ObjectiveTo observe the effect of phase Ⅱenzyme inducer 5, 6-dihydrocyclopenta 1, 2-dithiole-3-thione (CPDT) on nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signal pathway and oxidative stress in the retina of type 2 diabetic rats. MethodsThirty-five male Wistar rats were randomly divided into two group, normal group and model group. Model group were further randomly divided into two group, diabetic group and CPDT intervention group. There were 8 rats in the normal group and 27 rats in the model group. Diabetic group and CPDT intervention group were given high fat and high sugar diet for 2 months. After 12 hours of fasting, type 2 diabetic rat model was induced by intraperitoneal injection of low dose of streptozotocin. CPDT was added into the high fat and high sugar diets at 1 week after the diabetic model was established in the CPDT intervention group. Eight weeks after CPDT treatment, blood glucose, serum malondialdehyde (MDA), blood lipid, Nrf2 and hemeoxygenase-1 (HO-1) expression were evaluated. ResultsType 2 diabetic model was successfully established in 25 rats, the success rate was 92.6%.The level of blood lipid of diabetic group was higher than those of the normal group (FTC=65.866, FTG=25.441, FLDL-C=38.889; P=0.000). Blood glucose was significant different between all groups (χ2=25.812, P=0.000), and was significantly higher in diabetic group than that in normal group and CPDT intervention group. The serum MDA content was significant different between all groups (F=59.545, P=0.000), and was significantly higher in diabetic group than that in normal group (t=10.523, P=0.000) and CPDT intervention group (t=7.766, P=0.000). The mRNA level of retinal Nrf2 and HO-1 was significant different between all groups (FNrf2=19.503, PNrf2=0.000;FHO-1=9.737, PHO-1=0.001), and was higher in CPDT intervention group than the diabetic group (tNrf2=3.399, PNrf2=0.002;tHO-1=2.167, PHO-1=0.039). The protein level of retinal Nrf2 and HO-1 was significant different between all groups (FNrf2=112.823, FHO-1=119.361; P=0.000), and was higher in CPDT intervention group than the diabetic group (tNrf2=6.203, tHO-1=6.388; P=0.000). Immuno-staining showed that Nrf2 and HO-1 were mainly expressed in retinal ganglion cell layer, inner plexiform layer and inner nuclear layer, and were significant different between all groups (FNrf2=16.206, FHO-1=46.790; P=0.000). They also were higher in CPDT intervention group than the diabetic group (tNrf2=3.172, PNrf2=0.003;tHO-1=6.321, PHO-1=0.000), was higher in diabetic group than that in normal group (tNrf2=2.679, PNrf2=0.011;tHO-1=3.482, PHO-1=0.001). ConclusionCPDT may activate Nrf2/ARE pathway, induce Nrf2 and HO-1 expression, decrease serum MDA and blood glucose, and thus reduce oxidative stress injury in the retina of type 2 diabetic rats.
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 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.
ObjectiveTo observe the effect of tert-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 of tBHQ.MethodsRetinal 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.ResultsMüller 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 Müller 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).ConclusiontBHQ can inhibit the apoptosis of retinal Müller cells by up-regulating the expression of Nrf2, HO-1 and PI3K.
ObjectiveTo observe the protective effect of polypyrimidine bundle-binding protein-related splicing factor (PSF) over-expression on RPE cell injury induced by advanced glycation end products (AGEs).MethodsThe human RPE cells cultured in vitro were divided into three groups: normal control group (N group), blank control group (N + AGEs group), empty vector control group (Vec + AGEs group), and PSF high expression group (PSF + AGEs). group). RPE cells in N group were routinely cultured; RPE cells in N + AGEs group were only transfected but did not introduce any exogenous genes combined with AGEs induction; Vec +AGEs group and PSF + AGEs group were transfected with pcDNA The empty vector or pcDNA-PSF eukaryotic expression plasmid was introduced into RPE cells and induced by AGEs. Except the N group, the other 3 groups of cells were transfected accordingly, and were stimulated with 150 μg/ml AGEs for 72 h after 24 h. HE staining and Hoechst 33258 staining were used to observe the effect of high PSF expression on the morphological changes of RPE cells; ROS level detection was used to analyze the effect of PSF high expression on the ROS expression of RPE cells induced by AGEs; MTT colorimetric method was used to detect the high PSF expression Effects on the viability of RPE cells; Western blot was used to detect the effects of different time and dose of PSF on the expression of heme oxygenase 1 (HO-1).ResultsHE staining and Hoechst 33258 staining observation showed that the cells in group N were full in shape, the nucleus was round, the cytoplasm was rich, and the staining was uniform; the cells in N + AGEs group and Vec + AGEs group were reduced in size, the eosinophilic staining was enhanced, and the nucleus was densely densely stained. Pyrolysis and even fragmentation; the morphology of cells in the PSF + AGEs group was still full, the cytoplasm staining was more uniform, and the nucleus staining was uniform. The results of MTT colorimetry showed that high expression of PSF can effectively improve the viability of RPE cells, but this effect can be effectively antagonized by ZnPP, and the difference is statistically significant (F=33.26, P＜0.05). DCFH-DA test results showed that compared with the N + AGEs group and Vec + AGEs group, the ROS production in PSF + AGEs group decreased, the difference was statistically significant (F=11.94, P＜0.05). Western blot analysis showed that PSF protein up-regulated HO-1 expression in a time- and dose-dependent manner. The relative expression level of HO-1 at 24, 48, and 72 h after PSF protein was significantly higher than that at 0 h, and the difference was statistically significant (F=164.91, P＜0.05). The relative expression level of HO-1 under the action of 0.1, 0.5, 1.0, 1.5, and 2.0 μg PSF protein was significantly higher than 0.0 μg, and the difference was statistically significant (F=104.82, P＜0.05).ConclusionPSF may inhibit the production of ROS by up-regulating the expression of HO-1, thus protecting the RPE cells induced by AGEs.
ObjectiveTo investigate the inhibitory effect of lentivirus-mediated polypyrimidine bundle binding protein-associated splicing factor (PSF) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR).MethodsOne hundred and twelve 5-day-old C57BL/6J mice were randomly divided into normal control group, simple OIR model group, OIR model + lentivirus empty vector treatment group (Vec group) and OIR model + PSF lentivirus treatment group (PSF group), with 16, 32, 32 and 32 mice, respectively. When the mice were 7 days old, the mice in the normal control group were fed in a routine environment, and the mice in the OIR model group, Vec group and PSF group were established OIR model. The mice in the Vec group and PSF group were given an intravitreal injection of 1 μl of lentiviral vector and PSF lentivirus (titer 1×1011 TU/ml) at the age of 12 days. No injection was performed in the normal control group and simple OIR group. RNV was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR was applied to detect the mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Western blot analysis was applied to detect the protein expression of Nrf2, HO-1 and PSF. Results Of the normal control group, simple OIR model group, Vec group and PSF group, the number of pre-retinal neovascular cell nuclei were 0.00, 14.36±5.50, 15.67±4.96, 8.13±2.09, the non-perfusion area were 0.00%, (35.71±2.81)%, (36.57±4.53)%, (15.33±4.75)%, respectively. The differences of the number of pre-retinal neovascular cell nuclei and non-perfusion area among 4 groups were significant (F=24.87, 165.70; P＜0.05). Compared with the normal control group, there were more pre-retinal neovascular cell nucleis and larger non-perfusion area in the simple OIR model group and Vec group (P＜0.05). Compared with the simple OIR model group and Vec group, there were lower pre-retinal neovascular cell nucleis and smaller non-perfusion area in the PSF group (P＜0.05). Real-time quantitative PCR and Western blot showed that the mRNA expression of Nrf2, HO-1 (F=53.66, 83.54) and protein expression of Nrf2, HO-1 and PSF (F=58.38, 52.69, 24.79) among 4 groups were significant (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the simple OIR model group and Vec group decreased significantly than those in the normal control group (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the PSF group were increased significantly than those in the simple OIR model group and Vec group (P＜0.05). model group and Vec group (P＜0.05).ConclusionIntravitreal injection of lentivirus-mediated PSF inhibits RNV in mice model of OIR possibly through up-regulating the expression of Nrf2 and HO-1.