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Diabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus, with capillary occlusion, microaneurysms, and neovascularization as main pathological features, and its pathogenesis has not been fully understood.The existing clinical treatment can not cure DR, therefore, the study of the pathogenesis of DR is still a hot topic and difficult problem.Current studies have suggested that oxidative stress response is one of the important mechanisms for the development of DR, which promotes the production of excessive reactive oxygen species to induce retinal cell dysfunction and apoptosis.Imbalance of levels of nitric oxide (NO) from different types of sources in the retina under hyperglycemic stimulation also plays an important role in pathological changes, such as capillary damage, blood-retinal barrier disruption and neovascularization in DR.At the same time, NO interacts with oxides produced by oxidative stress to promote further retinal damage in DR, leading to retinal morphology abnormality and dysfunction, which ultimately cause irreversible blindness in patients.Research status of NO and oxidative stress in the pathogenesis of DR from pathophysiological changes of DR, DR and oxidative stress, DR and NO, and the role of NO in oxidative stress were reviewed in this article.
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Objective:To investigate the protective effects of an antioxidant tert-butylhydroquinone (tBHQ) on the morphology and function of retina in early-stage experimental diabetic rats, and to explore the mechanism of its protective effect.Methods:Forty-five healthy SD rats of clean degree were randomized into normal control group, diabetes model group and tBHQ intervention group, with 15 rats in each group according to a random number table.The diabetes model was established via a single intraperitoneal injection of streptozotocin (STZ) in diabetes model group and tBHQ intervention group.Normal control group was intraperitoneally administered with an equal-volume injection of sodium citrate buffer.Rats in the tBHQ intervention group maintained a diet with 1% tBHQ for 2 weeks before the STZ injection, and the other two groups were fed with normal rat food only.Blood from tail vein was collected to assay the blood glucose level at 72 hours, 2 weeks and 4 weeks following modeling.Rat electroretinogram (ERG) was detected at 4 weeks after modeling.Morphological changes of rat retina were observed by hematoxylin and eosin staining.The apoptosis of retinal cells in different layers was detected by TUNEL assay.The expression of protein kinase B (Akt), p-Akt, endothelial nitric oxide synthase (eNOS) and p-eNOS was detected by Western blot.Müller cell line rMC-1 cells cultured in vitro were divided into 5 groups, including normal control group (72-hour culturing in normal medium), mannitol control group (72-hour culturing in medium containing 5.5 mmol/L glucose and 24.5 mmol/L mannitol), high glucose group (72-hour culturing in high-glucose medium), tBHQ intervention group (24-hour culturing in normal-glucose medium containing 5 μmol/L tBHQ, 72-hour culturing in high-glucose medium containing 5 μmol/L tBHQ), and phosphoinositide 3-kinase (PI3K) inhibitor group (6-hour culturing in normal medium containing 5 μmol/L LY294002, 24-hour culturing in normal-glucose medium containing 5 μmol/L LY294002 and 5 μmol/L tBHQ, 72-hour culturing in high-glucose medium containing 5 μmol/L LY294002 and 5 μmol/L tBHQ). The expression of Akt, p-Akt, eNOS and p-eNOS in the cells was detected by western blot.The use and care of animals complied with Regulations for the Administration of Laboratory Animals in Southwest Medical University.The study protocol was approved by the Animal Ethics Committee of Southwest Medical University (No.201711189). Results:The blood glucose level at 72 hours, 2 weeks and 4 weeks after modeling was higher in diabetic model group than tBHQ intervention group and normal control group (all at P<0.01). Four weeks after modeling, the scotopic ERG a-wave and b-wave amplitudes of diabetic model group were lower than those of normal control group and tBHQ intervention group (all at P<0.05). With edema and thickening of inner plexiform layer, thinning of inner nuclear layer and outer nuclear layer, as well as loosely arrangement and disorder of retinal layers, the number of retinal ganglion cells was decreased in diabetic model group in comparison with normal control group, all of which were improved in tBHQ intervention group in comparison with diabetic model group.There were more apoptotic retinal cells in diabetic model group than normal control group and tBHQ intervention group (both at P<0.05), which mainly existed in the outer nuclear layer.The relative expressions of p-Akt/Akt and p-eNOS/eNOS in rat retina of normal control group, diabetic model group and tBHQ intervention group were 0.76±0.11 and 0.83±0.06, 0.52±0.10 and 0.52±0.08, 1.14±0.31 and 1.03±0.13, respectively.The relative expressions of p-Akt/Akt and p-eNOS/eNOS in diabetic model group were lower than those of normal control group and tBHQ intervention group (all at P<0.01). The relative expressions of p-Akt/Akt and p-eNOS/eNOS in normal glucose group, mannitol control group, high glucose group, tBHQ intervention group and PI3K inhibitor group were 0.95±0.38 and 0.86±0.11, 0.94±0.27 and 0.74±0.29, 0.33±0.25 and 0.45±0.29, 1.32±0.37 and 1.28±0.22, 0.24±0.09 and 0.73±0.29, respectively.The relative expressions of p-Akt/Akt and p-eNOS/eNOS were significantly lower in high glucose group than those in normal glucose group and tBHQ intervention group (all at P<0.05), which were significantly lower in PI3K inhibitor group compared with tBHQ intervention group (both at P<0.01). Conclusions:tBHQ has protective effects on the morphology and function of retina in early diabetic rats, and the mechanism may be related to the activation of Akt/eNOS signaling pathway.
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Objective To expore the impact of teniary butyl hydroquinone (tBHQ) on Müller cells in SD rats retina under high glucose condition,and discuss the mechanism of tBHQ.Methods The Müller cells of SD rats were cultured in vitro and the experiment was divided into normal control group,high glucose group and tBHQ intervention group.Western blot and quantitative real time PCR were used to determine the expression of nuclear factor erythroid 2-related factor 2 (Nrf2),hemeoxygenase-1 (HO-1),hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) protein and mRNA in each group.Results Müller cells cultured in vitro were flat with irregularly sharp.The nucleus was oval,while the cytoplasm was abundant.Adjacent cells were interwoven to a network.Western blot assay showed the overall expression of Nrf2,HO-1,HIF-1α,and VEGF in Müller cells of normal control group,high glucose group,and tBHQ intervention group were significantly different (F =73.831,148.618,152.269,91.217,all at P＜0.001);Among them,the relative expressions of Nrf2,HO-1,HIF-1α and VEGF proteins in the high glucose group were 0.17±0.02,0.47±0.02,0.67±0.07,and 0.6±0.05,which were increased in comparion with 0.06±0.01,0.19±0.03,0.06±0.00 and 0.07±0.02 in the normal control group,with statistically significant differences (t =4.114,9.275,16.479,13.353,all at P ＜ 0.001);the relative expressions of Nrf2 and HO-1 proteins in the tBHQ intervention group were 0.40±0.06 and 0.72±0.05,which were increased by comparion with those in the higher glucose group,with statistically significant differences (t =7.847,7.947,both at P＜0.001);the relative expressions of HIF-1α and VEGF proteins in the tBHQ intervention group were 0.18±0.04,0.26±0.07,which were decreased in comparion with those in the higher glucose group,but were increased in comparion with those in normal control group,with statistically significant differences (t =13.215,8.444,both at P＜ 0.001).Quantitative real time PCR showed that the relative mRNA expressions of Nrf2,HO-1,HIF-1α,and VEGF in Müller cells of normal control group,high glucose group,and tBHQ intervention group were significantly different (F =340.317,1 582.911,488.852,185.699,all at P＜0.001);the relative mRNA expressions of Nrf2,HO-1,HIF-1α,and VEGF proteins in the high-glucose group were 1.53 ± 0.06,1.50 ± 0.04,2.56 ± 0.09,and 3.04 ± 0.19,which were increased in comparion with 1.07±0.07,0.95±0.05,0.99±0.02,and 1.09±0.08 in the normal control group,with statistically significant differences (t =7.292,15.014,30.550,18.573,all at P ＜ 0.001);The relative mRNA expressions of Nrf2 and HO-1 proteins in the tBHQ intervention group were 2.68±0.09 and 2.94±0.05,which were increased in comparion with those in the high-glucose group,with statistically significant differences (t =18.046,39.458,both at P＜0.001);The relative mRNA expression of HIF-1α and VEGF protein in the tBHQ intervention group were 1.48±0.05 and 1.6±0.08,which were decreased by comparion with those in the higher glucose group were increased in comparion with those in normal control group,with statistically significant differences (t =21.036,13.739,both at P＜0.001).Conclusions tBHQ protects Müller cells from damage in high glucose condition by activating anti-oxidative stress signaling pathway of Nrf2/ARE.
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Objective@#To expore the impact of teniary butyl hydroquinone (tBHQ) on Müller cells in SD rats retina under high glucose condition, and discuss the mechanism of tBHQ.@*Methods@#The Müller cells of SD rats were cultured in vitro and the experiment was divided into normal control group, high glucose group and tBHQ intervention group.Western blot and quantitative real time PCR were used to determine the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) protein and mRNA in each group.@*Results@#Müller cells cultured in vitro were flat with irregularly sharp.The nucleus was oval, while the cytoplasm was abundant.Adjacent cells were interwoven to a network.Western blot assay showed the overall expression of Nrf2, HO-1, HIF-1α, and VEGF in Müller cells of normal control group, high glucose group, and tBHQ intervention group were significantly different (F=73.831, 148.618, 152.269, 91.217, all at P<0.001); Among them, the relative expressions of Nrf2, HO-1, HIF-1α and VEGF proteins in the high glucose group were 0.17±0.02, 0.47±0.02, 0.67±0.07, and 0.6±0.05, which were increased in comparion with 0.06±0.01, 0.19±0.03, 0.06±0.00 and 0.07±0.02 in the normal control group, with statistically significant differences (t=4.114, 9.275, 16.479, 13.353, all at P<0.001); the relative expressions of Nrf2 and HO-1 proteins in the tBHQ intervention group were 0.40±0.06 and 0.72±0.05, which were increased by comparion with those in the higher glucose group, with statistically significant differences (t=7.847, 7.947, both at P<0.001); the relative expressions of HIF-1α and VEGF proteins in the tBHQ intervention group were 0.18±0.04, 0.26±0.07, which were decreased in comparion with those in the higher glucose group, but were increased in comparion with those in normal control group, with statistically significant differences (t=13.215, 8.444, both at P<0.001). Quantitative real time PCR showed that the relative mRNA expressions of Nrf2, HO-1, HIF-1α, and VEGF in Müller cells of normal control group, high glucose group, and tBHQ intervention group were significantly different (F=340.317, 1 582.911, 488.852, 185.699, all at P<0.001); the relative mRNA expressions of Nrf2, HO-1, HIF-1α, and VEGF proteins in the high-glucose group were 1.53±0.06, 1.50±0.04, 2.56±0.09, and 3.04±0.19, which were increased in comparion with 1.07±0.07, 0.95±0.05, 0.99±0.02, and 1.09±0.08 in the normal control group, with statistically significant differences (t=7.292, 15.014, 30.550, 18.573, all at P<0.001); The relative mRNA expressions of Nrf2 and HO-1 proteins in the tBHQ intervention group were 2.68±0.09 and 2.94±0.05, which were increased in comparion with those in the high-glucose group, with statistically significant differences (t=18.046, 39.458, both at P<0.001); The relative mRNA expression of HIF-1α and VEGF protein in the tBHQ intervention group were 1.48±0.05 and 1.6±0.08, which were decreased by comparion with those in the higher glucose group were increased in comparion with those in normal control group, with statistically significant differences (t=21.036, 13.739, both at P<0.001).@*Conclusions@#tBHQ protects Müller cells from damage in high glucose condition by activating anti-oxidative stress signaling pathway of Nrf2/ARE.
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Objective To observe the effect oftert-Butylhydroquinone (tBHQ) on the expression of nuclear factor erythroid 2-related factor 2 (Nrf2),heme oxygenase (HO)-1 and phosphatidylinositol 3-kinase (PI3K) in high glucose cultured retinal Müller cells;and to investigate the anti-oxidative stress and anti-apoptotic effects oftBHQ.Methods Retinal Müller cells were divided into normal glucose group (5.5 mmol/L,N group),high glucose group (45 mmol/L,HG group) and tBHQ intervention group (HG+tBHQ group).After retinal Müller cells were cultured with high glucose for 48 hours,the pretreatment with tBHQ (20 μmol/L) induced the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1.The Müller cells were identified by immunofluorescence staining.The expressions of Nrf2,HO-1,PI3K,B-cell lymphoma-2 (Bcl-2) and Bax were detected by Western blot and real-time fluorescence quantitative PCR.Flow cytometry was used to detect the apoptosis of retinal Müller cells in rats.Results Müller cytoplasm and nucleus GS showed strong positive,large cell body,abundant cytoplasm,uniform green fluorescence;nuclear DAPI staining round or oval,clear boundary.The expression of Nrf2 protein (t=4.114,P=0.006),HO-1 protein (t=9.275,P=0.000),Nrf2 mRNA (t=7.292,P=0.000) and HO-1 mRNA (t=15.014,P=0.000) in the HG group were higher than those in the N group.The expressions of Nrf2 protein (t=7.847,P=0.000),HO-1 protein (t=7.947,P=0.000),PI3K protein (t=5.397,P=0.002),Bcl-2 protein (t=6.825,P=0.000),Nrf2 mRNA (t=18.046,P=0.000),HO-1 mRNA (t=39.458,P=0.000),PI3K mRNA (t=4.979,P=0.003) and Bcl-2 mRNA (t=9.535,P=0.000) in the HG+tBHQ group were significantly higher than those in the HG group.The protein and mRNA expressions of Bax protein in the HG+tBHQ group were significantly lower than those in the HG group (t=14.998,16.520;P=0.000,0.000).Flow cytometry showed that the apoptosis rate of Müiller cells in the HG group was significantly higher than that in the N group (t=39.905,P=0.000).The apoptosis rate of Müller cells in the HG+tBHQ group was significantly lower than that in the HG group (t=21.083,P=0.000).Conclusion tBHQ can inhibit the apoptosis of retinal Müller cells by up-regulating the expression ofNrf2,HO-1 and PI3K.
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Objective To observe the clinical effect of arthroscopic surgery on patients with rotator cuff tear injury. Methods One hundred and twenty patients with rotator cuff tears admitted to the Department of Orthopaedic of the First Hospital of Changsha from January 2013 to September 2015 were enrolled. Sixty patients treated with traditional conservative methods were assigned in a control group, the whole arthroscope or shoulder arthroscope was used to assist performing microsurgical incisions for another such 60 patients in the observation group. Visual analogue scale (VAS) of pain was applied to evaluate the pain of the patients with rotator cuff injury before and after treatment in both groups. The function of the shoulder joints and recovery situation were evaluated by using the shoulder functional system score and the American shoulder and elbow surgeon score (ASES). Results The results showed that all patients had no postoperative complications such as secondary fracture of the rotator cuff tear, infection, incision non-healing, etc. Compared with those before treatment, in both groups, the VAS scores were obviously lowered after treatment, while ASES and shoulder function system scores after treatment were significantly higher than those before treatment, and the changes in the observation group were more pronounced than those in the control group (VAS: 0.82±0.11 vs. 2.20±0.59, ASES: 82.21±10.81 vs. 70.53±6.21, pain score: 9.81±0.21 vs. 9.11±0.51, function score: 9.70±0.09 vs. 8.80±0.40, anterior flexion score: 4.59±0.41 vs. 4.20±0.61, all P ＜ 0.05). Conclusion The clinical effect of arthroscopic surgical treatment of patients with rotator cuff tear injury is relatively satisfactory and worthy to be deeply studied clinically.
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Objective To investigate the cellular viability and mitochondrial reactive oxygen species (ROS) production of the Müller cells under high glucose condition,and explore the protection role of the 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) on Müller cells.Methods Müller cells from Sprague Dawley rats were divided into 5 groups randomly,including 25 mmol/L normal glucose group (group A) and 65 mmol/L high glucose group (group B).High glucose group with 45,60,70 μmol/L CPDT and cultured them 72 hour was set as group C,D and E.Water soluble tetrazolium salt (WST)-8 was used to measure the cellular viability.Flow cytometry was used to measure the active oxygen and apoptosis index.The expression of nuclear factor erythroid 2-related factor 2 (Nrf2),hemeoxygenase-1 (HO-1),Bcl-2 and Bax protein were measured by Western blot.Results Compared with group A,the WST-8 showed that the viability of Müller cells apparently decreased in group B (t=39.59,P＜0.05).Compared with the group B,the viability of Müller cells had changes in group C (t=0.97,P＞0.05),but recovered in group D and E (t=-4.17,-7.52;P＜0.05).Compared with group A,the FCM showed that the mitochondrial ROS levels was higher in group B (t=-30.99,P＜0.05).Compared with group B,the mitochondrial ROS levels were decreased in group D (t=27.68,P＜0.05).Compared with group A,Bax,Nrf2 and HO-1 increased (t=-11.03,-63.17,-11.44;P＜0.05),while the bcl-2 decreased in group B (t=7.861,P＜0.05).Compared with the group B,Nrf2,HO-1 and Bax decreased (t=15.11,26.59,6.27;P＜0.05),while the bcl-2 increased in group D (t=-6.53,P＜0.05).Conclusions Under the high glucose,CPDT may reduce the mitochondrial ROS levels and the expression of Nrf2,HO-1 and Bax protein of Müller cells.It may inhibit apoptosis through activating the Nrf2/HO-1 pathway and balancing of level of Bcl-2 protein and mitochondrial ROS.
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Background Apoptosis is a primary clinical pathological mechanism of diabetic retinopathy (DR).Oxidative stress and high glucose can activate cell apoptosis pathway and thus leads to cellular damage.It is confirmed that tert-butyl hydroquinone (tBHQ) plays an antioxidation effect,however,whether it has a protective role on retinal cells in DR is still unelucidated.Objective This study was to investigate the effect of tBHQ on vascular endothelial growth factor (VEGF) and bcl-2 expressions in retina of type 2 diabetic rats and its possible mechanism via nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signal pathway.Methods Fifty clean healthy male SD rats were included in this experimental study.Ten rats were fed with normal diet as the normal control group,and other rats were fed with high fatty and high sugar food for 4 weeks.After 12 hours of fasting,streptozotoin (STZ) (30 mg/kg) was intraperitoneally injected to induce the type 2 diabetic models.The model rats were randomly divided into the diabetic control group and tBHQ group and 1％ tBHQ was added into the high fatty and sugar food 1 week after modeling in the tBHQ group.Fasting plasma glucose (FPG) level,blood total cholesterol (TC) level,blood triglyceride (TG) level,high density lipoprotein-cholesterol (HDL-C),low density lipoproteincholesterol (LDL-C) and fasting serum insulin (FINs) were detected 4 and 12 weeks after modeling,respectively,and radio immunoassay was used to detect the FIN levels of the rats.The relative expression of VEGF and bcl-2 in retinas of the rats were assayed by immunohistochemistry and fluorescence real-time quantitative PCR (qRT-PCR).The use of the animals complied with the Regulations for the Administration of Affairs Concerning Experimental Animals by State and Technology Commission.Results Type 2 diabetic models were successfully established in 35 rats with successful rate 92.1％.The FIN levels were significantly different among different groups and time points (Fgroup =22.480,P =0.000;Ftime =7.636,P =0.008).The FPG,TC,TG and LDL-C levels were significantly different among the groups (FPG:Fgroup =78.531,P =0.000;TC:Fgroup =28.049,P =0.000;TG:Fgroup =13.108,P =0.000;LDL-C:Fgroup =6.804,P＜0.05).Immunohistochemistry showed that VEGF and bcl-2 were mainly expressed in retinal ganglion cell layer,inner plexiform layer and outer plexiform layer.The expressions of VEGF and bcl-2 proteins were significantly different among different groups (VEGF:Fgroup =11.805,P =0.000;bcl-2:Fgroup =22.943,P =0.000);the expression level of bcl-2 protein was higher in 12 weeks after modeling than that in 4 weeks after modeling in the tBHQ group (P＜0.05).The expressions of VEGF and Bcl-2 mRNA in rat retinas were significantly different among different groups and time points (VEGF:Fgroup =79.220,P =0.000;Ftimo =6.090,P＜0.05;Bcl-2:Fgroup =105.000,P=0.000;Ftime =13.170,P=0.001).Four and eight weeks after modeling,the expressions of VEGF and Bcl-2 mRNA in the diabetic control group and tBHQ group were significantly higher than that in the normal control group,and the expressions of Bcl-2 mRNA in the tBHQ group were significantly higher than that in the model control group (all at P＜0.05);the expression of Bcl-2 mRNA was higher at 12 weeks after modeling than that at 4 weeks in the tBHQ group (P＜0.05).Conclusions tBHQ produces anti-oxidative-damage and anti-apoptosis effects on retinal cells by up-regulating VEGF expression and down-regulating bcl-2 expression in DR rats.In addition,tBHQ may have effects on lowering high blood sugar,regulating insulin and blood lipid levels.
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Objective To assess the association of vascular endothelial growth factor (VEGF) gene-460C/T and-634C/G polymorphism with diabetic retinopathy (DR) among patients in Asia and European by meta-analysis.Methods A systematic search of electronic databases (PubMed,Cochrane Library,EMBASE,VIP,Wanfang technological,CNKI,etc.) was carried out until Jun,2014.Case-control studies on the relationship between genetic polymorphism of VEGF-460C/T and VEGF-634C/G with diabetic retinopathy were included in this analysis.The data were quantitatively analyzed by RevMan 5.0 software after assessing the quality of included studies.The pooled odds ratios (OR) and their corresponding 95％ confidence intervals (CI) were used to assess the strength of the association.Results VEGF-460C/T (7 studies:899 cases and 786 controls) and VEGF-634C/G (10 studies:1615 cases and 1861 controls) were inclued in this meta-analysis.Significant association was found for-460C/T polymorphism in Aisa (C versus T:OR=1.52,95％CI was [1.22,1.90],Z=3.72,P=0.0002;CC versus CT+TT:OR=1.61,95％CI was [1.19,2.19],Z=3.05,P=0.002;TT versus CT+CC:OR=0.64,95％CI was [0.41,0.98],Z=2.07,P=0.04),and VEGF-634CC gene type was associated with DR in European (OR=1.56,95％CI [1.08,2.25],Z=2.37,P=0.02).No significant publication bias was found.Conclusions The metaanalysis demonstrated that DR was associated with VEGF-460C/T polymorphism in Asia,and C alleles and CC gene type was the risk polymorphism;VEGF-634C/G polymorphism was not associated with DR,but its CC genotype maybe the risk factor in European.Further case-control studies based on larger sample size are still needed,especially for-634C/G polymorphism.