Objective To observe the effect of melatonin (MT) on retinal apoptosis in rats with ischemia-reperfusion injury (RIRI). Methods A total of 54 male healthy Sprague-Dawley adult rats were randomly divided into the normal control (CON) group (6 rats), RIRI group (24 rats) and MT group (24 rats). The rats of RIRI and MT group were induced using suture-occluded methods to establish RIRI model. The rats of MT group were injected with MT in the left carotid artery 30 minutes after RIRI, and RIRI group was injected with the same amount of saline. On 6, 24 hours and 3, 7 days after RIRI, the morphological changes of retina were evaluated by hematoxylin and eosin (HE) staining; the effects of MT on retinal cell apoptosis and Nrf2, HO-1 proteins were examined by immunohistochemistry staining. The correlation between active Caspase-3 and Nrf2 protein, active Caspase-3 and HO-1 protein in MT group were analyzed by linear regression analysis. Results HE staining results showed that the morphology of retinal cells was regular and retinal cells were well arranged in the CON and MT group. In the RIRI group, both the thickness of inner retinal layer and the number of retinal ganglion cells (RGC) were decreased. On 6, 24 hours and 3, 7 days after RIRI, the thickness of inner retinal layer (F=16.710, 62.303, 68.389, 57.132; P＜0.01) and RGC number (F=24.250, 11.624, 14.155, 32.442; P＜0.05) in MT group were more than those in RIRI group. Immunohistochemistry staining results showed that less active Caspase-3+ cells were observed in MT group as compared with those in RIRI group at each time points (F=49.118, 134.173, 76.225, 18.385; P＜0.01). There were more Nrf2+ (F=11.041, 31.480, 59.246, 6.740; P＜0.05) and HO-1+ cells (F=128.993, 21.606, 51.349, 8.244; P＜0.05) in MT group as compared with those in RIRI group at each time points. Linear regression analysis results showed that the difference of active Caspase-3+ cells were all linearly correlated with the Nrf2+ cells and HO-1+cells in the MT group (r2=0.810, 0.730; P＜0.01). Conclusion MT could reduce retinal cell apoptosis in RIRI rats, and its mechanism may be associated with increased Nrf2 and HO-1 expression, reduced active Caspase-3 expression.
ObjectiveTo observe the expression of probucol on high glucose-induced specificity protein 1(SP1), kelchlike ECH associated protein1 (Keap1), NF-E2-related factor 2 (Nrf2) and glutamate-cysteine ligase catalytic (GCLC) in the cultured human müller cells and preliminary study the antioxidation of the probucol on müller cells.MethodsPrimary cultured human müller cells were randomly divided into four groups: normoglycaemia group (5.5 mmol/L glucose), normoglycaemia with probucol group (5.5 mmol/L glucose+100 μmol/L probucol), hyperglycemia group (25.0 mmol/L glucose), hyperglycemia with probucol group (25.0 mmol/L glucose + 100 μmol/L probucol). Immunofluorescence staining was used to assess distribution of SP1, Keap1, Nrf2, GCLC in human Müller cells. SP1, Keap1, Nrf2 and GCLC messenger RNA (mRNA) expression was evaluated by quantitative real-time RT-PCR (qRT-PCR). Independent sample t test was used to compare the data between the two groups.ResultsAll müller cells expressed glutamine synthetase (＞95%), which confirmed the cultured cells in vitro were the purification of generations of müller cells. The expressions of SP1, Keap1, Nrf2, and GCLC protein were positive in human müller cells. qRT-PCR indicated that SP1 (t=28.30, P＜0.000), Keap1 (t=5.369, P=0.006), and Nrf2 (t=10.59, P=0.001) mRNA in the hyperglycemia group increased obviously compared with the normoglycaemia group; GCLC (t=4.633, P=0.010) mRNA in the hyperglycemia group decreased significantly compared with the normoglycaemia group. However, SP1 (t=12.60, P=0.000) and Keap1 (t=4.076, P=0.015) in the hyperglycemia with probucol group decreased significantly compared with the hyperglycemia group; Nrf2 (t=12.90, P=0.000) and GCLC (t=15.96, P＜0.000) mRNA in the hyperglycemia with probucol group increased obviously compared with with the hyperglycemia group.ConclusionProbucol plays an antioxidant role by inhibiting the expression of SP1, Keap1 and up-regulating the expression of Nrf2, GCLC in müller cells induced by high glucose.
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-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.
Objective To explore the correlation between the levels of serum nuclear factor-erythroid 2-related factor 2 (Nrf2) as well as heme oxygenase-1 (HO-1) and cognitive dysfunction by determining the levels of Nrf2 and HO-1 in patients with obstructive sleep apnea (OSA) to different degrees and combining Montreal cognitive assessment (MoCA). Methods Serum levels of Nrf2 and HO-1 were determined in 32 patients with mild-moderate OSA, 23 patients with severe OSA and 20 healthy controls. The differences of Nrf2 and HO-1 levels among groups were compared. All subjects were evaluated by MoCA score. According to MoCA score, OSA patients were divided into two groups: OSA with mild cognitive impairment (MCI) group and OSA with normal cognition group. Serum Nrf2 and HO-1 levels were compared between the two groups, and the differences in the OSA patients with or without cognitive impairment were understood. Spearman correlation coefficient was used to explore the correlation between serum Nrf2 and HO-1 levels and cognitive function of OSA patients. The diagnostic value of serum Nrf2 and HO-1 in the OSA patients with cognitive impairment was determined by receiver operating characteristic curve. Results Serum levels of Nrf2 and HO-1 in the mild-moderate and severe OSA groups were higher than those in the control group, and those in the severe OSA group were higher than those in the mild-moderate OSA group (P<0.05). Compared with the OSA with normal cognition group, the serum HO-1 level in the OSA patients with MCI was higher (P<0.05), but the serum NRF2 level had no significant difference between the two groups (P>0.05). There was a negative correlation between serum HO-1 level and total MoCA score in the OSA patients (r=–0.495, P=0.000), but there was no significant correlation between serum Nrf2 and total MoCA score in the OSA patients (P>0.05). Serum Nrf2 and HO-1 were 0.791 and 0.818 for predicting OSA patients with cognitive impairment. The sensitivity was 84.20% and 86.80%, and the specificity was 67.60% and 73.00%, respectively. Conclusions Serum Nrf-2 and serum HO-1 play important role in the pathogenesis of OSA. Serum HO-1 level may be closely related to cognitive dysfunction in OSA patients. Detection of serum HO-1 may be helpful in early identification of cognitive dysfunction in OSA patients, which has potential clinical application value.
Objective To explore the regulation of peroxisome proliferator-activated receptor γ coactivator 1α( PGC-1α) and NF-E2-related factor 2( Nrf2) on expression of γ-glutamylcysteine synthetase ( γ-GCS) , and their roles in chronic obstructive pulmonary disease( COPD) . Methods Twenty-four SD rats were randomly divided into a COPD group and a normal control group. COPD model was established by intratracheal instillation of lipopolysaccharide ( LPS) and daily exposure to cigarette smog in the COPD group. The lung function was measured and the pathological changes were observed. The protein and mRNA expressions of PGC-1α, Nrf2, and γ-GCS in lung tissue were measured by immunohistochemistry, Western blot, in site hybridization ( ISH) , and reverse transcription-polymerase chain reaction ( RT-PCR ) ,respectively. Results In the COPD group, the pulmonary function ( FEV0. 3, FEV0. 3 /FVC, PEF) damage and lung pathological changes were conformed as morphological characteristics of COPD. The mRNA of PGC-1α and Nrf2 expressed in lung tissues of two group rats in the region consistent with γ-GCS mRNA. The protein and mRNA expressions of PGC-1αand γ-GCS were markedly increased in the COPD group( all P lt;0. 05) ,and the protein expression of Nrf2 was obviously up-regulated ( P lt; 0. 01) , while Nrf2 mRNA had no significant difference between the two groups( P gt;0. 05 ) . Linear correlation analysis showed that the level ofPGC-1αprotein was positively correlated with the levels of Nrf2 protein and mRNA ( r = 0. 775, 0. 515, all P lt; 0. 01) , and the levels of PGC-1αand Nrf2 protein were positively correlated with the levels of γ-GCS protein ( r = 0. 531, 0. 575, all P lt; 0. 01) and mRNA ( r = 0. 616, 0. 634, all P lt; 0. 01) . Conclusions PGC-1α, which may serve as a co-activator of Nrf2, can up-regulate the expression of γ-GCS gene cooperatively with Nrf2 through a common pathway, which might involve in the oxidative and antioxidative mechanism in the pathogenesis of COPD.
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 therapeutic effect of vitrectomy on Eales disease and the correlated factors affecting the visual prognosis and disease outcomes. Methods The clinical and follow-up data from 128 patients (142 eyes) with Eales disease undergone vitrectomy were retrospec tively analyzed. The statistical methods including t test,chi;2test, one-way Anova method of square-deviation(SD), and logistic regression were used to analyze the relationship between the general data of the patients (including age, sex, laterality of the eye, visual acuity before the treatment, stages of disease, duration from vitreous hemorrhage to vitrectomy, neovascularization and proliferative vitreoretinopathy, and whether or not combined with retinal detachment and other complications) and the prognosis of the visual acuity after surgery (including the surgical method, techniques, and times and complications after the surgery). The patients were 18-45 years old (mean 28.5 years) with single vitreous hemorrhage in 28 and proliferative changes in 114 in whom 59 had retinal detachment. The follow-up period after the surgery was more than 3 months (mean 35.8 months). The success criteria of the surgery were complete or part retinal reattachment, and failure of retinal reattachment, eye-ball atrophy or excis ion of the affected eye were the failure criteria. Results Successful vitrectomies had been performed on 129 eyes (90.8%) and unsuccessful ones on 13 eyes (9.2%). The difference between the stages of the disease and prognosis of visual acuity after the surgery was significant (chi;2=64.0, Plt;0.01); the duration of vitreous hemorrhage obviously affected the prognosis of visual acuity (OR=11.6，Plt;0.01); the degree, quality, curable possibility, and recurrent probability of combined retinal detachment were the key factors affecting the visual acuity after vitrectomy; the visual acuity before and after the surgery was interrelated; the method and techniques of the surgery and the different filling matters affected the visual acuity after the surgery; the difference between multiple times and once of surgery was significant (chi;2=66.84，Plt;0.01); the degree of complexity of the operative procedure, especially repetitious vitrectomies obviously affected the surgical prognosis and the improvement of visual acuity; the possibility of fa ilure of the surgery differs 7 times in patients with or without post-operative complications ( chi;2=67.23，Plt;0.01); whether the post-operative complications occurred or not significantly affected the prognosis of the visual acuity a-fter the surgery. Conclusions Vitrectomy is effective for Eales disease. The important factors affecting the prognosis of visual acuity after the operation include stages of disease, degree and extent of proliferative vitreoretinopathy, whether or not combined with retinal detachment and other complic ations, duration from vitreous hemorrhage to vitrectomy, the degree of complexity of the operation, and the complications during or after the operation. (Chin J Ocul Fundus Dis, 2006, 22:291-294)
Objective To investigate the expression of transcriptional factors zinc finger Krüppel like transcription factor 2 ( Klf2) and NF-E2 related factor 2 ( Nrf2) /BTB CNC homology 1 ( Bach1) in rat bronchial epithelial cells stimulated by cigarette smoke extract ( CSE) , and explore the regulatingmechanism of γ-glutamylcysteine synthetase ( γ-GCS) expression in the oxidative condition. Methods Rat bronchial epithelial cells were harvested using enzyme digestion method, and intervened by 10% CSE for 6 hours. Then γ-GCS activity was detected by two enzymes method, and the nuclear transfer of Nrf2 /Bach1 in cells was detected by immunohistochemistry. Western blot and reverse transcription-polymerase chain reaction ( RT-PCR) techniques were used for detecting the protein and mRNA expressions of Klf2, Nrf2, Bach1, and γ-GCS in the cells. Results The γ-GCS activity was elevated in the CSE group. Immunohistochemical results showed that Nrf2 translocated from cytoplasm to nucleus in response to stimulation by CSE. On the contrary, Bach1 translocated from nucleus to cytoplasm in the same condition. Western blot results showed that protein levels of Klf2, Nrf2, Bach1, and γ-GCS were higher in the CSE group than those in the control group ( Plt;0.05) . RT-PCR results were the same as the Western blot results ( Plt;0.05) . Linear correlation analysis showed that γ-GCS expression was positively correlated with Klf2, Nrf2, and Bach1 ( Plt;0. 05) . Conclusion CSE might regulate the expression of γ-GCS through the transcription factors of Klf2, Nrf2, and Bach1.
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.