ABSTRACT
Objective To explore repressive effects of transthyretitin (TTR) on the growth of human retinal endothelial cells (hREC) under high glucose and hypoxia environment. Methods hRECs were divided into 8 groups, including normal glucose group (5.5 mmol/L glucose), hypoxia group, high glucose group (25.0 mmol/L glucose), high glucose and hypoxia group, normal glucose group+TTR, normal glucose and hypoxia group+TTR, high glucose group+TTR, high glucose and hypoxia group+TTR. Flow cytometry was used to analyze cellular apoptosis. The expression level of Akt, p-Akt, eNOS, Bcl-2 and Bax protein were measured by Western blot. Results Hypoxia could induce apoptosis as the apoptosis rate of normal and hypoxia group was higher than normal group (χ2=25.360, P<0.05), high glucose and hypoxia group was higher that high glucose group (χ2=17.400, P<0.05). The cell apoptosis rate of high glucose and hypoxia group+TTR were increased significantly as compared with high glucose and hypoxia group (χ2=9.900, P<0.05). There was no statistically significant difference on the cell apoptosis rate between normal group and high glucose group, normal group+TTR and normal group, high glucose group+TTR and high glucose group, normal and hypoxia group+TTR and normal and hypoxia group (P>0.05). Western blot showed that the expression of Akt did not change significantly in all eight groups(F=2.450, P>0.05). Compared to normal group, the expression of p-Akt, eNOS, Bcl-2 in normal and hypoxia group were decreased (t=9.406, 5.306, 4.819), and the expression of Bax (t=-4.503) was increased (P<0.05). Compared to high glucose group, same trend was found in high glucose and hypoxia group (t=8.877, 7.723, 6.500, -14.646; P<0.05). The expression of p-Akt in normal and hypoxia group+TTR was higher than normal and hypoxia group (t=-5.024, P<0.05) ,but there was no difference on the expression of eNOS, Bcl-2, Bax between these two groups (t=-2.235, -2.656, -0.272;P>0.05). Compared to high glucose and hypoxia group, the expression of p-Akt and Bcl-2 in high glucose and hypoxia group+TTR were decreased (t=4.355, 4.308; P<0.05), the expression of Bax was increased (t=-4.311, P<0.05), and there was no difference on the expression of eNOS between these two groups (t=-1.590, P>0.05). There was no statistically significant difference in the expression of p-Akt, eNOS, Bcl-2, Bax between high glucose group and normal group (t=-3.407, -4.228, -4.302, -2.076; P>0.05), normal group+TTR and normal group (t=-4.245, -4.298, -2.816, -1.326; P>0.05), high glucose group+TTR and high glucose group (t=4.016, -0.784, 0.707, -0.328; P>0.05). Conclusion Under high glucose and hypoxia, transthyretitin suppress the growth of hREC through Akt/Bcl-2/Bax, but not Akt/eNOS signaling pathway.
ABSTRACT
Objective To investigate the effect of arginase (Arg) inhibitor N-ω-Hydroxy-L norArginine (nor-NOHA) on high glucose cultured rhesus macaque retinal vascular endothelial cell line (RF/6A) in vitro.Methods The RF/6A cells were divided into the following 4 groups:normal control group (5.0 mmol/L of glucose,group A),high glucose group (25.0 mmol/L,group B),high glucose with 125 mg/L nor-NOHA group (group C),and high glucose with 1％ DMSO group (group D).The proliferation,migration ability and angiogenic ability of RF/6A cells were measured by Methyl thiazolyl tetrazolium (MTT),transwell chamber and tube assay respectively.The express of Arg Ⅰ,eNOS,iNOS mRNA of RF/6A cells were measured by real-time polymerase chain reaction (RT-PCR),Enzyme-linked immuno sorbent assay (ELISA) was used to detect the expression of NO and interleukine (IL)-1b of RF/6A cells.Results The proliferation,migration,and tube formation ability of group A (t=2.367,5.633,7.045;P＜0.05) and group C (t=5.260,6.952,8.875;P＜0.05)were significantly higher than group B.RT-PCR results showed the Arg Ⅰ and iNOS expression in group B was higher than that in group A (t=6.836,3.342;P＜0.05) and group C (t=4.904,7.192;P＜0.05).The eNOS expression in group B was lower than that in group A and group C (t=4.165,6.594;P＜0.05).ELISA results showed NO expression in group B was lower than that in group A and group C (t=4.925,5.368;P＜0.05).IL-1b expression in group B was higher than that in group A and group C (t=5.032,7.792;P＜0.05).Conclusions Nor-NOHA has a protective effect on cultured RF/6A cells in vitro and can enhance its proliferation,migration and tube formation.The mechanism may be inhibiting the oxidative stress by balancing the expression of Arg/NOS.
ABSTRACT
Objective To explore repressive effects of transthyretitin (TTR) on the growth of human retinal endothelial cells (hREC) under high glucose and hypoxia environment. Methods hRECs were divided into 8 groups, including normal glucose group (5.5 mmol/L glucose), hypoxia group, high glucose group (25.0 mmol/L glucose), high glucose and hypoxia group, normal glucose group+TTR, normal glucose and hypoxia group+TTR, high glucose group+TTR, high glucose and hypoxia group+TTR. Flow cytometry was used to analyze cellular apoptosis. The expression level of Akt, p-Akt, eNOS, Bcl-2 and Bax protein were measured by Western blot. Results Hypoxia could induce apoptosis as the apoptosis rate of normal and hypoxia group was higher than normal group (χ2=25.360, P<0.05), high glucose and hypoxia group was higher that high glucose group (χ2=17.400, P<0.05). The cell apoptosis rate of high glucose and hypoxia group+TTR were increased significantly as compared with high glucose and hypoxia group (χ2=9.900, P<0.05). There was no statistically significant difference on the cell apoptosis rate between normal group and high glucose group, normal group+TTR and normal group, high glucose group+TTR and high glucose group, normal and hypoxia group+TTR and normal and hypoxia group (P>0.05). Western blot showed that the expression of Akt did not change significantly in all eight groups(F=2.450, P>0.05). Compared to normal group, the expression of p-Akt, eNOS, Bcl-2 in normal and hypoxia group were decreased (t=9.406, 5.306, 4.819), and the expression of Bax (t=-4.503) was increased (P<0.05). Compared to high glucose group, same trend was found in high glucose and hypoxia group (t=8.877, 7.723, 6.500, -14.646; P<0.05). The expression of p-Akt in normal and hypoxia group+TTR was higher than normal and hypoxia group (t=-5.024, P<0.05) ,but there was no difference on the expression of eNOS, Bcl-2, Bax between these two groups (t=-2.235, -2.656, -0.272;P>0.05). Compared to high glucose and hypoxia group, the expression of p-Akt and Bcl-2 in high glucose and hypoxia group+TTR were decreased (t=4.355, 4.308; P<0.05), the expression of Bax was increased (t=-4.311, P<0.05), and there was no difference on the expression of eNOS between these two groups (t=-1.590, P>0.05). There was no statistically significant difference in the expression of p-Akt, eNOS, Bcl-2, Bax between high glucose group and normal group (t=-3.407, -4.228, -4.302, -2.076; P>0.05), normal group+TTR and normal group (t=-4.245, -4.298, -2.816, -1.326; P>0.05), high glucose group+TTR and high glucose group (t=4.016, -0.784, 0.707, -0.328; P>0.05). Conclusion Under high glucose and hypoxia, transthyretitin suppress the growth of hREC through Akt/Bcl-2/Bax, but not Akt/eNOS signaling pathway.
ABSTRACT
Objective To explore transthyretin (TTR) effect on retinal vascular endothelial cells (hREC) under high glucose and hypoxia environment.Methods hREC and human retinal pigment epithelial cell (hRPEC) were cultured at low-glucose (LG),high glucose (HG) and hypoxia.The glucose concentration was increased from 5.5 mmol/L up to 25 mmol/L,and hypoxia was induced by 200 μmol/L CoCl2.The cells were divided into LG group,LG-hypoxia group,HG group,HG-hypoxia group according to the different cell culture environment.The growth index was detected at 0,4,8,16,24,36,48,60,72 hours after cultured.Furthermore,hREC and hRPEC were also cultured with additional TTR (4 μmol/L),respectively.Then transwell co-culture system was employed to reveal the effects of hRPEC on the growth of hREC.Results At 72 hours after cultured,the growth index of hREC and hRPEC in LG group were increased as compared with LG-hypoxia group and HG group (hREC:F=17.098,22.970;P＜0.05.hRPEC:F=45.442,9.011;P＜0.05);the growth index of hREC and hRPEC were decreased in HG group and HG-hypoxia group (hREC:F=146.184,P＜0.05;hRPEC:F=27.907,P＜0.05).Additionally,hREC could be significantly repressed by added TTR during culture with high concentration of glucose (F=161.430,24.106;P＜0.05).hREC could be significantly increased by added TTR during culture with low concentration of glucose (F =200.486,48.662;P ＜ 0.05).In co-culture process,hRPEC revealed inhibition activity against hREC under both natural and abnormal environment (LG group:F=15.711,P＜ 0.05;LG-hypoxia group:F =45.659,P＜0.05;HG group:F =7.857,P ＜0.05;HG-hypoxia group:F=6.348,P＜0.05).Conclusion Under high glucose and hypoxia environment,the growth of hREC from neovascular could be inhibited by TTR.