Metabolic memory means if the hyperglycemia can't be controlled at early stage of diabetes, chronic complications such as diabetic retinopathy (DR) will continue to develop even if the blood glucose level maintains normal level at later stage. Oxidative stress plays an important role in the "metabolic memory" of DR, which interacts with the nitrative stress, advanced glycation end products, genetic modification and endoplasmic reticulum stress in the pathogenesis of DR. Further elucidation of the relationship between oxidative stress and "metabolic memory" of DR can open the way for the discovery of novel therapeutic targets to prevent DR progression.
ObjectiveTo predict as well as bioinformatically analyze the target genes of has-miR-451. MethodsmiRBase, miRanda, TargetScan and PicTar were used to predict the target genes of hsa-miRNA-451. The functions of the target genes were demonstrated by Gene Ontology and pathway enrichment analysis. P < 0.05 was set as statistically significant. Results18 target spots of hsa-miRNA-451 were predicted by 3 databases or prediction software at least. The functions of the target genes were enriched in proliferation and development of epithelial cells and regulation of kinase activity (P < 0.05). Pathway analysis showed that transforming growth factor-beta signaling pathway, mitogen-activated protein kinase signaling pathway, epidermal growth factor signaling pathway, Wnt signaling pathway and mammalian target of rapamycin signaling pathway were significantly enriched (P < 0.05). Conclusionhsa-miRNA-451 might be involved in various signaling pathways related to proliferation and development of epithelial cells.
Objective To observe the effect of netrin-1 on retinal Müller cells in diabetes mellitus (DM) rats. Methods Fifty Sprague-Dawley rats were randomly divided into the normal control group (group A), normal + balanced salt solution (BSS) group (group B), normal+netrin-1 group (group C), DM+BSS group (group D) and DM+netrin-1 group (group E), with 10 rats in each group. DM rats were induced by intraperitoneal injection of Streptozotocin (60 mg/kg). The expression level of glial fibrillary acidic protein (GFAP) on retinal Müller cells was determined by immunohistochemistry, the level of GFAP mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Results Immunohistochemistry showed that GFAP was distributed in retinal ganglion cells and retinal nerve fiber layer in group A, B and C. Compared to group B, GFAP staining was brighter in the group D. There were significant differences in the expression of GFAP protein and mRNA among groups A-E (F=203.43, 72.91; P=0.00, 0.00), they were higher in group D than group A (t=?26.01, 22.26; P=0.00, 0.00), and group E (t=?10.78, 3.93; P=0.00, 0.00). They were higher in group E than group A (t=7.00, ?9.82; P=0.00, 0.00). There were no significant differences in between group A and group C (t=?0.29, 0.50; P=0.77, 0.62). Conclusion The expression of GFAP in Müller cells of DM rats could be decreased by injecting netrin-1 into vitreous.
ObjectiveTo observe the expression of glutamate (Glu) andγ-aminobutyric acid (GABA) in the retina of diabetic rats which were intervened later by insulin intensive therapy, and to investigate the mechanism of metabolic memory of hyperglycemia which induced the retina neuropathy in diabetic rats. Methods60 Brown Norway rats were randomly divided into normal control (NC) group, diabetes mellitus (DM) group (6 weeks at DM1, 12 weeks at DM2) and metabolic memory (MM) group, 15 rats in each group. Diabetes was induced by intraperitoneal injection of streptozocin. After 6 weeks, MM group was treated with insulin intensive therapy for 6 weeks. DM1 group was sacrificed at the end of 6 weeks and other groups were sacrificed at the end of 12 weeks. High performance liquid chromatography was used to detect the amount of Glu and GABA in the rat retina. Real-time polymerase chain reaction was applied to quantify the mRNA expressions of Glutamate decarboxylase (GAD). TdT mediated dUTP nick ending labelling was used to detect cell apoptosis. ResultsThe concentration of Glu (t=6.963), GABA (t=4.385) and the ratio of Glu/GABA (t=4.163) in MM group were significantly higher than DM1 group, but the concentration of Glu (t=3.411) and GABA (t=3.709) were significantly lower than DM2 group (P < 0.05). And there was no significant difference in the ratio of Glu/GABA between MM and DM2 groups (t=1.199, P > 0.05). The level of expressions of GAD mRNA in MM group was significantly lower than DM1 group (t=3.496, P < 0.05), but higher than DM2 group (t=8.613, P < 0.05). The number of nerve cells apoptosis in MM group was significantly higher than DM1 group (t=2.584, P < 0.05), but lower than DM2 group (t=3.531, P < 0.05). ConclusionsIntensive therapy later by insulin can partially reduce the content of Glu and GABA and the rate of nerve cells apoptosis, which cannot return to normal levels, and has no effect on the rise in the ratio of Glu/GABA caused by the hyperglycemia. The disorders of Glu and GABA may participate in the metabolic memory of hyperglycemia.
The pathogenesis of diabetic retinopathy (DR) is more complex. For the upstream of traditional pathogenesis, to looking for unifying mechanism theory which proposed in foundation of common promoters and the latest view of DR may be the result of chronic inflammation. Both of them provide the basic and clinical theraby of DR with new direction. Therefore, there are many related issues still needs to intensive study. （Chin J Ocul Fundus Dis，2008，24：237-239）
Objective To observe the effect of different concentration netrin-1 on retinal vascular permeability in diabetes mellitus (DM) rats. Methods Eighty adult Sprague-Dawley rats were randomly divided into 8 groups, 10 rats in each group, including normal control group (group A), normal＋balanced salt solution (BSS) group (group B), normal＋netrin-1 (500 μg/ml) group (group C) and DM group (50 rats in 5 sub-groups). DM rats were induced by intraperitoneal injection of streptozocin. Three months after intraperitoneal injection, 10 DM rats in the control group were injected with BSS (group D). Forty DM rats were injected with 5 μl of different concentrate netrin-1, and were divided into DM+netrin-1 10 μg/ml group (group E), DM+netrin-1 50 μg/ml group (group F), DM＋netrin-1 100 μg/ml group (group G), DM＋netrin-1 500 μg/ml group (group H) according to the different concentration. Non-DM rats in group C were injected with netrin-1 500 μg/ml. The expression of occludin was determined by immunohistochemistry for protein, and by real-time fluorescence quantitative reverse transcription polymerase chain reaction for mRNA level. Retinal vascular permeability was measured by Evans blue infusion. Results The expression of occludin protein and mRNA in group D were less than group A (t=27.71, 8.59;P=0.00, 0.00). However, the retinal vascular permeability increased in group D (t=?42.72,P=0.00). The expression of occluding protein, occludin mRNA and retinal vascular permeability showed significant differences between group D, E, F, G and H (F=146.31, 16.54, 67.77;P=0.00, 0.00, 0.00). Compared the group B with group C, there was no significant differences between the expression of occludin protein, occludin mRNA and the retinal vascular permeability (t=?1.13, 0.93, 1.04;P=0.27, 0.36, 0.31). The concentrate of netrin-1 showed a significant positive correlation to the expression level of occludin and occludin mRNA (r=0.73, 0.81;P=0.00, 0.00), but negative correlation to the vascular permeability (r=?0.61,P=0.00). Conclusion Netrin-1 can reduce the DM rats' retinal vascular permeability, which depended on the concentration of netrin-1.
ObjectiveTo observe the effect of tert-butyl hydroquinone (tBHQ) on type 2 diabetic rats retinal nuclear factor E2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Methods60 Sprague Dawley rats were randomly divided into normal control group (NC group, n=20) and model group (n=40). The rats in model group were intraperitoneal injected with streptozotocin (30 mg/kg) to establishing type 2 diabetic mellitus (DM). There were 35 rats successfully established and they were randomly divided into diabetic group (DM group, 17 rats) and tBHQ group (18 rats). The rats in tBHQ group were fed with high fat and sugar diet with 1% tBHQ. After 4 weeks and 12 weeks of tBHQ intervention, hematoxylin eosin staining of retinal sections, immunohistochemical staining and quantitative polymerase chain reaction (PCR) of Nrf2 and HO-1 were performed. ResultsIn tBHQ control, the retina of rats was normal and individual cells showed slightly edema at 4 weeks; the retinal structure of rats was clear and part of cells showed edema at 12 weeks. At 4 and 12 weeks, the expression of Nrf2 (t=3.115, 3.781) and HO-1 (t=3.485, 3.785) protein in DM group were higher than that in NC group (P < 0.05); the expression of Nrf2 (t=2.473, 2.576) and HO-1 (t=2.785, 2.879) protein in tBHQ group were higher than that in DM group (P < 0.05). In DM group, the expression of Nrf2 protein at 12 weeks was higher than that at 4 weeks (t=0.276, P < 0.05). In tBHQ group, the expression of Nrf2 (t=2.516) and HO-1 (t=2.776) protein at 12 weeks were higher than that at 4 weeks (P < 0.05). 4 and 12 weeks, the expression of Nrf2 (t=4.758, 4.285) and HO-1 (t=5.114, 4.514) mRNA in DM group were higher than that in NC group (P < 0.05); the expression of Nrf2 (t=5.133, 4.976) and HO-1 (t=4.758, 4.251) mRNA in tBHQ group were higher than that in DM group (P < 0.05). In DM gruop, the expression of Nrf2 protein at 12 weeks was higher than that at 4 weeks (t=5.114, P < 0.05). In tBHQ group, the expression of Nrf2 (t=4.292) and HO-1 (t=4.974) protein at 12 weeks were higher than that at 4 weeks (P < 0.05). ConclusiontBHQ intervention can increased the expression of Nrf2, HO-1 significantly in the retina of type 2 diabetic rats.
Objective To observe the morphological changes of dendrite and soma in retinal ganglion cells (RGCs) which subsisted in early diabetic rats. Methods The RGCs of 3-months-course diabetic rats and coeval normal rats were marked by gene gun techniques. To collect RGCs photographs by Leica microscope with Z axis and CCD camera；to observe the changes of diameter, variance of structural features in dendritic field and somata after classification which according to the size and morphology. Thy-1 antibody marks on the retinal RGCs, taking a photograph under fluorescent microscope, counting the changes of retinal RGCs density in early diabetic rat. Results In three-month diabetic rats,the density of retinal RGCs was decreased obviously. Morphological changes of RGCs in the dendritic fields were observed with gene gun technique. There was no severe variation in all kinds of the bole of cell dendrite, in which some only showed crispation partially and sparseness also twisting in the dendritic ramus. The mean diameter of dendritic field and soma in class A of diabetic rats was (401plusmn;86) mu;m, the mean diameter of dendritic field in control group was (315plusmn;72) mu;m,compared with each other, there is statistically significant differences (t=21.249，Plt;0.001); the mean diameter of soma in class A of diabetic rats was (24plusmn;6) mu;m, the mean diameter of soma in control group was (22plusmn;5) mu;m, compared with each other, there is no statistically significant differences (t=0.927，Pgt;0.05); the mean diameter of dendritic field and soma in class B of diabetic rats were (170plusmn;36)、(14plusmn;2) mu;m respectively, in control group were (165plusmn;36)、(16plusmn;2) mu;m, the mean diameter of dendritic field and soma in class C of diabetic group were(265plusmn;78)、(17plusmn;5) mu;m respectively, in control group were (251plusmn;57)、(17plusmn;4) mu;m , compared with each other, there are on statistically significant differences(t=1.357，0.798，0.835，1.104，Pgt;0.05). Conclusions In short-term diabetes, the survived RGCs show good plasticity in adult diabetic rats, especially in class A. The changes of dendrites were more sensitive than the soma, which could be the leading index of the morphologic changes of RGCs in the early stage. The good plasticity showed by the RGCs and the time window from changing in dendrite to cell death provide us many evidences not only for the research but also for the nerve protection in clinic. (Chin J Ocul Fundus Dis,2008,24:249-254)
Objective To observe the effect of high glucose on the expression of activating transcription factor 4 (ATF4) in cultured retinal Muuml;ller glia cells. Methods The retinal tissue of Sprague-Dawley (SD) rats was collected, and Muuml;ller cells were isolated and cultured. The glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) of Muuml;ller cells were identified by streptavidin-biotin-peroxidase complex. Cultured rat Muuml;ller cells were divided into control group (5.5 mmol/L glucose), group A (20 mmol/L glucose), group B (30 mmol/L glucose) and group C (40 mmol/L glucose). ATF4 protein expressions in Muuml;ller cells of four groups were measured by Western blot four days after cultured. Results GFAP and GS expressed in more than 95% of Muuml;ller cells. Over 95% of Muuml;ller cells of group A, B and C were positive for GFAP and GS. Western blots indicated that ATF4 protein in group A, B and C increased obviously compared with the control group (q=0.293, 0.754,0.484;P＜0.05). Conclusion High glucose can increase the expression of ATF4 protein and cause endoplasmic reticulum stress in retinal Muuml;ller glia cells in vitro.
Objective To observe the effects of high concentr at ion glucose on the calcium-activated potassium channel of rabbits′ retinal Müller cells. Methods The rabbits′retinal Müller cells were cultured in vitro under the condition of high concentration glucose, and identified by immunohistochemical staining and transmission electron microscopy. Patch-clamp technique was used to observe the changes of the calcium-activated potassium channel of retinal Müller cells caused by high concentration glucose at different time.Results High concentration glucose could inhibit the calcium-activated potassium channel of cultured retinal Müller cells in a time-dependent manner. Conclusion High concentration glucose may reduce the biological functions of Müller cells by inhibiting calcium-activated potassium channel. (Chin J Ocul Fundus Dis，2003，19：164-167)