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 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.
ObjectiveTo investigate the effects and mechanisms of G protein-coupled receptor 91 (GPR91) on blood-retinal barrier (BRB) in diabetic rats. MethodsA lentiviral vector of shRNA targeting rat GPR91 and scrambled shRNA were constructed. Healthy male Sprague-Dawley (SD) rats were selected in this study. The 60 rats were randomized into 4 groups and treated as follows:(1) control group (Group A, n=15), the rats received injections of an equal volume of 0.1% citrate buffer; (2) streptozocin (STZ) group (Group B, n=15), the rats received injections of STZ; (3) LV.shScrambled group (Group C, n=15), diabetic rats received an intravitreal injection of 1 μl 1×108 TU/ml scrambled shRNA lentiviral particles at 2 weeks after the induction of diabetes; (4) LV.shGPR91 group (Group D, n=15), diabetic rats received an intravitreal injection of 1 μl 1×108 TU/ml pGCSIL-GFP-shGPR91 lentiviral particles. At 12 weeks after intravitreal injection, immunohistochemistry and Western blot were used to assess the expression of GPR91, p-extracellular signal-regulated kinase(ERK)1/2, t-ERK1/2, p-Jun N-terminal kinase (JNK), t-JNK, p-p38 mitogen-activated protein kinase (MAPK) and t-p38 MAPK. Haematoxylin and eosin (HE) staining and Evans blue dye were used to assess the structure and function of the retinal vessel. Immunohistochemistry enzyme-linked immunosorbent assay (ELISA) was used to test the protein level of VEGF. ResultsImmunohistochemistry staining showed that GPR91 was predominantly localized to the cell bodies of the ganglion cell layer. Western blot showed that GPR91 expression in Group D decreased significantly compared with Group C (F=39.31, P < 0.01). HE staining showed that the retina tissue in Group B and C developed telangiectatic vessels in the inner layer of retina, while the telangiectatic vessels attenuated in Group D. It was also demonstrated in Evans blue dye that the microvascular leakage in Group D decreased by (33.8±4.11)% compared with Group C and there was significant difference (F=30.35, P < 0.05). The results of ELISA showed the VEGF secretion of Group B and C increased compared with Group A and the VEGF expression in Group D was significantly down regulated after silencing GPR91 gene (F=253.15, P < 0.05).The results of Western blot indicated that compared with Group A, the expressions of p-ERK1/2, p-JNK and p-p38 MAPK were significantly upregulated (q=6.38, 2.94, 3.45;P < 0.05). Meanwhile, the activation of ERK1/2 was inhibited by GPR91 shRNA and the difference was statistically significant (F=22.50, P < 0.05). ConclusionsThe intravitreal injection of GPR91 shRNA attenuated the leakage of BRB in diabetic rats. GPR91 regulated the VEGF release and the leakage of BRB possibly through the ERK1/2 signaling pathway.
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 observe the effect of interleukin (IL) 10 modified endothelial progenitor cells (EPC) in diabetic retinopathy (DR). MethodsEPC cells were collected and cultivated from the bone marrow of rats and identified by immuno-fluorescence staining. EPC cells were infected with lentivirus (LV) of EPC-LV-IL10-GFP (EPC-LV-IL10-GFP group) or EPC-LV-NC-GFP (GFP group). EPC cells without lentivirus infection was the EPC group. Enzyme-linked immuno sorbent assay (ELISA) was used to measure the concentrations of tumor necrosis factor (TNF)-α, IL10, IL8 and vascular endothelial growth factor (VEGF) in the supernatant of these three groups. 168 male Wistar rats were divided into normal control group (28 rats), diabetes mellitus (DM) group (28 rats), DM-blank control group (56 rats) and DM-intervention group (56 rats). DM was introduced in the latter 3 groups by streptozotocin intravenous injection. Three months later, the rats in the DM-blank control group and DM-intervention group were injected with EPC-LV-NC-GFP or EPC-LV-IL10-GFP by tail vein, respectively. Immunohistochemistry was used to observe the GFP expression in rat retinas. The blood-retinal barrier breakdown was detected by Evans blue (EB) dye. The retinal histopathologic changes were observed by transmission electron microscope. The mRNA level of VEGF, matrix metallproteinases-9 (MMP-9), angiopoietin-1 (Ang-1), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in retina were measured by reverse transcription-polymerase chain reaction (RT-PCR). ResultsELISA showed that the levels of TNF-αand IL8 in the supernatant significantly decreased, while the levels of IL10 and VEGF increased (P < 0.05) in EPC-LV-IL10-GFP group. GFP expressed in the retina of blank control group and intervention group, mainly in the ganglion cell layer, inner nuclear layer and outer plexiform layer. The retinal blood vessel pathological change and EB permeability significantly decreased in intervention group compared with DM group (P < 0.05), and blank control group (P < 0.05). RT-PCR revealed that the mRNA level of VEGF, MMP-9 and Ang-1 significantly increased, and eNOS decreased in DM group compared to the normal control group (P < 0.05). The mRNA level of VEGF and iNOS decreased, eNOS increased while Ang-1 and MMP-9 had not changed in DM-blank control group and DM-intervention group compared with DM group (P < 0.05). ConclusionsIL10 modified EPC can improve the inflammative microenvironment and suppressed the pathogenesis of DR. Furthermore, EPC transplantation can increase the number of EPC and exerted their effect.
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.
Objective Methods Ninety male Wister rats were randomly divided into normal control group, diabetic group and FTY720 group, thirty rats in each group. Diabetes was induced by giving a single intraperitoneal injection of streptozocin. FTY720 group was administered with FTY720 at a dose of 0.3 mg/kg by oral gavage daily for 3 months after establishment of diabetes. All rats were used for experiments following intervention for 3 months in FTY720 group. Immunohistochemical staining was used to observe the expression and distribution of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1), and the positive cells were counted. Real-time reverse transcription PCR was used to measure mRNA expression of ICAM-1 and VCAM-1. Fluorescein isothiocyanate-Concanavalin A perfusion was used to detect retinal leukocytes adhesion. Evans blue (EB) perfusion was used to analyze retinal vascular permeability. Immunofluorescence staining was used to detect retinal inflammatory cells infiltration. Results In diabetic group, both ICAM-1(t=12.81) and VCAM-1 (t=11.75) positive cells as well as their mRNA expression (t=16.14, 9.59) were increased compared with normal control group, with statistical significance (P < 0.05). In FTY720 group, both ICAM-1(t=-9.93) and VCAM-1 (t=-6.61) positive cells as well as their mRNA expression (t=-15.28, -6.10) were decreased compared with diabetic group, with statistical significance (P < 0.05). Retinal leukocytes adhesion (t=16.32) and EB permeability (t=17.83) were increased in diabetic group compared with normal control group, while they were decreased in FTY720 group compared with diabetic group(t=-9.93, -11.82),with statistical significance (P < 0.05). There were many CD45 positive leukocytes infiltration in retina of diabetic group, including CD11b positive macrophage/activated microglia, while both of them were little in FTY720 group. Conclusions FTY720 can decrease retinal leukocytes adhesion, reduce retinal vascular permeability and inflammatory cells infiltration, which is associated with down-regulation of ICAM-1 and VCAM-1.
ObjectiveTo observe the protective effect of Zhicao Tea Mixture on Müller cells and the expression of inflammatory factors in mice with diabetic retinopathy.MethodsSeventy-five C57BL/6J mice were randomly divided into the normal control group, diabetes mellitus (DM) group, low concentrations group, medium concentrations group and high concentrations group, with 16 mice in each group. The diabetes model of mice in all groups except the normal control group were established by intraperitoneal injection of STZ (60 mg/kg). Four weeks after the successful modeling, the Zhicao Tea Mixture with low (30 ml/kg), medium (60 ml/kg) and high concentrations (120 ml/kg) were respectively administered by gavage. Weight and blood glucose of mice in each group were measured every two weeks. After 8 weeks, Western blot method was used to detect the mice retina Müller cells activation marker gelatinous fibrous acidic protein (GFAP). Immunofluorescence was performed to detect the expression GFAP and glutamine synthetase (GS). Real-time quantitative PCR (RT-qPCR) and ELISA were used to determine the mRNA and protein expression levels of mouse retinal VEGF, TNF-α, IL-1β and IL-6 respectively.ResultsThe weight of mice in the DM group was lower than that of the normal control group, and the blood glucose was increased. Zhicao Tea Mixture had no effect on the weight of DM mice, but had a significant hypoglycemic effect. The GFAP expression (t=38.318, P＜0.001) in the retina of mice in the DM group was increased and GS expression (t=29.737, P＜0.001) was decreased compared with the control group. The GFAP expression (t=13.677, 19.387, 16.305; P＜0.05) in the retina of mice in the low, medium and high concentrations group were decreased and GS expression (t=5.170, 19.399, 6.705; P＜0.05) were increased compared with the DM group. The expressions of retinal inflammatory factors VEGF, TNF-α, IL-1β and IL-6 in DM group all increased, while the expressions of the above-mentioned inflammatory factors in the retina of mice decreased in the low, medium and high concentrations group.ConclusionZhicao Tea Mixture can decrease the blood glucose of DM mice and reduces the diabetic retinal inflammatory response.
Purpose To investigate the status of proliferation and activation of vascular endothelial cells in preretinal neovascular membranes from patients with insulin dependent diabetetes mellitus(IDDM)by means of immunohistochemical techniques. Methods Status of vascular endothelial cells in 18 preretinal neovascular membranes from 18 patients with IDDM was studied by double-immunofluorescence technique and the alkaline phosphataes-anti-alkaline phosphatase(APAAP)technique and compared the findings with the main clinical features of the patients. Results Of 18 vascularized membranes,16(88.9%)contained proliferating endothelial cells (positive for proliferating vascular endothelial cell marker EN 7/44) and 14 (77.8%) were positive for endothelial cell activation marker anti-VCAM-1;furthermore,by using a double staining technique,we found that in 14 of the 16 cases(87.5%) the proliferating vascular endothelial cells were activated (expressing VCAM-1). Conclusion The proliferation and activation of the vascular endothelial cells of the newly formed vessels in preretinal neovascular membranes suggests the significance of the vascular endothelial cells in the pathophysiology and the progress of proliferative diabetic retinopathy. (Chin J Ocul Fundus Dis,1998,14:141-143)
Objective To explore the relationship between the diabetic retinopathy (DR) and the changes of erythrocyte deformability(ED),erythrocyte membrane phospholipid and spectrin. Methods One hundred and eight patients with non-insulin dependent diabetes mellitus were divided into DR group(55 cases)and nonDR(NDR)group(53 cases).The changes of erythrocyte filtration index(EFI),erythrocyte membrane phospholipid and spectrin dimers(SP-D)and spectrin tetramers (SP-T)were measured in patients of DR and NDR groups and compared with the results of 53 cases of normal control group. Results The EFI,SP-D, SP-D/SP-T,sphingomyelin (SM) /phophatidylcholine(PC)were higher,and SPT,SM,PC,phophatidylserine(PS)and phatidylethanolamine(PE)were lower in patients with DR than those in control and NDR patients (F=8.467～18.925,q=6.845～12.627,Plt;0.001).The changes of all indicators in proliferative DR(PDR) patients were more obvious than those in background DR(BDR) patients(t=5,825-15.443,Plt;0.001).The EFI in DR patients was positively correlated to SM/PC,SP-D and SP-D/SP-T(Plt;0.01)，negatively correlated to SM,PC,PE,PS and SP-T(Plt;0.01). Conclusions The decrease of ED caused by the abnormalities of erythrocyte membrane phospholipid and spectrin might participate in the occurance and development of DR,and correlated to the degree of pathologic changes. (Chin J Ocul Fundus Dis, 1999, 15: 160-162)