The inhibitory effect of FoxF2 shRNA on the expression of extracellular matrix of human trabecular meshwork / 中华实验眼科杂志

Objective To explore the role of forkhead box F2 (FoxF2) in the extracellular matrix of trabecular meshwork.Methods The cultured human trabecular meshwork cells (HTMCs) were divided into Scramble control group and FoxF2 small hairpin RNA (shRNA) group,then FoxF2 shRNA,the FoxF2 restructuring interference carrier was built,HTMCs were infected with FoxF2 shRNA lentivirus.Western blot assay was used to detect the expression of FoxF2 protein and extracellular matrix.Furthermore,Transwell counting experiment was used to analyze the migration ability of HTMCs.Results The cultured HTMCs grew well and showed a long spindle shape.The growth status of HTMCs was well,and their morphological characteristics were consistent with the HTMCs in vivo.The relative expression level of FoxF2 protein in the FoxF2 shRNA group was lower than that in the Scramble control group,with a significant difference between them (0.72 ± 0.02 vs.1.27 ± 0.05;t =16.68,P ＜ 0.01).The relative expression level of fibronectin (FN),collagen type Ⅰ (COL Ⅰ) and α-smooth muscle actin (α-SMA) were 0.43±0.03,0.53 ±0.08 and O.86±0.15 in the FoxF2 shRNA group,and 0.87±0.04,1.66±0.06 and 1.73 ±0.13 in the Scramble control group,respectively,the relative expression levels of FN,COL Ⅰ and α-SMA in the FoxF2 shRNA group were significantly lower than those in the Scramble control group (t =15.08,18.81,7.50,all at P＜0.01).The migration number of HTMCs in the FoxF2 shRNA group was significantly lower than that in the Scramble control group (117.30±11.41 vs.251.00±10.37;t =8.72,P＜0.01).Conclusions The FoxF2 shRNA lentivirus are successfully constructed,which can decrease the expression of FoxF2 in HTMCs.Low expression of FoxF2 can reduce the expression level of extracellular matrix protein in HTMCs and inhibit the migration ability of HTMCs.

Effects of Krüppel-like factor 6 overexpression towards apoptosis of human lens epithelial cells with ultra violetradiation B treatment / 中华实验眼科杂志

Objective To investigate the effect of the overexpression of Krüppel-like factor 6 (KLF6)towards the apoptosis of human lens epithelial cells (HLECs) induced by ultraviolet B (UVB) radiation.Methods The eukaryotic expression plasmid pEGFP-C2-KLF6 which was successfully constructed were transfected into HLECs,followed by the detection of KLF6 level by using Western blot,and then companied by UVB stimulation.Cell viability was measured by methyl thiazolyl tetrazolium (MTT) assay.The morphology of the cells was observed by using hematoxylin-eosin staining method.The cell damage was examined by Live/Dead staining.The apoptotic markers bax and bcl-2 were detected by Western blot.Quantitative apoptotic levels were measured with the apoptosis detection kit;the expression level of reactive oxygen species (ROS) was analyzed by DCFH-DA probe.Results The cell viability of the 0.5 μg transfection group and the 1.0 μg transfection group was significantly lower than that of the blank vector control group (both at P＜0.05).In high KLF6 expression group,the cells were sparse,long and narrow in size and shape,and the cytoplasm was concentrated.The cells in the normal control group were green living cells with stable morphology and even quantity.The number of red dead cells was increased significantly in the KLF6 highexpression group.After UVB irradiation,the apoptosis value,relative bax expression,bax/bcl-2 ratio and ROS expression of HLECs cells in the KLF6 high-expression group were all higher than those in the blank vector control group,with statistically significant differences between them (all at P＜0.05).Conclusions Overexpression of KLF6 can exacerbate apoptosis of HLECs caused by UVB,by regulating the expression of apoptosis-related proteins and promoting the accumulation of ROS in the endoplasmic reticulum.Down-regulation of KLF6 expression by biological tools may play a protective role on LECs to a certain extent.

Regulation of Krüppel-like factor 6 via activating transcription factor 4 pathway to apoptosis of human lens epithelial cells / 中华实验眼科杂志

Objective To investigate the regulating effects of Krüppel-like factor 6 (KLF6) on the apoptosis of human lens epithelial cells (HLECs) by activating transcription factor 4 (ATF4) pathway and explore the bio-molecular mechanism of KLF6/ATF4-induced HLECs apoptosis.Methods HLECs (HLE-B3) were cultured using high glucose DMEM medium.The eukaryotic expression plasmid pEGFP-C2-ATF4 was transfected into the cells by liposome 2000 in the ATF4-transfected group,and pEGFP-C2 was transfected in the empty plasmid group.Then the cells were exposed to 20 mJ/cm2 ultraviolet ray B (UVB) for 200 seconds,The morphological changes of the cells were observed by hematoxylin & eosin staining and Hoechst33258 fluorescein staining.Cultured cells were transfected using pEGFP-C2-KLF6 and pEGFP-C2 plasmid and pSilencer-KLF6 (siKLF6) and pSilencer plasmid,respectively,and the expression of ATF4 protein in the cells was detected by Western blot assay.Culture cells were divided into four groups.pEGFP-C2 and pSilencer plasmids were co-transfected into the cells in the empty plasmid group;pEGFP-C2-KLF6 and pSilencer empty plasmid were co-transfected into the cells of the KLF6 + pSilencer group;pEGFP-C2 empty plasmid and pSilencer-ATF4 were co-transfected in the cells of the siATF4 + pEGFP-C2 group;pEGFP-C2-KLF6 and pSilencer-ATF4 plasmids were co-transfected in the cells of the KLF6 + siATF4 group,and then the cells were exposed to UVB.The apoptosis of the cells were detected by ELISA assay.Results Cultured cells grew well in the normal control group with the uniform morphology and regular arrangement.The karyopyknosis,karyorrhexis and enlargement of intercellular space were found in the cells exposed to UVB.In the ATF4 transfected group,the number of cells was decreased.The relative expression level of the ATF4 protein in the cells was 0.99±0.06 and 0.13±0.02 in the UVB+ATF4 transfected group and UVB+pEGFP-C2 plasmid group,respectively,with a significant difference between them (t =23.13,P＜0.01).The relative expression levels of KLF6 and ATF4 proteins in the KLF6 transfected group were higher than those in the empty plasmid group,and the relative expression levels of KLF6 and ATF4 proteins in the siKLF6 group were significantly lower than those in the empty plasmid group (all at P＜0.01).ELISA assay showed that the apoptotic rate in the ATF4 transfected group was 1.37± 0.11,which was significantly higher than 0.31 ±0.11 in the normal control group (t =8.034,P =0.001);the apoptotic rate of the cells was increased in the KLF6+pSilencer group and decreased in the siATF4+pEGFP-C2 group in comparison with the empty plasmid group (P＜0.01,P=0.02).In addition,the apoptotic rate in the KLF6+ siATF4 group was remarkably lower than that in the KLF6 + pSilencer group (P＜ 0.01).Conclusions KLF6 promotes the apoptosis of HLECs induced by UVB radiation.Silence of ATF4 gene reduces the apoptotic rate of the cells.ATF4 is probably a target factor in the regulating oathwav of KLF6 to apoptosis.

Effect of dl-3-n-Butylphthalide on apoptosis of retinal müller cells induced by hydrogen peroxide / 中华眼底病杂志

Objective To observe the protective effect of dl-3-n-Butylphthalide (NBP) on apoptosis of retinal Müller cells induced by hydrogen peroxide (H2O2).Methods Human retinal Müller cells cultured in vitro were divided into normal control group,model group (H2O2 group) and experimental group (H2O2+NBP group).The cells in the H2O2 group and H2O2+NBP group were cultured with 200 μ mol/L H2O2 for 2 h.Then the culture solution of the H2O2 group replace with complete medium and the H2O2+NBP group replace with complete medium containing 1 tmol/L NBP.The normal control group was a conventional cultured cells.Müller cells were identified by immunofluorescence staining.Hematoxylin-eosin (HE) staining was used to observe the apoptosis morphological changes.MTT assay was used to detect the activity of of retinal Müller cells after after 24 h and 48 h of NBP intervention.Hoechst33258 staining was used to observe the apoptosis.LIVE/DEAD (R)cell activity/cytotoxicity kit was used to detect cell viability.Dichlorofluorescein diacetate (DCFH-DA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) double staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells.One-way ANOVA combined with Dunnett statistical method were used for data analysis.Results HE staining showed that the number of cells in H2O2+NBP group was higher than that in H2O2 group.MTT assay showed that after 24 h and 48 h of NBP intervention,the differences in cell viability between the normal control group and the H2O2 group,the H2O2 group and the H2O2+NBP group were statistically significant (t=28.96,3.658,47.58,20.33;P＜0.001,0.022).The results of Hoechst33258 showed that the nuclear nucleus of a few cells in the H2O2+NBP group was crescent-shaped and the nuclear fragmentation was reduced,and the blue fluorescence of the remaining cells was uniform.The LIVE/DEAD ~ cell activity/cytotoxicity kit showed that the number of dead cells with red fluorescence in the H2O2 group increased significantly,and the number of viable cells with green fluorescence decreased significantly.In the H2O2+NBP group,the number of viable cells with green fluorescence increased,and the number of dead cells with red fluorescence decreased.The double staining results of DCFH-DA+ER-Tracker Red showed that the green fluorescence intensity of H2O2 group was significantly enhanced;the green fluorescence intensity of H2O2+NBP group was lower than that of H2O2 group.Conclusion NBP alleviates H2O2-induced apoptosis of human retinal Müller cells by inhibiting ROS production.

The synergistic effect of metformin and anti-vascular endothelial growth factor in the treatment of diabetic retinopathy / 中华眼底病杂志

Objective To observe the synergistic effect of metformin and anti-vascular endothelial growth factor (VEGF) in the treatment of diabetic retinopathy.Methods This study was composed of clinical data review and in vitro cell experiment.Ten patients (12 eyes) with diabetic macular edema treated with antiVEGF drugs were included in the study.Patients were randomly divided into the VEGF group (anti-VEGF drug therapy) and the combined treatment group (anti-VEGF drug combined with metformin).The changes of visual acuity and central retinal thickness (CRT) were compared between the two groups.As far as the in vitro experiment was concerned,vascular endothelial cells were divided into the control group (normal cells),the VEGF group (50 ng/ml VEGF),the anti-VEGF group (50 ng/ml VEGF+2.5 μg/ml of conbercept),and the combined group (50 ng/ml VEGF +2.5 μg/ml of conbercept +2.0 mmol/L of metforrnin).And then MTT cell viability assay,scratch assay and real-time quantitative polymerase chain reaction assay were performed to analyze the cell viability,cell migration and mRNA level of VEGFR2,protein kinase C (PKC)-α and PKC-β successively.Results Review of clinical trial shows that the CRT recovery rates in the combined treatment group were much higher than that in the VEGF group at 3 month after the operation,while the difference was statistically significant (t=-2.462,P＜0.05).In vitro cell experiment results showed that VEGF induction upregulated the viability and mobility of vascular endothelial cells obviously compared with control group,at the same time,the use of anti VEGF drugs can effectively reverse the trend,in contrast,combination of metformin and anti-VEGF showed a more superior effect to some extent (P＜0.05).In the VEGF group,the mRNA expression of VEGFR2,PKC-αand PKC-[β were significantly increased compared with the control group (P＜ 0.01);while the mRNA expression of VEGFR2,PKC-αand PKC-β in the combination group decreased significantly compared with the VEGF group and the control group (P＜0.05).However,in the anti-VEGF group,the mRNA expression of VEGFR2,PKC-αand PKC-β were decreased,but has failed to reach the level of statistical learn the difference.Conclusions The combination ofmetformin and anti-VEGF drugs can reduce the CRT of diabetic retinopathy patients and inhibit the proliferation and migration of retinal vascular endothelial cells which induced by VEGF.The synergistic mechanism may be related to the inhibitory effect of metformin on the expression of VEGFR and PKC.

RNA-Seq analysis of gene expression profiling in retinal vascular endothelial cells under high glucose condition / 中华眼底病杂志

Objective To observe RNA-Seq analysis ofgene expression profiling in retinal vascular endothelial cells after anti-vascular endothecial growth factor (VEGF) treatment.Methods Retinal vascular endothelial cells were cultured in vitro,and the logarithmic growth phase cells were used for experiments.The cells were divided into the control group and high glucose group.The cells of two groups were cultured for 5 hours with 5,25 mmol/L glucose,respectively.And then,whole transcriptome sequencing approach was applied to the above two groups of cells through RNA-Seq.Now with biological big data obtained as a basis,to analyze the differentially expressed genes (DEGs).And through enrichment analysis to explain the differential functions of DEGs and their signal pathways.Results The gene expression profiles of the two groups of cells were obtained.Through analysis,449 DEGs were found,including 297 upregulated and 152 downregulated ones.The functions of DEGs were influenced by regulations over molecular biological process,cellular energy metabolism and protein synthesis,etc.Among these genes,ITGB 1BP2,NCF 1 and UNC5C were related to production of inflammation;AKR1C4,ATP 1A3,CHST5,LCTL were related to energy metabolism of cells;DAB 1 and PRSS55 were related to protein synthesis;SMAD9 and BMP4 were related to the metabolism of extracellular matrix.GO enrichment analysis showed that DEGs mainly act in three ways:regulating biological behavior,organizing cellular component and performing molecular function,which were mainly concentrated in the system generation of biological process part and regulation of multicellular organisms.Pathway enrichment analysis showed that gene expressions of the two cell groups were differentiated in transforming growth factor-β (TGF-β) signaling pathway,complement pathway and amino acid metabolism-related pathways have also been affected,such as tryptophan,serine and cyanide.Among them,leukocyte inhibitory factor 9 and bone morphogenetic protein 4 play a role through the TGF-β signaling pathway.Conclusions High glucose affects the function of retinal vascular endothelial cells by destroying transmembrane conduction of retinal vascular endothelial cells,metabolism of extracellular matrix,and transcription and translation of proteins.

The effect of polypyrimidine tract binding protein-associated splicing factor on hydrogen peroxide induced apoptosis of retinal pigment epithelial / 中华眼底病杂志

Objective To observe the effect ofpolypyramidine tract binding protein-associated splicing factor (PSF) on hydrogen peroxide (H2O2) induced apoptosis of retinal pigment epithelial (RPE) cells in vitro.Methods RPE cells were cultured and divided into a normal group,normal+H2O2 group,Vec+H2O2 group,PSF+H2O2 group according to the experimental design.Overexpression of PSF in RPE cells were achieved by pEGFP-PSF plasmid transient transfection into RPE cells,then RPE cells were exposed to H2O2.The morphological changes were observed by hematoxylin-eosin (HE) staining and Live/Dead staining while the survival rate of cells was detected by MTT assay.The effect of PSF on H2O2-induced RPE apoptosis was analyzed by Cell Death Detection ELISA kit.Meanwhile,intracellular reactive oxygen species (ROS) level was detected by using DCFH-DA method.Results Overexpression of PSF could effectively alleviate the morphological changes induced by H2O2 stimulation shown by HE staining,and effectively reduce dead cells number shown by Live/Dead staining.After H2O2 stimulation,the survival rate,apoptosis rate and ROS production level in PSF overexpression group were 0.68± 0.12,0.44± 0.08 and 18 616± 3 382.54 respectively,showing significant difference in comparison with the vector plasmid group and normal group (P＜0.05).Conclusion PSF overexpression plays a protective role in H2O2-induced apoptosis by inhibiting the production of ROS in RPE cells.