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)

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.

The protection of salidroside on oxidative stress induced in human lens epithelium cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To explore the effect of different concentrations of salidroside on H2 O2 induced oxidative stress damage in human lens epithelium cells ( HLEC) . · METHODS: HLEC were cultured and divided into negative control group: cultured in normal cultivation;oxidative damage group: treated with 100μmol/L H2 O2 for 12h; Salidroside low concentration group: 10μmol/L salidroside treated for 24h and H2 O2 treated for 12h;Salidroside high concentration group: 100μmol/L salidroside treated for 24h and H2 O2 treated for 12h. MTT method was applied to observe the effect of salidroside on HLEC survival rate. Morphological change of each group were observed and recorded under inverted microscope. DCFH-DA fluorescent probe was applied to detect intracellular ROS changes; content of malondialdehyde ( MDA ) , superoxide dismutase ( SOD ) and glutathione peroxidase ( GSH-Px ) in supernatants were detected by pectrophotometer. · RESULTS: Salidroside obviously inhibited H2 O2 -induced HLEC vitality decline, inhibited ROS generation in cells, causing SOD, GSH-Px levels increased and MDA levels decreased. ·CONCLUSION:Salidroside inhibited H2 O2 induced HLEC injury by decreasing the intracellular MDA content levels and increasing SOD, GSH-Px content levels, which conclude that salidroside may have a certain role in the treatment of HLEC damage.

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.

MicroRNA profiling on oxidative stress induced apoptosis of human lens epithelium / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To identify the changes in microRNA ( miRNA ) profile of human lens epithelium ( HLE) induced by H2 O2 and the role of miRNA in oxidative stress induced apoptosis. METHODS: HLE cell line HLE - B3 was treated by 100μmol/L H2 O2 for 24h and the total RNA were isolated by Trizol reagent. miRNA profile was generated by miRCURYTM LNA microRNA Array. The target genes of differentially expressed miRNAs were predicted by bioinformatics software. RESULTS:Twenty-eight miRNAs showed significantly differential expression after H2 O2 treatment, 18 miRNAs upregulated and 10 miRNAs downregulated. The differentially expressed miRNAs may involve in apoptosis of lens epithetium and development of cataract through targeting BCL2L2 and MIP. CONCLUSION: H2 O2 can induce dramatically changes in miRNA profile of HLE, which may play a pivotal role in the pathogenesis and development of cataract.

Wikstroelide M potently inhibits HIV replication by targeting reverse transcriptase and integrase nuclear translocation / 中国天然药物

AIM@#To evaluate the anti-HIV activity and mechanism of action of wikstroelide M, a daphnane diterpene from Daphne acutiloba Rehder (Thymelaeaceae).@*METHODS@#The anti-HIV activities of wikstroelide M against different HIV strains were evaluated by cytopathic effect assay and p24 quantification assay with ELISA. The inhibitory effect of wikstroelide M on HIV reverse transcription was analyzed by real-time PCR and ELISA. The effect of wikstroelide M on HIV-1 integrase nuclear translocation was observed with a cell-based imaging assay. The effect of wikstroelide M on LEDGF/p75-IN interaction was assayed by molecular docking.@*RESULTS@#Wikstroelide M potently inhibited different HIV-1 strains, including HIV-1IIIB, HIV-1A17, and HIV-19495, induced a cytopathic effect, with EC50 values ranging from 3.81 to 15.65 ng·mL⁻¹. Wikstroelide M also had high inhibitory activities against HIV-2ROD and HIV-2CBL-20-induced cytopathic effects with EC50 values of 18.88 and 31.90 ng·mL⁻¹. The inhibitory activities of wikstroelide M on the three HIV-1 strains were further confirmed by p24 quantification assay, with EC50 values ranging from 15.16 to 35.57 ng·mL⁻¹. Wikstroelide M also potently inhibited HIV-1IIIB induced cytolysis in MT-4 cells, with an EC50 value of 9.60 ng·mL⁻¹. The mechanistic assay showed that wikstroelide M targeted HIV-1 reverse transcriptase and nuclear translocation of integrase through disrupting the interaction between integrase and LEDGF/p75.@*CONCLUSION@#Wikstroelide M may be a potent HIV-1 and HIV-2 inhibitor, the mechanisms of action may include inhibition of reverse trascriptase activity and inhibition of integrase nuclear translocation through disrupting the interaction between integrase and LEDGF/p75.

Expression of matrix metalloproteinases-3 gene in lens epithelial cell and its significance / 中华实验眼科杂志

Background Posterior capsular opacification(PCO) is common complication after extrecapsular extract of cataract.Matrix metalloproteinases-3 (MMP-3) can degrade all the extracellular matrix except polyose.The gene therapy of PCO upon MMP-3 is the researching hot topic.Fibronectin ( FN ) is a degrade gelatin,so its expression can reflect the effect of MMP-3 on LECs indirectly. Objective The aim of this study was to construct MMP-3 eukaryotic recombination plasmid and transfect to lens epithelium cells(LECs) for the observation of MMP3 expression,and to explore the feasibility of gene therapy for after cataract. Methods Six fresh lenses were obtained from pigs.LECs were cultured using explant method.The eukaryotic expression vector pEGFP-N1-MMP-3 was reconstructed with MMP-3 and pEGFP-N1 plasmids.The accuracy of MMP-3 gene fragment was confirmed by double enzyme digestion and DNA sequencing analysis.After transfecting pEGFP-N1-MMP-3 into LECs of pig,the expression of MMP-3 protein in the cells was indirectly observed by green fluorescent protein.The expression of FN in LECs was detected using Western blot. Results The result of double enzyme digestion was consistent with the base number of pEGFP-N1 plasmids and target fragment.By enlacing the result of DNA sequencing analysis with software,the resemblance of the DNA sequence of MMP-3 from recombination plasmid pEGFP-N1-MMP-3 and that of homo MMP-3 was 99.6％,indicating that the target fragment was inserted to pEGFP-N1 plasmids successfully.Green fluorescence for GFP was seen in the LECs in pEGFP-N1-MMP-3 transfected group,but absent response for GFP was in empty vector group.Western blot revealed that the relative expression level of FN in LECs was 0.666±0.008 in pEGFP-N1-MMP-3 trasfected group and 0.326 ±0.071 in empty vector group,with a significant difference between these two groups(P=0.000). Conclusions Eukaryotic recombination plasmid pEGFP-N1-MMP-3 is successfully constructed,and MMP-3 can be expressed in LECs after transfected.These results lay a foundation for the further research of MMP-3 gene therapy for PCO.

Mechanisms of Apoptosis on Human Lens Epithelium after Ultraviolet Light Exposure

PURPOSE: The purpose of this study is to understand the mechanism of apoptosis occurring on a cultured human lens epithelial cell line after exposure to ultraviolet (UV) light. We intended to confirm the presence of cellular toxicity and apoptosis and to reveal the roles of p53, caspase 3 and NOXA in these processes. METHODS: Cells were irradiated with an ultraviolet lamp. Cellular toxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining and fluorescent anti-caspase 3 antibodies were used for apoptosis investigation. The quantities of p53, caspase 3, and NOXA were measured by Western blotting for to investigate the apoptosis pathway. RESULTS: Cellular toxicity on the human lens epithelium markedly increased with time after UV exposure. On Hoechst staining, we found that apoptosis also remarkably increased after exposure to ultraviolet light, compared with a control group. In the immunochemical study using anti-caspase 3 antibodies, active caspase 3 significantly increased after exposure to ultraviolet light. On Western blotting, p53 decreased, while caspase 3 and NOXA increased. CONCLUSIONS: Exposure of cultured human lens epithelial cell lines to ultraviolet light induces apoptosis, which promotes the expression of NOXA and caspase 3 increases without increasing p53. This may suggest that UV induced apoptosis is caused by a p53-independent pathway in human lens epithelial cells.

The effects of TiO_2 nano thin film on the migration and adherence of BLEC in vitro / 眼科研究

Objective The generate of posterior capsular opacification(PCO)is associated with the adhensive and migration of residual subcapsular lens epithelial cells(LECs).Titanium dioxide(TiO_2)nanometer is proved to have the ability of killing tumor cells and cultured bovine LECs.This study tried to observe the effects of TiO_2 nanometer thin film provoked by light on adhesiveness and migration of bovine LECs in vitro.MethodsThe fresh bovine lenses were obtained and cultured in DMEM containing 10% of newborn bovine serum.The second to fifth generation of cells were used in this experiment.The slide modified by TiO_2 photocatalyst film was prepared by sol-gel method.Cultured cells were seeded in filmed or unfilmed slides respectively and exposed to ultraviolet(wavelength 365 nm)for 20 or 40 minutes.The contact angle between water drop and slide was measured by droping method and the cells adhered to slides were calculated after 24 and 48 hours of culture.The growth status and migration distance of bovine LECs were assayed and compared between filmed and unfilmed groups.ResultsThe contact angle between water drop and slide was 0°±2°and 18.825°±2.342° in filmed and unfilmed group respectively,indicating a obviously smaller contact angle in TiO_2 filmed group than unfilmed one.The numbers of bovine LECs adhered to filmed slide was considerably reduced in TiO_2 filmed group compared with unfilmed group in different UVA exposure time(t_(0 min)=5.492,P=0.001;t_(20 min)=6.031,P=0.000;t_(40 min)=6.828,P=0.000).However,no significant difference was found in the numbers of adhensive cells among 3 UVA irradiation time points(F=1.278,P=0.297).The migration distance of the cells was significantly shorter in TiO_2 filmed group in comparison with unfilmed group in 24 and 48 hours after UVA irradiation(F_(group)=14.965,P=0.000;F_(time)=38.033,P=0.000).ConclusionThe TiO_2 nanometer thin film is characterized by the superhydrophilic property.So it can effectively impede the adhesion and migration of bovine LECs in vitro.

Accumulation and Aberrant Modifications of alpha-Crystallins in Anterior Polar Cataracts

Crystallins are the major proteins found in the lens, and the localization of specific crystallins is well known. Overexpression and accumulation of alphaB-crystallin has been observed in response to stress conditions or in certain diseases, such as brain tumors and neurodegenerative diseases. The purpose of this study was to examine whether alpha-crystallins are modified during pathological myofibroblastic changes in lens epithelial cells. Lens epithelial cells attached to the anterior capsules of patients with nuclear or anterior polar cataracts were analyzed quantitatively for alpha-crystallin proteins and mRNAs using Western blot and RT-PCR analysis., respectively. The degree of modification of alpha-crystallins was determined by 2-dimensional gel electrophoresis followed by Western blotting. Higher molecular weight protein bands that were immunoreactive to anti-alphaA- and anti-alphaB-crystallin antibodies around 45 kDa accumulated more in the anterior polar cataract samples than in those with the nuclear type of cataracts. Also monomeric alphaB-crystallins accumulated more in lens epithelial cells of patients with anterior polar cataracts. By comparison, no significant changes were found in the levels of the mRNAs encoding alphaA- and alphaB-crystallins in the different types of cataracts. Both alphaA- and alphaB-crystallin proteins seemed to undergo more extensive modification in anterior polar cataracts. Conclusion. In addition to fibrotic changes, which accompany increased levels of extracellular matrix molecules, accumulation and abnormal modification of alpha-crystallins might be implicated in the pathogenic mechanism of this type of cataract.

Detection of Epidermal Growth Factor Receptor mRNA by in Situ Hybridization in Cataractous Lens Epithelium

In this study, we investigated the correlation between the expression of EGFR-mRNA in situ hybridization method and the clinical feature of the cataract. The results were as followed. Nineteen out of fourty six cases(41.3%) were showed the expression of EGFRmRNA in lens epithelium. No correlation between the age of the lenses and the expression of EGFR-mRNA could be demonstrated. The expression of EGFR-mRNA in lens epithelium correlated significantly (P<0.01) with the clinical stage of the cataract. No correlation between the diabetes mellitus and the expression of EGFRmRNA could be demonstated. These results suggest that the EGFR-mRNA status in lens epithelium could be important for the development of the cataract.

Inhibition of wound healing of lens epithelial monolayer wounds by an applied electric field / 眼科

Objectives To investigate the effects of electric field on the healing of lens epithelial cell wounds. Design Experimental study. Participants The cultured bovine lens epithelial cells and monolayers. Methods Small circle wounds were made by stabbing the cultured bovine lens epithelial monolayers with a glass needle and exposed to an applied electric field at a physiological strength of 200 mV/mm for 2 hours (n=37), and the wounds with no electric field exposure were used as control (n=29). With an image analyzer (Leica), the wound areas at 0, 1 and 2 hours after wounding were measured. Wounds were stained with F-actin fluorescence antibodies to observe the arrangement of microfilament cytoskeleton. Main Outcome Measures The wound areas and the arrangement of microfilament cytoskeleton of wounds. Results Compared to control wounds, the healing of wounds exposed to electric field was reduced, the mean area of field-exposed wounds at 2 hours after wounding (19106?m2?2167?m2) was significantly more than that of control wounds (8555?m2?1911?m2) (t=2.942, P=0.0045). The arrangement of microfilament cytoskeleton in control wounds was centripetally and a purse string-like arrangement of microfilament cytoskeleton was showed in field-exposed wounds. Conclusion Applied electric field at a physiological strength inhibits the healing of lens epithelial wounds.

The Effect of Saponin on the Na+ - K+ Activated ATPase of Cattle Lens Epithelial Cell

The effect of saponin on the Na+ - K+ ATPase of anterior capsule and epithelium of cattle lens has been investigated. The experiment also designed to determine the cation of saponin on the Na+ - K+ ATPase activity in anterior capsule and epithelium of cattle lens. The following results are observed. 1. The Na+ - K+ ATPase activity of cattle lens epithelial cell membrane is inhibited by low concentration of saponin, but increased by high concentration. The strongest activity showed at the dose of 50mg% saponin concentration. 2. The activating effect of saponin on the cattle epithelial cell membrane Na+ - K+ ATPase activity is inhibited by ouabain, but the stimulation of the Mg++ ATPase by high concentration of saponin is not inhibited by ouabain. 3. The activity ratio of Na+ - K+ ATPase by high concentration of saponin is increased by raising the sodium concentration but decreased by raising the potassium concentration. 4. The Na+ - K+ ATPase activity is increased by small amount of calcium but completely inhibited by over 5mM concentration of calcium.