-containing serum group(200μmol/L CoCl2+20% medicated serum); CCK-8 detects cell viability; Detect the levels of reduced glutathione(GSH)and malondialdehyde(MDA)in the cell supernatant with a kit; ELISA was used to detect the content of hypoxia-inducible factor-1α(HIF-1α)and vascular endothelial growth factor(VEGF)in cell culture medium; Real-time quantitative PCR(qPCR)to detect the mRNA levels of VEGF, HIF-1α and Prolyl hydroxylase-2(PHD-2); The expressions of VEGF, HIF-1α and PHD-2 were detected by Western Blot.<p>RESULTS: Hypoxia model of ARPE-19 can successfully establish by CoCl2 at 200μmol/L. Low-dose and high-dose astragalus-containing serum could inhibit hypoxia-induced ARPE-19 proliferation(<i>P</i><0.05), increase the GSH level and reduce the MDA content in ARPE-19 with hypoxic injury(<i>P</i><0.05). Low-dose and high-dose astragalus-containing serum could inhibit the expression of HIF-1α and VEGF in ARPE-19 hypoxic injury supernatant(<i>P</i><0.05), as well as the mRNA and protein expressions of VEGF, HIF-1α and PHD-2 in ARPE-19(<i>P</i><0.05).<p>CONCLUSION: Low-dose and high-dose astragalus-containing serum alleviates the hypoxia injury of ARPE-19 induced by CoCl2 through anti-oxidant effect.
ABSTRACT
@#AIM: To investigate the effect of miRNA-147 targeted regulation of vascular endothelial growth factor(VEGF)on the proliferation, apoptosis and migration of human retinal pigment epithelial cells, and to explore its molecular mechanism. <p>METHODS: Human retinal pigment epithelial(ARPE-19)cells were selected and divided into 7 groups: blank control group(untreated), nonsense miRNA group(transfected with mimic NC), miRNA-147 simulant group(transfected with miRNA-147 mimic), inhibitor negative control group(transfected with shRNA NC), VEGF inhibitor group(transfected with shRNA VEGF), miRNA-147 simulant+empty viral vector group(transfected with miRNA-147 mimic and pcDNA3.1)and miRNA-147 simulant+VEGF overexpression group(transfected with miRNA-147 mimic and pcDNA3.1 VEGF). RT-qPCR was used to detect the expression of miRNA-147 and VEGF mRNA. Dual luciferase experiments were used to verify the targeting relationship between miRNA-147 and VEGF. Western blot was used to detect the expression of VEGF protein. MTT method was used to detect the proliferation. Flow cytometry to detect the apoptosis level and cell cycle changes. Cell scratch test to detect the level of cell migration. <p>RESULTS: Compared with the blank control group and the nonsense miRNA group, the expression level of miRNA-147 in miRNA-147 simulant group was significantly increased, while the expression levels of VEGF mRNA and protein were significantly reduced(<i>P</i><0.05). Compared with the inhibitor negative control group, the expression levels of VEGF mRNA and protein in the VEGF inhibitor group were significantly reduced(<i>P</i><0.05). Compared with the miRNA-147 simulant+empty viral vector group, the expression level of VEGF mRNA in the miRNA-147 simulant+VEGF overexpression group was significantly increased(<i>P</i><0.05). The dual luciferase report shows that VEGF is the target gene of miRNA-147. Transfection of miRNA-147 mimic and shRNA VEGF can reduce the proliferation and migration of ARPE-19 cells and promote apoptosis can reduce the proliferation and migration of ARPE-19 cells and promote apoptosis(<i>P</i><0.05). Transfection VEGF overexpression reverses the effect of miRNA-147 mimics on proliferation, migration and apoptosis of ARPE-19 cells(<i>P</i><0.05). <p>CONCLUSION: miRNA-147 can inhibit ARPE-19 cell proliferation, migration and promote cell apoptosis by targeting VEGF.
ABSTRACT
@#AIM: To explore the effect of lycium barbarum polysaccharides(LBP)on inflammatory response of human retinal pigment epithelial cells(ARPE-19)induced by lipopolysaccharide(LPS)and its possible signal pathway.<p>METHODS: ARPE-19 cells were stimulated by LPS <i>in vitro</i> to construct the inflammatory injury cell model. Primarily, the cells were divided into five groups randomly. The blank group was cultured in complete medium, and the LPS group was stimulated with complete medium containing 10μg/mL LPS for 24h. The low, medium and high concentration LBP groups were incubated with complete medium importing 0.1, 0.5 and 1mg/mL LBP for 24h separately, and then stimulated with complete medium containing 10μg/mL LPS for 24h. We used the CCK-8 method to observe the cell survival rate, real-time fluorescent quantitative PCR to detect the mRNA expression of inflammatory factors and Western blot to test the changes of phosphorylated protein within the signaling pathway of NF-κB/MAPK.<p>RESULTS: Compared with normal cells, the survival rate of ARPE-19 cells was decreased after the LPS stimulation. With the increase of exogenous LBP concentration, the survival rate of ARPE-19 cell was gradually increased, while the inflammatory factors expression of cytokines IL-1β, IL-6 and MCP-1 were reduced accompany with the phosphorylated proteins(p-p65, P-IκBα, p-JNK, p-ERK and p-p38)of NF-κB/MAPK signaling pathway were decreased.<p>CONCLUSION: LBP prevents LPS-induced inflammatory response of ARPE-19 by inhibiting the intracellular inflammatory factors and the phosphorylation of the related protein within NF-κB/MAPK signaling pathway.
ABSTRACT
AIM: To study the protective effects and mechanism of resveratrol (RES) on the injury of human retinal pigment epithelial cells (ARPE-19) induced by high glucose (HG). METHODS: ARPE-19 cells were cultured in HG to simulate injury. Cell viability, apoptosis, ROS generation and miR-26a level were examined by CCK-8 assay, flow cytometry assay, DCFH-DA staining and RT-qPCR, respectively. Expression of proteins associated with viability, apoptosis and oxidative stress was measured by Western blot analysis. In addition, the involvements of the ERK and Wnt/β-catenin pathways were analyzed by Western blot analysis.RESULTS:HG reduced cell viability while promoted apoptosis and oxidative stress in ARPE-19 cells. RES ameliorated HG-induced cell injury. The expression of miR-26a was up-regulated by RES in HG-treated cells, and miR-26a inhibition obviously reversed the effects of RES on HG-treated cells. Finally, we found the ERK and Wnt/β-catenin pathways were inhibited by RES through up-regulation of miR-26a. CONCLUSION: RES protected ARPE-19 cells against HG-induced injury through up-regulating miR-26a, along with inhibition of the ERK and Wnt/β-catenin pathways. RES might be a potential therapeutic drug for diabetic retinopathy.
ABSTRACT
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.
ABSTRACT
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 1000 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.
ABSTRACT
Objective To investigate the effects of bevacizumab on cell morphology,apoptosis rate and apoptosis-related factors in human retinal pigment epitheliumcells.Methods Human retinal pigment epithelial cells (ARPE-19) were cultured in DMEM/F1 2 medium containing 0.25 g · L-1 bevacizumab and divided into 1-week dosing group and 2-week dosing group according to the different incubation time,respectively.Meanwhile,the control group was set up.Then,the cell morphology and apoptosis of each group were observed by light microscope and flow cytometry,accordingly.The expression levels of apoptosis-promoting genes P53,TP53INP1,Bax and apoptosisinducible gene Bcl-2 mRNA and protein were detected by RT-PCR and Western blot,respectively.Results Light microscopic observation revealed that the cells in the 1-week control group and the 2-week control group showed typical epithelial cell morphology,and were in stone-like,single-layer adherent-like growth.Compared with the control group,part of the cell morphology after bevacizumab treatment changed slightly,and the cells became rounded,the cell body was elongated and the boundary was poor.The apoptotic rates of the 1-week control group,l-week dosing group,2-weekcontrol group and 2-week dosing group were (5.57 ± 1.46) ％,(6.39 ± 1.25) ％,(6.88 ± 1.10)％ and (13.34 ± 1.94)％,respectively,and there was no significant difference in the apoptotic rate between 1-week control group,1-week dosing group and 2-week control group (both P ＞ 0.05),but the apoptotic rate of the 2-week dosing group was significantly higher than that of the 2-week control group,the 1-week dosing group and 1-week control group,and the differences were statistically significant (all P ＜0.01).RT-PCR and Western blot results showed that compared with the 1-week control group,the expression of P53,TP53INP1 and Bax mRNA was up-regulated in 1-week dosing group,but the expression of Bcl-2 mRNA was down-regulated,and the differences were statistically significant (all P ＜ 0.05).The expression levels of P53,TP53INP1 and Bax mRNA in 2-week dosing group were higher than those in 2-week control group and 1-week dosing group,while Bcl-2 mRNA expression was down-regulated,and the differences were statistically significant (all P ＜ 0.05).There was no significant difference in the expression of the above factors in 1-week control group and 2-week control group(all P ＞ 0.05).The protein expressions of above factors were similar to their mRNA expression.Conclusion Bevacizumab can alter the morphology of ARPE-19 ceils,increase the apoptotic rate and up-regulate the expression of apoptosis-promoting factors,but down-regulate the expression of apoptosis-suppression factors in ARPE-19 cells,which may be the reason for the loss of RPE layer in the macula after anti-VEGF therapy.
ABSTRACT
Objective To investigate the effects of conbercept combined with platelet-derived growth factor (PDGF) receptor inhibitor on the proliferation and migration of human retinal pigment epithelial cells (ARPE-19),as well as mRNA and protein expression of vascular endothelial growth factor (VEGF).Methods ARPE-19 cells were cultured in in vitro and the cells in logarithmic growth phase were obtained and induced by different concentrations of CoCl2.Then cell proliferation and toxicity test kit (CCK-8) was used to detect the proliferation activity of ARPE-19 cells for screening the optimal concentration of CoCl2 to construct hypoxic model.The cultured ARPE-19 cells were divided into normal group,hypoxic group and hypoxia + conbercept group (different concentrations),and CCK-8 assay was applied to analyze the effects of conbercept with different concentrations on cell proliferation activity for screening the best concentration of conbercept.The ARPE-19 cells in logarithmic phase were divided into normal group,hypoxic group,hypoxia + PDGF receptor inhibitor group (different concentrations),and CCK-8 assay was performed to detect the effects of PDGF receptor inhibitor on cell proliferation with hypoxic damage activity for screening the best concentration of PDGF receptor inhibitor.The logarithmic growth phase cells were divided into normal group,hypoxic group,hypoxia + 20 mg · L-1 conbercept group,hypoxia + 200 μmol · L-1 PDGF receptor inhibitor group,hypoxia + conbercept + PDGF receptor inhibitor group (both optimal concentration),and then the cell proliferation in each group was detected by CCK-8 assay,and the migration was detected by transwell chambers.The level of VEGF protein in the supernatant of each group was detected by ELISA methods,and VEGF mRNA expression was measured by RT-PCR.Results CCK-8 assay results showed that the A value of ARPE-19 in the 100 μmol · L-1 CoCl2 group was the highest (1.063 ± 0.031),which was set to be the optimal concentration for preparation of hypoxic model.CCK-8 assay results showed the cell proliferation rate of 20 μg · mL-1 conbercept and 20 μmol · L-1 PDGF receptor inhibitor group was (94.58 ± 3.80) ％ and (96.72 ± 5.44) ％,respectively,which could achieve the most satisfying efficacy,thereby both concentrations were selected for follow-up experiments.The cell proliferation and migration ability and VEGF protein level decreased in the conbercept group,PDGF receptor inhibitor group and conbercept + PDGF receptor inhibitor group when compared with the hypoxic group,and the decrease in the combined group was the most significant.Moreover,VEGF mRNA expression in the conbercept group and combined group were decreased when compared with the hypoxic group.Conclusion Hypoxia can enhance cell proliferation and migration ability and induce the up-regulation of VEGC protein and mRNA expression.In addition,PDGF receptor inhibitor combined with conbercept has inhibitory effects on the migration and proliferation of ARPE-19 cells as well as VEGF protein and mRNA expression following hypoxia injury,which is superior to conbercept treatment alone.
ABSTRACT
AIM: To investigate the influence and mechanism of blue light on the proliferation of human retinal pigment epithelial cells.METHODS: Cells were divided into two groups,including blue light group and control group.The 35W white light lamp with blue filter was used to establish damaged RPE cell model in vitro.Blue ray wavelength ranged between 470nm and 520nm.And the light intensity was about 2000Lx.After exposure to blue light,we tested the proliferation of human retinal pigment epithelial cells by CCK-8 kit.And then expression of miR-103 was measured by the real-time PCR.RESULTS: Exposure to blue light inhibited the proliferation of human retinal pigment epithelial cells and increased the expression of miR-103.Moreover,up-regulation of miR-103 inhibited the proliferation of human retinal pigment epithelial cells,and down-regulates miR-103 promoted the proliferation of human retinal pigment epithelial cells.CONCLUSION: Blue light inhibits the proliferation of human retinal pigment epithelial cells by the up-regulation of miR-103.
ABSTRACT
Objective To investigate the effects of bevacizunab on the antioxidative function of human retinal pigment epithelium (RPE),in order to explore the possible mechanism of macular atrophy induced by the application of anti-vascular epithelial growth factor (VEGF) agents in age-related macular degeneration.Methods Human RPE cells were incubated in DMEM/F12 medium containing 0.25 g · L-1 bevacizumab and divided into 5 groups according to incubation period:0 hour(control),12 hours,24 hours,48 hours and 72 hours,and then the oxidative stress was induced by adding H2O2.Cell viability was measured by the CCK8 assay.MitoSox Red was used to determine mitochondrial reactive oxygen species (mtROS) production.Mitochondrial membrane potential was measured using the JC-1 assay.The expression levels of NOX4 and HO-1 were detected by RT-PCR and Western blot,respectively.Results CCK8 assay determination showed that the above treatment had no significant effect on cell viability,the cell viability of 0 hours,12 hours,24 hours,48 hours and 72 hours were (100.2 ±3.3)％,(99.2 ±2.7)％,(102.5 ±6.4)％,(103.9 ±3.7)％,(103.6 ±3.3)％,the difference was not statistically significant (P ＞ 0.05).Compared with the control group,the levels of mtROS increased at 12 hours,24 hours,48 hours and 72 hours,the difference was statistically significant (P ＜ 0.05).Mitochondrial membrane potential at 12 hours,24 hours,48 hours,72 hours were lower than the control group,the difference was significant,48 hours reached the lowest,72 hours significantly increased,but still lower than the control group.RT-PCR and western blot results demonstrated that the expression of NOX4 mRNA and protein increased at 12 hours,24 hours,48 hours and 72 hours,and reached the highest at 24 hours,then decreased significantly,but still higher than the control group,the difference was statistically significant (P ＜0.01).Compared with the control group,the expression of HO-1 mRNA decreased at 24 hours,48 hours and 72 hours,while the expression of HO-1 protein decreased at 48 hours and 72 hours,the difference was statistically significant (P ＜ 0.05).Conclusion The clinical concentration of bevacizumab can reduce the anti-oxidative function of RPE cells,which may be one of the causes of progressive macular atrophy after long-term anti-VEGF therapy.
ABSTRACT
PURPOSE: To investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmic reticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluated in the human retinal pigment epithelial cell line, ARPE-19. METHODS: PTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatment by enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blot analysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measured in the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfected ARPE-19 cells. RESULTS: The protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-κB inhibitor abolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNA expression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells. CONCLUSIONS: These results suggest that PTX3 production increased in the presence of tunicamycin-induced ER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinal pigment epithelial cells. Inositol-requiring enzyme 1α and the NF-κB signaling pathway may serve as potential targets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to be further investigated.
Subject(s)
Humans , Anti-Bacterial Agents/pharmacology , Apoptosis , Blotting, Western , C-Reactive Protein/biosynthesis , Cells, Cultured , Endoplasmic Reticulum Stress/drug effects , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation , Polymerase Chain Reaction , RNA, Messenger/genetics , Retinal Pigment Epithelium/metabolism , Serum Amyloid P-Component/biosynthesis , Tunicamycin/pharmacologyABSTRACT
PURPOSE: To evaluate the protective effects of Epigallocatechin gallate (EGCG) against UV irradiation in cultured human retinal pigment epithelial (RPE) cells. METHODS: UV irradiation was produced by a UV lamp for 30 seconds with an irradiance of 3.3 mW/cm2. After 5 minutes and 1 hour, we administered different concentrations of EGCG (0, 5, 10, 15, 25, 50, 100 uM). The cell count was determined under a microscope using a counting chamber and the cell activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: The cell count of cultured human RPE cells after UV irradiation was markedly increased in the EGCG administration group, compared with the non-administrated group. The cell activity of the cultured human RPE cells after UV irradiation was markedly increased in the EGCG administration group and was increased in a dose-dependent way as determined by the MTT assay. CONCLUSIONS: The administration of EGCG increased the cell count and the cell activity after UV irradiation in cultured human retinal pigment epithelial cells; this suggests that EGCG provided protection against UV damage in cultured human retinal pigmented epithelial cells.
Subject(s)
Humans , Antioxidants/pharmacology , Catechin/analogs & derivatives , Cell Count , Cells, Cultured , Dose-Response Relationship, Radiation , Pigment Epithelium of Eye/cytology , Radiation Injuries/pathology , Radiation-Protective Agents , Spectrophotometry , Ultraviolet RaysABSTRACT
PURPOSE: To investigate the effects of glucose concentrations on formation of reactive oxygen products and cellular activity in human retinal pigment epithelial cells. METHODS: Human retinal pigment epithelial cells were cultured with high glucose (200 mg/100 ml, 300 mg/100 ml, 400 mg/100 ml) and normal glucose (100 mg/100 ml). The amounts of reactive oxygen products were assayed with dihydroethidium (DHE). Paraquat-induced cellular activity was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) method. RESULTS: Reactive oxygen products of human retinal pigment epithelial cells were increased 120%, 250% and 390% in high glucose (200 mg/100 ml, 300 mg/100 ml, 400 mg/100 ml) media compared to those of normal glucose (100 mg/100 ml) media. Paraquat-induced cell toxicity was increased by high glucose concentrations. CONCLUSIONS: High glucose increased formation of reactive oxygen products in human retinal pigment epithelial cells. These results suggest that high glucose can make human retinal pigment epithelial cells more sensitive to oxidative cellular injury.
Subject(s)
Humans , Epithelial Cells , Glucose , Oxygen , RetinaldehydeABSTRACT
In order to investigate the effects of TGF-β1 on the expression of MMP-2, -9 and TIMP1 in human retinal pigment epithelial (RPE) cells, the third-sixth passage cultured RPE cells were treated with TGF-β~ 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-quantitative 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-β1, 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.
ABSTRACT
Objective To study the inhibitive effect of adenovirus mediated CD gene and 5-FC on proliferative human retinal pigment epithelial (HRPE) cells, and to search for an effective method to take precautions against proliferative vitroretinopathy (PVR). Method Different concentrations of CD and 5-FC were added respectively to the cultured third-growth-generation HRPE cells.Transferance rate was detected by positive HRPE cells marked by X-gal and LacZ. The number of HRPE cells were counted and evaluated by methylthiazol-tetrazollium (MTT) method. Results The adenovirus mediated CD gene could be transfered into HRPE cells with a dose-dependent manner. Positive HRPE cells with CD gene could transform 5-FC to 5-Fu,which could inhibit the increase of HRPE cells effectively. No obvious bystander effect on the growth of HRPE cells was detected. Conclusions The adenovirus may introduce a foreign gene into cultured HRPE cells efficiently. It could be a good method to treat and prevent PVR by medication.