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Down regulation of UCP2 expression in retinal pigment epithelium cells under oxidative stress: an in vitro study

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International Journal of Ophthalmology(English edition) 2019年第7期12卷 1089-1094页

作者： Ying Liu Yuan Ren Xia Wang Xu Liu Yun Xu Yuan He Department of Ophthalmology the Second Affiliated Hospital of Xi’an Medical UniversityOcular Immunology and Inflammation InstituteShaanxi Provincial Clinical Research Center for Ophthalmology

AIM: To evaluate the expression of uncoupling protein 2(UCP2) in a retinal pigment epithelium cell line(ARPE-19), under oxidative stress(OS).METHODS: ARPE-19 cells were divided into groups treated with various concentrations of hydrogen peroxide(H2 O2;0, 150, 300, 500, 700, and 900 μmol/L) for 24 h, to induce oxidative damage and cell viability was assessed by MTT assay. UCP2 mRNA expression in cells treated with H2 O2 was investigated by reverse transcription-polymerase chain reaction(RT-PCR). UCP2 protein expression was assessed by Western blotting and ROS levels analyzed by flow cytometry(FCM). Further, UCP2-siRNA treated cultures were exposed to H2 O2(0, 75, 150, and 300 μmol/L) for 2 h and cell viability determined by MTT ***: Cells treated with higher concentrations of H2 O2 appeared shrunken;their adhesion to adjacent cells was disrupted, and the number of dead cells increased. The results of cell viability assays demonstrated that the numbers of cells were decreased in a dose-dependent manner following treatment with H2 O2. Compared with untreated controls, cell viability was significantly reduced after treatment with >300 μmol/L H2 O2(P<0.05). Cell metabolic activity was decreased with increased concentrations of H2 O2 as detected by MTT assay. Levels of OS were further decreased in cells treated with UCP2-siRNA compared with those treated with H2 O2 alone(P<0.05). The results of RT-PCR and Western blotting demonstrated that UCP2 expression was reduced in H2 O2-treated groups compared with controls(P<0.05). FCM analysis showed that cell reactive oxygen species(ROS) levels were increased in H2 O2-treated groups and further upregulated by UCP2-si RNA treatment(P<0.05).CONCLUSION: Expression levels of UCP2 are decreased in ARPE-19 cells treated with H2 O2. ROS levels are further increased in cells treated with UCP2-siRNA relative to those treated with H2 O2 alone. UCP2 may have a protective role in ARPE-19 cells during oxidative injury.

关键词： uncoupling protein 2 retinal pigment epithelium cells oxidative stress

Autophagy: a new mechanism for regulating VEGF and PEDF expression in retinal pigment epithelium cells

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International Journal of Ophthalmology(English edition) 2019年第4期12卷 557-562页

作者： Rong Li Jun-Hui Du Guo-Min Yao Yang Yao Jin Zhang Department of Ophthalmology the First Affiliated Hospital of Xi'an Medical University Department of Ophthalmology Xi'an Ninth Hospital Affiliated to Medical College of Xi'an Jiaotong University Department of Central Laboratory the First Affiliated Hospital of Xi'an Medical University Department of Ophthalmology the First Hospital of Yulin

AIM: To investigate the regulation of vascular endothelial growth factors(VEGF) and pigment epithelium-derived factor(PEDF) expression by autophagy in retinal pigment epithelium(RPE) cells on exposure to hypoxia. METHODS: ARPE-19, an RPE cell line, was treated as following: the control group was kept in a normoxic incubator; the hypoxia group was incubated in a hypoxic incubator with 1% O_2/5% CO_2/94% N_2 for 24 h; the hypoxia + 3-methyladenine(3-MA) group was pretreated with 10 mmol/L 3-MA for 1 h and then in the hypoxic incubator for 24 h; and the hypoxia + chloroquine(CQ) group was pretreated with 50 μmol/L CQ for 1 h and then in the hypoxic incubator for 24 h. The morphology and ultrastructure of the cells was observed by an inverted microscope or a transmission electronic microscope(TEM). Western blot was performed to assay the expression of autophagy-associated markers, including microtubule associated protein 1 light chain 3 B(LC3 B), Beclin-1, Atg5 and p62. The concentration of VEGF and PEDF in the culture supernatant was determined by ELISA, and the ratio of VEGF/PEDF was calculated. RESULTS: There were no obvious differences in cell morphology among different groups and autolysosomes could be observed in the cytoplasm in all groups. Compared to the control cells, the LC3 B-II/I ratio and levels of Beclin-1 and Atg5 were significantly increased and p62 level was significantly decreased in the hypoxia group. With the increase of VEGF and decrease of PEDF concentration, the VEGF/PEDF ratio was significantly increased in the hypoxia group compared to the control cells. The LC3 B-II/I ratio was significantly reduced by 3-MA treatment and increased by CQ treatment. The expressions of Beclin-1 and Atg5 were significantly reduced by 3-MA or CQ treatment, while expression of p62 was increased in the 3-MA or CQ treated cells. The concentration of VEGF was significantly decreased and PEDF increased, thereby the VEGF/PEDF ratio was decreased in the hypoxia + 3-MA gro

关键词： autophagy retinal pigment epithelium cells vascular endothelial growth factors pigment epithelium-derived factor angiogenesis

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

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Current Medical Science 2019年第3期39卷 442-448页

作者： Lei-lei YANG Hao CHEN Jun WANG Ting XIA Hong SUN Chun-hui YUAN Shi-liang LIU Jian-bin CHEN Department of Ophthalmology Wuhan Fourth Hospital (Puai Hospital) Huazhong University of Science and Technology Wuhan 430030China Department of Pathology Zhongnan Hospital of Wuhan University Wuhan 430070China Department of Laboratory Medicine Wuhan Medical and Health Center for Women and Children Huazhong University of Science and Technology Wuhan 430016 China Department of Ophthalmology Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan 430030 China

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.

关键词： 4-hydroxy-2-nonenal HSP70 XIAP human retinal pigment epithelial cells apoptosis

Beta-adrenergic agonist protects retinal pigment epithelium against hydroxycholoroquine toxicity via c AMP-PKA signal pathway

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International Journal of Ophthalmology(English edition) 2020年第4期13卷 552-559页

作者： Ruihua Zhang Dan-Ning Hu Richard Rosen Eye and Vision Research Institute Department of OphthalmologyIcahn School of Medicine at Mount SinaiNew York 10029USA Department of Ophthalmology New York Eye and Ear Infirmary of Mount SinaiNew York 10003USA

AIM: To test our hypothesis that activation of protein kinase A(PKA) signal pathway by β-adrenergic agonist plays an important role in the protecting of cultured retinal pigment epithelial(RPE) cells against the hydroxychloroquine(HCQ) toxicity. METHODS: Cultured human RPE cells were treated with 1) HCQ, 2) HCQ with salbutamol(a β2-adrenergic receptor agonist), and 3) HCQ with salbutamol and a PKA inhibitor, and compared these to 4) untreated cells(controls). After treated for 24 h, cell vacuolation, cells viability, PKA and PKA kinase activity levels were determined by the measurement of the size of vacuoles using Image J software, the cell counting with a dye-exclusion testing, Western blot and PKA kinase detection, respectively. RESULTS: Cell vacuolation and cell death of cultured RPE cells were significantly increased by the treatment of HCQ. Salbutamol significantly elevated PKA and PKA activity levels and this was associated with the inhibition of the vacuolation and cell death. The PKA inhibitor significantly decreased the PKA levels and eliminated the protective effects of salbutamol on HCQ-treated RPE cells. CONCLUSION: The PKA pathway plays an important role in the protective effects of β2-adrenergic agonist on the RPE cells against HCQ toxicity. These findings reveal a novel potential strategy against HCQ retinopathy by treatment with PKA activating medications.

关键词： hydroxycholoroquine retinal pigment epithelial cells retinopathy protein kinase A vacuolation

Decreased uncoupling protein 2 expression in aging retinal pigment epithelial cells

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International Journal of Ophthalmology(English edition) 2019年第3期12卷 375-380页

作者： Yuan He Xia Wang Xu Liu Zhi Ji Yuan Ren Department of Ophthalmology the Second Affiliated Hospital of Xi'an Medical UniversityOcular immunology and Inflammation InstituteShaanxi Provincial Clinical Research Center for Ophthalmology

AIM: To analyze the expression of uncoupling protein 2(UCP2) in retinal pigment epithelium(RPE) cells at the different human age, further explore the possible new target of RPE cells ***: Adult retinal pigment epithelial-19(ARPE-19) cells and the primary RPE cells at the different age(9-20 y,50-55 y, 60-70 y, >70 y) were cultured and harvested. The expression of UCP2 in these cells was detected by reverse transcription-polymerase chain reaction(RT-PCR), Western blot and confocal ***: Cells from the donors more than 60 y are larger and more fibroblastic in appearance compared to ARPE-19 cells and those primary cultures obtained from the younger individuals by using phase-contrast micrographs. Results of RT-PCR, Western blot and confocal microscopy all showed that UCP2 was highly expressed in ARPE-19 cells and in the younger primary cultured human RPE cells at the age of 9-20 y and 50-55 y, whereas lower expression of UCP2 was measured in the older primary cultured human RPE cells at the age more than 60 ***: Expression of UCP2 gene is decreased in aged RPE cells, promoting the lower ability of anti-oxidation in these cells. It is indicated that UCP2 gene might be a new target for protecting the cells from oxidative stress damage.

关键词： retinal pigment epithelium cells aging uncoupling protein 2 oxditive stress anti-oxidation

Protective effects of lipoic acid-niacin dimers against blue light-induced oxidative damage to retinal pigment epithelium cells

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International Journal of Ophthalmology(English edition) 2019年第8期12卷 1262-1271页

作者： Xiu-Lan Zou Yong-Zhen Yu Hong-Hua Yu Guan-Feng Wang Rong-Biao Pi Zhe Xu Chu Zhang Wen-Jie Zhou Dan-Dan Li Xuan-Ge Chen Yu-Ping Zou Department of Ophthalmology General Hospital of SouthernTheatre Command of PLAGuangzhou510010GuangdongProvinceChina Department of Ophthalmology Guangdong ProvincialPeople’s HospitalGuangzhou510000Guangdong ProvinceChina Department of Ophthalmology Third Affiliated Hospitalof Guangzhou Medical UniversityGuangzhou510000Guangdong ProvinceChina School of Pharmaceutical Sciences Sun Yat-sen UniversityGuangzhou510000Guangdong ProvinceChina Department of Ophthalmology the First Affiliated HospitalGuangdong Pharmaceutical UniversityGuangzhou510000Guangdong ProvinceChina Zhongshan Ophthalmic Centre of Sun Yat-sen University Guangzhou510000Guangdong ProvinceChina

AIM: To evaluate the protective effects of lipoic acid-niacin(N2 L) dimers against blue light(BL)-induced oxidative damage to human retinal pigment epithelium(hRPE) cells in ***: h RPE cells were divided into a control group(CG), a BL group, an N2 L plus BL irradiation group, an α-lipoic acid(ALA) plus BL group, an ALA-only group, and an N2 L-only group. hRPE cellular viability was detected by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium(MTT) bromide assays, and apoptosis was evaluated by annexin-V-PE/7-AAD staining followed by flow cytometry. Ultrastructural changes in subcellular organelles were observed by transmission electron microscopy. Reactive oxygen species formation was assayed by flow cytometry. The expression levels of the apoptosis-related proteins BCL-2 associated X protein(BAX), B-cell leukmia/lymphoma 2(BCL-2), and caspase-3 were quantified by Western blot ***: BL exposure with a light density of 4±0.5 mW/cm2 exceeding 6 h caused hRPE toxicity, whereas treatment with a high dose of N2 L(100 mol/L) or ALA(150 mol/L) maintained cell viability at control levels. BL exposure caused vacuole-like degeneration, mitochondrial swelling, and reduced microvillus formation;however, a high dose of N2 L or ALA maintained the ultrastructure of hRPE cells and their organelles. High doses of N2 L and ALA also protected hRPE cells from BL-induced apoptosis, which was confirmed by Western blot analysis: BCL-2 expression significantly increased, while BAX and caspase-3 expression slightly decreased compared to the ***: High-dose N2 L treatment(>100 mol/L) can reduce oxidative damage in degenerating hRPE cells exposed to BL with an efficacy similar to ALA.

关键词： lipoic acid-niacin dimers retinal pigment epithelium cell lipoic acid oxidative stress reactive oxygen species apoptosis

Effects of pigment glands and gossypol on somatic cell culture of upland cotton (Gos-sypium hirsutum L.)

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Chinese Science Bulletin 2001年第23期46卷 1975-1980页

作者： Zhu, SJ Ji, DF Zhejiang Univ Fac Agr & Biotechnol Dept Agron Hangzhou 310029 Peoples R China

The effects of pigment glands and gossypol on the somatic cell culture of upland cotton were studied, using the materials as follows: three pairs of glanded and gland less upland cotton near isogenic lines, TM-1, and Coker 312. The results showed that the pigment glands and gossypol contents in the explants had great inhibiting effect on the induction and growth of callus in somatic cell culture of upland cotton, and the induction rate of callus and the single callus weight of glandless cotton were much higher than those of their glanded near isogenic lines. It was easier to obtain regeneration plants from glandless cotton than from their glandednear isogenic lines. There was a significant inverse correlation between the gossypol contents in the explants and callus induction rate, with the correlation coefficient of -0.84. The vitro gossypol in the medium had some inhibiting effect on the induction and growth of callus, especially for the gland-less cotton. However, a certain concentration of vitro

关键词： pigment gland gossypol G. hirsutum somatic cell cul- ture.

Determination and thermal stability experiments of red pigment from large-scale silver deposit hosted in the Sinian strata in South China and their significance

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Chinese Science Bulletin 1995年第16期40卷 1347-1350页

作者：卢家烂庄汉平 Guangzhou Institute of Geochemistry. Chinese Academy of Sciences Guangzhou 510640. China Institute of Geochemistry Chinese Academy of Sciences Guiyang 550002 China

Over the past years a kind of red organic pigment was found in various strata in Chi-na. Recently, the authors have discovered the similar red pigment in a large-scale sil-ver deposit hosted in the Sinian black shale,... 详细信息

关键词： silver deposit red pigment thermal stability.

The Effect of Zinc on the Apoptosis of Cultured Human Retinal pigment Epithelial Cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2003年第4期23卷 414-417页

作者：陈雯王智张缨 Department of Ophthalmology

To clarify the effects of zinc on the proliferation and apoptosis of cultured human retinal pigment epithelia (RPE) and the expression of caspase 3 in RPE cells. The effect of Zinc on theproliferation of RPE were examined with MTT method. TUNEL method was used to detect the apoptosis of RPE cells. Caspase 3 was detected by immunohistochemistry. A concentration of zinc higher than 0.001 μM could inhibit the proliferation of RPE. And the relationship between concentration of zinc higher than 10 μM and growth prohibition rate of RPE cells was dose dependent. All concentrations of zinc including 0.001 μM enhanced the expression of caspase 3 of RPE. But only the concentration of zinc higher than 0.01 μM could induce apoptosis of RPE. It is concluded that zinc could enhance the expression of caspase 3 of RPE cells and induce apoptosis of RPE cells. Caution should be taken when using zinc supplements for the treatment of ARMD patients without deficiency of zinc.

关键词： pigment epithelium eyes cell culture apoptosis zinc