Mechanism of blue light-induced ferroptosis in retinal pigment epithelium cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the occurrence and possible mechanism of blue light-induced ferroptosis in retinal pigment epithelial cells.METHODS: ARPE-19 cells cultured in vitro were irradiated by 405 nm blue light at 50 mW/cm2 irradiance with different duration and were divided into control, 16.3J/cm2, 32.6J/cm2, and 65.2J/cm2 groups; the 65.2J/cm2 group was defined as the high-level blue light irradiation group and cells were further divided into control, high-level blue light irradiation group and high-level blue light irradiation + ferroptosis inhibitor group. CCK-8 assay was used to detect cell viability, commercial kits were used to detect intracellular glutathione(GSH), ferrous iron and malondialdehyde(MDA)concentration, and Western blot was used to detect the relative expression of glutathione peroxidase 4(GPX4)and xCT proteins in cells.RESULTS: The decrease of ARPE-19 cell viability caused by blue light irradiation was dose-dependent, and the reduction of intracellular GSH concentration, the increase of ferrous iron concentration and MDA concentration were all caused by high-level blue light irradiation(all P<0.05); the ferroptosis inhibitor partially restored cell viability and recovered intracellular GSH, reduced concentrations of MDA and ferrous iron in the blue light irradiation group(all P<0.05). The relative expressions of GPX4 and xCT proteins were significantly decreased in the blue light irradiation group, and such change was alleviated by the ferroptosis inhibitor(P<0.05).CONCLUSION: Blue light irradiation may induce ferroptosis in RPE cells by targeting the xCT and GPX4-associated antioxidant pathways.

Overexpression of SARS-CoV-2 spike protein mediates growth inhibition in human retinal pigment epithelial cells / 中华眼底病杂志

Objective:To observe the inhibition of SARS-CoV-2 spike protein (S-protein) on the proliferation of human retinal pigment epithelium (RPE) cells.Methods:SARS-CoV-2 S-protein gene fragment expression plasmid (p3xflag-S) was constructed and transfected into human RPE, HEK293 cells. DNA sequencing was used for identification, and the expression of Flag-S was detected by Western blot. HEK293 cells were divided into the cells 1, 2, 3 and 4 and transfected with GFP11 plasmid and vector, GFP1-10 plasmid and vector, transfected with GFP11 and pCMV-HA-ACE2 plasmid, GFP1-10 and p3xflag-S plasmid. Cell 1 was co-cultured with cell 2 (control group 1), cell 2 with cell 3 (control group 2), cell 3 with cell 4 (observation group), and cell 1 mixed with cells 2, 3 and 4 (control group 3). Bright-field microscopy and fluorescence microscopy were used to observe cell fusion. RPE cells were divided into control group and overexpression S-protein group. The cell cycle was detected by flow cytometry; the cell proliferation level was detected by Counting Kit 8 (CCK-8); and the S-protein expression level in RPE cells was detected by Western blot. The Student’s t-test was performed for comparison between groups. Results:DNA sequence assay showed that S-protein cDNA was fused with flag-tagged protein. Western blot assay showed that S-protein-related expression was elevated in transfected HEK293 cells compared with untransfected p3xflag-S cells. Large, multinucleated fused cell clusters were visible under bright-field microscopy; multiple nuclear with distinct green fluorescence were visible in the fused cells under fluorescence microscopy. Western blot assay showed elevated S-protein-related expression in transfected p3xflag-S plasmid RPE cells compared to untransfected p3xflag-S plasmid RPE cells. CCK-8 results showed that the proliferative capacity of RPE cells in the S-protein overexpression group was significantly reduced compared with the control group, with statistically significant differences ( t=22.70, 16.75, 23.38; P<0.000 1). The results of flow cytometry showed that the G1 phase cells in the control and overexpression S-protein groups were 41.1 % and 67.0%, respectively; compared with the control group, the G1 phase cells in the overexpression S-protein group were significantly higher, and the difference was statistically significant ( t=4.76, P=0.018). The apoptosis rate was significantly increased in the S-protein overexpression group compared with the control group, and the difference was statistically significant ( t=4.91, P=0.008). Conclusion:Overexpression of the SARS-CoV-2 spike protein reduced the proliferation of human RPE cells.

Study on the apoptosis of human retinal pigment epithelium cells induced by EGCG / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To investigate the effect of epigallocatechin gallate(EGCG)on the apoptosis of human retinal pigment epithelium(ARPE-19)cells and its mechanism. METHODS:The ARPE-19 cells were cultured in vitro and treated with 0,40,80 and 160 μg/mL EGCG, respectively. At the proposed time of treatment the morphological changes were detected by hoechst 33258 staining. The apoptosis rate was detected by flow cytometry. The expression of apoptosis-related factors B lymphocytoma-2 gene(bcl-2), BCL2-Associated X protein(Bax),caspase-3 and p53 were detected by quantitative RT-PCR and Western blotting.RESULTS: Hoechst 33258 staining showed that the ARPE-19 cells with the increase of EGCG drug concentration, the number of apoptotic cells gradually increased and the apoptotic bodies were observed. Flow cytometry showed that the apoptosis rate increased gradually with the increase of EGCG drug concentration. The apoptosis rates at 40, 80 and 160 μg/mL were 4.95%±0.071%, 11.75%±0.075% and 21.25%±0.919% respectively, which was significantly different compared with the control group(2.8%±1.556%)(P<0.01), presented with a drug concentration-dependent. The results of quantitative PCR and Western blotting showed that EGCG could significantly up-regulate the expression of apoptosis-promoting factors Bax, caspase-3 and the mRNA and protein expression of p53, and down-regulate the apoptosis-inhibiting factor bcl-2, all of these showed concentration-dependent effects.CONCLUSION:EGCG can obviously induce the apoptosis of ARPE-19 cells. The mechanism is related with the inhibition of bcl-2 and increase the expression of Bax, caspase-3 and p53.

Inhibitory effect of melatonin on pyroptosis of lens epithelium cells and its mechanism / 中华实验眼科杂志

Objective:To investigate the inhibitory effect of melatonin on pyroptosis of lens epithelium cells (HLECs) induced by hydrogen peroxide (H 2O 2) and its mechanism. Methods:The cultured HLECs were divided into normal control group, model control group, melatonin group, vitamin E group, and vitamin E solvent group.Cells in melatonin group, vitamin E group and vitamin E solvent group were pre-cultured with 1×10 -6 mol/L melatonin, 100 μmol/L vitamin E or equal volume of vitamin E solvent, then cultured with 100 μmol/L H 2O 2, respectively, and the cells in the normal control group and model control group were cultured with normal condition or 100 μmol/L H 2O 2, respectively.The HLECs transfected with nuclear factor erythroid 2-related factor 2 short hairpin RNA (shNrf2) or shNrf2 negtive control lentivirus and following with 100 μmol/L H 2O 2 treatment were served as shNrf2 group and shNrf2 negative control group, respectively; and the transfected cells treated with 1×10 -6 mol/L melatonin and subsequent 100 μmol/L H 2O 2 treatment were served as melatonin+ shNrf2 group and melatonin+ shNrf2 negative control group.The activity of lactic dehydrogenase (LDH) and the expression levels of interleukin (IL)-1β and IL-18 in the culture supernatant were detected by the enzyme linked immunosorbent assay (ELISA) kit.The concentration of reactive oxygen species (ROS) was assessed by flow cytometry.The expression level of Nrf2, NLRP3, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), caspase-1 p20 and GSDMD-N proteins were evaluated by Western blot. Results:Compared with model control group, the activity of LDH and the concentrations of IL-1β and IL-18 were significantly decreased in melatonin group and vitamin E group, showing statistically significant differences (all at P<0.05). The ROS fluorescence intensities were 13 040.67±1 550.66 and 12 593.67±1 677.06 in melatonin group and vitamin E group, respectively, which were significantly lower than 18 310.33±1 248.01 in model control group (both at P<0.05). The relative expression levels of Nrf2 protein were 4.24±0.44 and 3.73±0.38 in melatonin group and vitamin E group, respectively, which were significantly higher than 2.28±0.34 in model control group, and the relative expression levels of NLRP3, ASC, caspase-1 p20 and GSDMD-N in melatonin group and vitamin E group were significantly decreased than model control group (all at P<0.05). The relative expression level of Nrf2 protein in shNrf2 group and melatonin+ shNrf2 group was significantly reduced, and the expression levels of LDH, IL-1β, IL-18, ROS content, NLRP3, ASC, caspase-1 p20 and GSDMD-N were significantly increased in comparison with shNrf2 negative control group and melatonin+ shNrf2 negative control group, respectively (all at P<0.05). Conclusions:Melatonin can inhibit the release of NLRP3 inflammasome by activating Nrf2, and has an inhibitory effect on the pyroptosis of HLECs.

NLRP3 inflammasome and its role in age-related macular degeneration / 中华实验眼科杂志

Age-related macular degeneration (AMD), the leading cause of central vision loss among people aged 50 years and older, is one of the major eye diseases causing blindness in the world.Clinically, advanced AMD is divided into two types, non-exudative AMD with manifestation of geographic atrophy and exudative AMD with manifestation of choroidal neovascularization.The pathogenesis of AMD is complex, and the para-inflammation is recognized as an important risk factor.Nucleotide-binding oligomerization domain like receptors 3 (NLRP3) inflammasome is a cytoplasmic pattern recognition receptor and is expressed in several kings of cells, including retinal pigment epithelium (RPE) cells, microglial cells, Müller glia cells and retinal vascular endothelial cells.Recent studies have suggested that NLRP3 inflammasome plays an important role in the pathophysiology of both non-exudative and exudative AMD.The role of the NLRP3 inflammasome and its effector cytokines interleukin (IL)-1β and IL-18 in AMD were reviewed in this article to provide guidance on future prevention and therapy of AMD.

Role of vascular endothelial growth factor and epithelial-mesenchymal transition in the pathogenesis of proliferative vitreoretinopathy / 国际眼科杂志(Guoji Yanke Zazhi)

@#Proliferative vitreoretinopathy(PVR)is a serious complication that occurs in the natural history of rhegmatogenous retinal detachment(RRD)or after retinal detachment surgery, often resulting in vision loss. Currently, there has no effective treatment. The pathological characteristics of PVR are the excessive inflammatory response and abnormal proliferation of various cells under the action of cytokines, which eventually form a layer of proliferative membrane around the retinal surface, and further lead to traction retinal detachment(TRD). In-depth studies on the pathogenesis of PVR will help to find promising molecular targets for its treatment. Recent studies have found that vascular endothelial growth factor(VEGF)and the epithelial-mesenchymal transition(EMT)of retinal pigment epithelium(RPE)cells play an important role in the pathogenesis of PVR. This article summarizes the roles of VEGF and RPE cell EMT in the pathogenesis of PVR and the interaction mechanism between them, with the aim to provide new ideas for the treatment and clinical research of PVR.

Endoplasmic reticulum stress in the pathogenesis of age-related macular degeneration / 国际眼科杂志(Guoji Yanke Zazhi)

@#Age-related macular degeneration(ARMD)is a main cause of irreversible visual impairment in the elderly. The major pathological features are drusen formation, macular pigment disorder, geographic atrophy and abnormal neovascularization. Retinal pigment epithelium(RPE)function is impaired in ARMD. Endoplasmic reticulum(ER)is an organelle in eukaryotes responsible for protein synthesis, modification, integration and quality control. ER also participates in the maintenance of calcium homeostasis and lipid biosynthesis. Stimuli from the external and internal environment may trigger ER stress and therefore activate the intracellular signal transduction pathway-the unfolded protein response(UPR), to restore cell homeostasis. However, prolonged ER stress may lead to apoptosis. The pathogenesis of ARMD has not been fully elucidated, nevertheless, compelling evidence demonstrates that ER stress is involved. In this article, we summarize recent advances in UPR pathways, as well as the role of ER stress in the physiological function of RPE and in the pathogenesis of ARMD.

Evaluation of programmed cell death processes on the lens epithelium of older dogs with diabetic and hypermature cataracts / Avaliação dos processos de morte celular programada no epitélio da lente de cães idosos com catarata diabética e hipermadura

It is well known that posterior capsule opacification (PCO), one of the most common late postoperative complications of cataract surgery, is mainly caused by proliferation and differentiation of remaining lens epithelial cells (LECs) on the posterior lens capsule. Many authors suggest that alterations induced by the pathophysiology of cataracts, its metabolism and the use of 0.1% trypan blue (TB) must cause some degree of cellular damage on these cells, wicht would help to prevent and/or reduce the incidence of PCO after cataract surgery in humans. Therefore, the aim of this study was to evaluate the expression of cell death markers on LECs of older dogs with diabetic and hypermature cataracts, after capsulorhexis, both using 0.1% TB. Twenty samples collected from 13 dogs of different breeds, with ages varying from 8 to 12 years-old, with diabetic and hypermature cataracts, which had been subjected to phacoemulsification surgery (Phaco) using 0.1% TB for staining were studied. Animals were classified as dogs with diabetic (DC) and hypermature cataracts (HC), and expression of molecular markers for apoptosis and autophagy (caspase-3 and beclin-1) on LECs were obtained by immunofluorescence technique. The expression of caspase-3 and beclin-1 was observed in every studied sample and did not differ between groups. In conclusion, our findings suggest that apoptosis and autophagy processes occur to LECs in older dogs presenting diabetic and hypermature cataracts after Phaco utilizing 0.1% TB. Our results may be helpful to future studies of PCO in post-phacoemulsification surgery patients.(AU)

A opacificação da cápsula posterior da lente do globo ocular é a complicação mais observada após a remoção da lente. Essa patologia é causada principalmente pela proliferação e diferenciação das células do epitélio anterior da lente em sua cápsula posterior. Muitos autores sugerem que alterações induzidas pelo metabolismo e/ou patofisiologia da catarata e o uso do corante de azul de tripan a 0,1% devam causar algum dano a essas células, o que supostamente ajudaria a prevenir e reduzir a incidência de tal complicação em humanos. Este trabalho avaliou a expressão de marcadores de morte celular no epitélio anterior da lente de cães idosos com catarata diabética e hipermadura, após capsulorrexe realizada com o emprego do azul de tripan a 0,1%. Foram estudadas vinte amostras colhidas de treze cães de diferentes raças, com idades variando de oito a doze anos, que apresentavam catarata diabética ou hipermadura e que foram submetidos à facoemulsificação utilizando corante de azul de tripan a 0,1%. Foram designados dois grupos: com catarata diabética (DC) e com catarata hipermadura (HC). A expressão molecular dos marcadores de morte celular por apoptose a autofagia (caspase-3 e beclina-1) no epitélio anterior da lente foi avaliada pela técnica de imunofluorescência. Observou-se que a expressão de caspase-3 e beclina-1 ocorreu em todas as amostras e não foi diferente entre os grupos. Os achados deste estudo sugerem que o processo de morte celular por apoptose e autofagia ocorre no epitélio anterior da lente de cães idosos com catarata diabética e hipermadura submetidos à facoemulsificação com o corante de azul de tripan a 0,1%. Este resultado pode ser útil para estudos futuros da opacidade da cápsula posterior da lente em cães submetidos à facoemulsificação.(AU)

Protective effect of Ghrelin against oxidative stress induced by high glucose in human retinal pigment epithelium cells / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM: To investigate the effect of Ghrelin on oxidative stress induced by high glucose in human retinal pigment epithelium (RPE) cells. ·METHODS: RPE cells were cultured and divided into the negative control group, high sugar group, Ghrelin low dose group ( 10-9mol/L ) and high dose group (10-6mol/L). Cells survival rate were detected by CCK-8 colorimetry, cells oxidative damage were observed by oxygen sensitive fluorescence probe H2DCFDA staning, changes of intracellular reactive oxygen species ( ROS ) were detected by H2DCFDA staining, super oxide dismutase ( SOD) activity and malondialdehyde ( MDA) content were detected by spectrophotometer colorimetry. ·RESULTS: CCK-8 results showed that RPE cells survival rate increased to 54.79%±3.43% and 79.16%±3.29% after treated with 10-9mol/L, 10-6mol/L Ghrelin, the difference was statistically significant compared with high glucose group (41.65%±3.42%)(P<0.05). H2DCFDA fluorescent probe dying showed that Ghrelin reduced ROS generation in RPE cells and decreased oxidative damage cells. Spectrophotometer colorimetric method showed that according to the high sugar group, SOD activity increased and MDA content decreased in Ghrelin group. ·CONCLUSION: Ghrelin could inhibit high glucose - induced oxidative damage in human RPE cells, which has protective effect on the process of the occurrence and development of diabetic retinopathy.

Protective effect of pyridoxamine on RPE cells treated with AGEs / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To observe the effects of pyridoxamine(PM)on RAGE, ROS and apoptosis in RPE cells treated with advanced glycation end products(AGEs), and to investigate the protective effect of PM on RPE cells in diabetic retinopathy. <p>METHODS:Primary cultured human RPE cells, the third generation of cells were synchronized with serum-free Dulbecco-modified Eagle medium for 24h, and then grouped: 1)Control group: cultured with 100mg/L BSA for 48h; 2)AGEs-treated group: cultured with 200mg/L AGEs for 48h; 3)PM group: PM1 group: cultured with 16mg/L PM+200mg/L AGEs for 48h; PM2 group: cultured with 32mg/L PM+200mg/L AGEs for 48h. The expression of RAGE protein was detected by immunohistochemistry. The formation of ROS was observed by fluorescence microscopy. The apoptosis of cells was detected by TUNEL. <p>RESULTS:The expression of RAGE protein, ROS and apoptosis of RPE cells in PM group were significantly lower than those in AGEs-treated group, and decreased with the increase of PM concentration. <p>CONCLUSION:Pyridoxamine can inhibit the expression of RAGE and the production of ROS, reduce apoptosis, and have a protective effect on RPE cells.

Rapamycin protects human lens epithelial cells against oxidative stress induced by high glucose / 眼科新进展

Objective To investigate the effects of rapamycin on human lens epithelial cells (HLECs) against high glucose-induced oxidative stress.Methods A oxidative damage model of HLECs induced by high glucose was established,and intervention with different concentrations of rapamycin for the cells was performed for detecting the survival rate by CCK-8 assay,observing cell morphology by inverted microscope,and measuring the level of intracellular reactive oxygen species (ROS) by H2DCFDA staining,as well as determining the expression of apoptosis related Bcl-2 and Bax proteins by Western blot in all groups.Results CCK-8 results showed that the survival rate of HLECs was increased to 62.00％ ± 3.74％ and 79.57％ ± 5.26％ after treated with 10 nmol · L-1 and 100 nmol · L 1 rapamycin,and the difference was statistically significant when compared with the high glucose group (42.32％ ± 3.10％) (all P ＜ 0.05);H2DCFDA fluorescent probe staining demonstrated that rapamycin could suppress ROS generation in HLECs and alleviate oxidative damage to cells;besides,rapamycin could downregulate the expression of Bcl-2 and Bax protein in HLECs and the inhibitory effects were positively correlated with the concentration of rapamycin.Conclusion Rapamycin can inhibit high glucose-induced oxidative damage and apoptosis m human HLECs,which provides a prevention effect for diabetic cataract.

Protective effects of resveratrol on human retinal pigment epithelium cells against H2O2 induced apoptosis / 实用医学杂志

Objective To observe the effects of resveratrol on human retinal pigment epithelium cells against H2O2-induced cell apoptosis and cell cycle. Methods Human RPE cells were divided into 3 groups ran-domly including normal control group,H2O2injury group and resveratrol protected group. H2O2injury group was treated with 200 μmol/L H2O2,and resveratrol protected group were treated with 50 mg/L resveratrol and then treat-ed with 200 μmol/L H2O2.CCK-8 was used to observe inhibitory rate of cell,and the flow cytometry was applied to detect the apoptosis and cell cycle distribution. Results CCK-8 assay showed that the inhibitory rate of cell treat-ed with resveratrol(50 mg/L)were obviously decreased,which was significantly different with H2O2injury group (P < 0.05),the apoptosis levels of cell treated with resveratrol for 2,8,24 h were significantly decreased(P <0.01).Moreover,the cell cycle distribution in the G0/G1 phases was also significantly decreased(P<0.01),the cell cycle distribution in the S phases was significantly increased(P<0.01).Conclusion Resveratrol can inhibit the cell apoptosis on human retinal pigment epithelium cells induced by H2O2,which is related to cell cycle regula-tion.

Clinicopathological significance of atypical squamous epithelium cells which cannot exclude high-grade squamous intraepithelial lesions in cervical cytology / 肿瘤研究与临床

Objective To investigate clinicopathological significance of atypical squamous epithelium cells which cannot exclude high grade squamous intraepithelial lesions (ASC-H) in the diagnosis of cervical diseases. Methods The results of age, high-risk human papillomavirus DNA (hrHPV DNA) and cervical biopsy in 496 patients with ASC-H from March 2012 to December 2015 in Shanxi Dayi Hospital were analyzed. Results Among 496 ASC-H cases, the proportion of the patients between 40 and 49 years old was the highest [30.8 ％ (153/496)]. HrHPV DNA was detected in 154 cases, and the positive rate was 79.2 ％(122/154), and the positive rate of patients at the age of 18 to 29 years old was the highest [84.2 ％ (16/19)]. The detection rate of cervical intraepithelial neoplasia (CIN) Ⅱand above lesions in 124 cases with cervical biopsy was 66.9 ％ (83/124), including 100 cases with hrHPV DNA positive (80.6 ％) and 24 cases with hrHPV DNA negative (19.4 ％). There were 71 cases (71.0 ％) of CINⅡ, CIN Ⅲ, early squamous carcinoma and squamous cell carcinomas in 100 hrHPV DNA positive patients with cervical biopsy. There were 12 cases (50.0 ％) of CINⅡ/Ⅲ changes in 24 hrHPV DNA negative patients with cervical biopsy, but none in early squamous carcinoma and squamous cell carcinomas was detected, and there was a significant difference between hrHPV DNA positive and negative patients (χ2=3.857, P< 0.05). The positive predictive value, negative predictive value, sensitivity and specificity of hrHPV DNA detection for diagnosis of CINⅡand above lesions were 85.5 ％, 29.3 ％, 71.0 ％ and 50.0 ％, respectively. Conclusions ASC-H strongly predicts CINⅡand above lesions in cervical cytology. The detection of hrHPV DNA has a high positive predictive value for CINⅡand above lesions.

Effects of plasma containing invigorating blood and dissipating masses Chinese medicine compound on proliferation of rabbit RPE cell by PDGF intervention / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To invesitigate the effect of invigorating blood and dissipating masses traditional Chinese medicine compound drug-containing plasma on the proliferation of rabbit retinal pigment epithelium(RPE)cells treated with platelet derived growth factor(PDGF). <p>METHODS: Primary cells of RPE cells were obtained by enzymatic digestion, and the primitive culture and subculture of RPE cells were proceeded; prepared blood plasma-contained traditional Chinese medicine compound drug-containing plasma; the fourth genertation rabbit RPE cells were selected as the experimental cells by PDGF of low, medium and high doses(5μg/L, 10μg/L, 20μg/L)for 48h. Suitable concentration was detected and selected in cells experiment by using CCK-8 method. Establishing rabbit model of RPE cell proliferation treated with PDGF. The experimental groups were blank control group(DMEM), normal plasma group, PDGF(10 μg/L)group, PDGF(10 μg/L)+ AG1296(10 μmol/L)group, PDGF(10 μg/L)+ 10% drug-containing plasma, PDGF(10 μg/L)+ 20% drug-containing plasma. Respectively, transwell method was used to determine the migration of rabbit RPE cells after 24h intervention in each group; CCK-8 was used to determine the cell viability OD value of rabbit RPE cells after 48h of intervention in each group.<p>RESULTS: The plasma containing 10% and 20% concentration of invigorating blood and dissipating masses traditional Chinese medicine compound drug can effectively inhibit cell viability and cell migration of RPE cells treated with PDGF. <p>CONCLUSION: We found the certain concentration of invigorating blood and dissipating masses traditional Chinese medicine compound drug can effectively inhibit cell viability and cell migration of RPE cells treated with PDGF, which may be an effective treatment for proliferative vitreoretinopathy and provide a new way to study the mechanism of proliferative vitreoretinopathy.

Gintonin, an exogenous ginseng-derived LPA receptor ligand, promotes corneal wound healing

Ginseng gintonin is an exogenous ligand of lysophosphatidic acid (LPA) receptors. Accumulating evidence shows LPA helps in rapid recovery of corneal damage. The aim of this study was to evaluate the therapeutic efficacy of gintonin in a rabbit model of corneal damage. We investigated the signal transduction pathway of gintonin in human corneal epithelium (HCE) cells to elucidate the underlying molecular mechanism. We next evaluated the therapeutic effects of gintonin, using a rabbit model of corneal damage, by undertaking histochemical analysis. Treatment of gintonin to HCE cells induced transient increases of [Ca²⁺](i) in concentration-dependent and reversible manners. Gintonin-mediated mobilization of [Ca²⁺](i) was attenuated by LPA1/3 receptor antagonist Ki16425, phospholipase C inhibitor U73122, inositol 1,4,5-triphosphate receptor antagonist 2-APB, and intracellular Ca²⁺ chelator BAPTA-AM. Gintonin facilitated in vitro wound healing in a concentration-dependent manner. When applied as an eye-drop to rabbits with corneal damage, gintonin rapidly promoted recovery. Histochemical analysis showed gintonin decreased corneal apoptosis and increased corneal cell proliferation. We demonstrated that LPA receptor activation by gintonin is linked to in vitro and in vivo therapeutic effects against corneal damage. Gintonin can be applied as a clinical agent for the rapid healing of corneal damage.

Effect of iopromide on glomerular visceral epithelium cells / 局解手术学杂志

Objective To investigate the mechanism of contrast-induced nephropathy (CIN) caused by Iopromide.Methods Two-four female SD rats were randomly divided into two groups which were control group and CIN group .The rats in CIN group were injected Io-promide via caudal vein ,the rats in control group were injected the equal amount of solvent .After 24 hours,all the rats were euthanized and tested.The excretion of 24 h urinary protein was detected using biochemistry assay .The expression of related cell cycle regulatory protein such as P21,P27 and TGF-β1 in glomerular visceral epithelium cells were measured using immunohistochemical technique .A semiquantitative score was used to evaluate the injure degree of glomerular and tubulointerstitium .Renal glomerular cell apoptosis was evaluated by TUNEL . Results Compared with control group ,CIN group rat glomerular epithelial cells of P 21,P27 and TGF beta 1 positive expression rate signifi-cantly increased,[(12.86 ±0.98) %vs (0.46 ±0.21)%,P=0.004 5],[(21.76 ±2.75)% vs (9.57 ±1.86)%,P =0.0071], [(12.85 ±5.54) vs (7.63 ±0.84),P=0.003 7)] respectively,24 h urine protein significantly increase [(23.44 ±5.22) mg/d vs (2.13 ±0.52) mg/d,P=0.007 0,P=0.005 0],CIN pathological damage of rat glomerular epithelial cells and apoptotic rate significantly more serious [(52.5 ±6.4)%vs (4.2 ±0.3) %,P =0.007 5].In addition,the renal pathologic scores were positively correlated with the excretion of 24hr urinary protein and the expression of P 21,P27,and TGF-β1(r=0.765,0.701,0.842,0.651,P<0.01).Conclusion Io-dine amine via increased glomerular epithelial cells P 27 and TGF-beta 1expression and urinary protein excretion , aggravating pathological damage and apoptosis .

A pilot study on indunction and differentiation of human fibroblasts to iPS cells and iPS-RPE cells mediated by retrovirus infection / 中华实验眼科杂志

Background Retinal pigment epithelium (RPE) cell transplantation is the primary means of human trial for the treatment of retinal degeneration.Induced pluripotent stem cells (iPSCs) will become an important source for cell transplantation.In addition,iPS-RPE cells may provide a personalized treatment platform for the patient's own cells treatment.Objective This study was to evaluate the feasibility of human fibroblasts differentiate toward iPSCs from retinitis pigmentosa (RP) patients and toward iPSC-RPE cells from non-RP individual by retroviral transfection of Oct4,Sox2,c-Myc and KLF4 genes.Methods Human thigh skin tissues were obtained from a RP patient with hotspot mutation of SNRNP200 p.S1087L and individual without SNRNP200 p.S1087L mutation,respectively,with the size 2 c m×3 cm.Human dermal fibroblasts were isolated and cultured by trypsin digestion and explant method.The fibroblasts were transfected by a series of retrovirus and cultured by human embyonic cellconditioned medium to generate and induce iPSCs,and then the iPSCs were identified by morphology,alkaline phosphatase (AP) staining and immunofluorescence assay of specific markers of pluripotent stem cells.iPSCs suspension were injected into SCID mouse to observe the tumorigenesis.The iPSCs from non-RP subject were induced to differentiate toward iPS-RPE cells by embryonic body (EB) inducing method,and iPS-RPE cells were identified by detecting the expression of RPE65,zonula occludens protein 1 (ZO-1) and lecithin retinol acyltransferase (LRAT).Results Cultured human dermal fibroblasts showed fusiform or polygon shape and intercellular close arrangement,and Vimentin was positively expressed in the cells.Small cell colonies were harvested 5-7 days after infected by retroviruses,and the morphology changed from spindle into round mass.The hESC-like iPSCs clonies appeared 20 days after cultivation,and the positive expressions of hESC-specific surface antigens including SSEA3,SSEA4,TRA-1-60,TRA-1-81 and Nanog were found in the cells 25-30 days after cultivation,and the positive staining of AP was obtained 12 weeks after cultivation.A teratoma was formed at the injection site of iPSCs suspension in SCID mouse.Immunofluorescence technique showed that RPE cell-specific proteins including RPE65,ZO-1 and LRAT proteins were positively expressed in iPS-RPE cells at 30 days after differentiation.Conclusions Mutation SNRNP200 p.S1087L of RP patient-specific iPSCs can be induced from human dermal fibroblast by retrovirus infection method.The function and morphology of the iPSCs were similar to hESCs.Human iPSCs cell line generated from the dermal fibroblasts of non-RP individuals can differentiate into iPS-RPE cells.

Effect of epigallocatechin - 3 - gallate against oxidative stress induced by high glucose in human lens epithelium cells / 国际眼科杂志(Guoji Yanke Zazhi)

Abstract?AIM: To investigate the effect of epigallocatechin-3-gallate ( EGCG ) against oxidative stress induced by high glucose in human lens epithelium ( HLE) cells.? METHODS: The HLE cell oxidative damage model induced by high concentration glucose was established, and was intervented with different concentrations of EGCG. Cell viability was determined by MTT assay, cell morphology was investigated by convert microscope, cells apoptosis was assayed by flow cytometry with Hoechst-PI staining. Moreover, the levels of super oxide dismutase ( SOD) , glutathione peroxidase ( GSH-Px) and malondialdehyde ( MDA) in supernatant were also tested after different treatment either with high concentration glucose or with different concentrations of EGCG.?RESULTS: MTT results showed that HLE cells activity increased to 50. 33%± 3. 52% and 63. 33%± 4. 63% after treated with 10 μmol/L and 100 μmol/L EGCG respectively, the difference was statistically significant compared with oxidative injury group(32. 67%±3. 10%)(P<0. 05 ); HLE cells maintained better morphology intervented with EGCG under high glucose conditions, the number of apoptotic cells reduced, SOD and GSH-Px level within HLE cells increased and MDA levels decreased.?CONCLUSION:EGCG plays its strong antioxidant effect by increasing SOD, GSH-Px content and decreasing MDA content in cells, therefore provides a reliable experimental basis for the search for effective prevention and treatment of cataract drug.

Effects of N-acetylcysteine, glutathione, and dexamethasone on oxidative stress of human thyrocyte / 中华内分泌代谢杂志

[Summary] The human thyroid epithelium cells were obtained from normal para-adenoma tissues of patients with thyroid adenoma or nodule.Cells were treated with 1 000 IU/ml interferon-γ (IFN-γ) + 10 ng/ml tumor necrosis factor-α (TNF-α),1 mmol/L N-acetylcysteine (NAC),1 mmol/L glutathione (GSH),and 10 μmol/L dexamethasone (DEX) respectively.Malondialdehyde(MDA),glutathioneperoxidase(GSH-Px),and superoxidedismutase(SOD) levels in the cell supernatant were measured.The results showed that IFN-γ+TNF-α significantly increased MDA level (P＜0.05) while decreased GSH-Px and SOD levels (P＜0.05).After NAC,GSH,and DEX intervention,MDA levels all were significantly lowered (all P＜0.05) while GSH-Px and SOD levels were significantly increased compared with IFN-γ+TNF-α stimulation(all P＜0.05).These results suggest that IFN-γ and TNF-α can induce oxidative stress in the human thyrocyte,and this effect is antagonized by NAC,GSH,and DEX via increasing GSH-Px,SOD activity and decreasing MDA content.

Caracterização morfológica e frequência dos estádios do ciclo do epitélio seminífero em preás (Galea spixii Wagler, 1831) criados em cativeiro / Morphological characterization and frenquency of stages of the seminiferous epithelium cycle in captive bred Spix's yellow-toothed (Galea spixii Wagler, 1831)

Estudos baseados nas características testiculares estão altamente relacionados com a eficiência reprodutiva de varias espécies. Assim, o projeto desenvolvido teve como objetivo identificar as células do epitélio seminífero, caracterizar histologicamente suas associações, que formam os estádios, e determinar a frequência destes. Os fragmentos de testículos, com 30, 45, 60, 75, 90, 105, 120, 150 dias foram coletados no Centro de Multiplicação da Universidade Federal Rural do Semi-árido, Mossoró/ RN. Passando pelos processos de fixação, lavagens em soluções de concentrações crescentes de álcoois (70-100 por cento), desidratação em xilol, inclusão em Histosec®, preparação das lâminas histológicas, colorações em Hematoxilina e Eosina (HE) e suas fotomicrografias para a caracterização dos núcleos celulares do epitélio germinativo e a definição dos oitos estágios do ciclo do epitélio seminífero (CES) baseados no Método da Morfologia Tubular. Das faixas etárias analisadas todos os animais de 90-150 dias de idade apresentaram todos os estádios do CES. Os estádios I e III foram os que apresentaram maior e menor freqüência, respectivamente. Os animais caracterizados como pré-púberes (30 dias), púberes (45-90 dias de idade) e pós-púberes (105150 dias de idade) apresentaram os estádios I, VIII e IV com uma maior freqüência, respectivamente.

Studies based on the testicular characteristics are strongly associated with the reproductive efficiency of various species. Thus, the developed project aimed to identify the cells of the seminiferous epithelium, histologically characterized their associations, which form stages, and determine the frequency of these. The fragments of testes, 30, 45, 60, 75, 90, 105, 120, 150 days were collected Multiplication Center of Universidade Federal Rural do Semi-Árido, Mossoró, RN. Through the process of fixing, washing in solutions of increasing concentrations of alcohols (70-100 percent), dehydration in xylene, inclusion in Histosec ®, preparation of histological slides, stained with hematoxylin and eosin (HE) and their photomicrographs for the characterization of cell nuclei of the germinal epithelium and the definition of the eight stages of the seminiferous epithelium cycle (CES) based on the tubular morphology method. The different age groups all animals at 90 to 150 days of age showed all stages of the CES. Stages I and III showed the highest and lowest frequency, respectively. Animals categorized as prepubertal (30 days), pubertal (45 to 90 days old) and postpubertal (105 to 150 days of age) had stage I, IV and VIII with a higher frequency, respectively.