| Objective Diabetic retinopathy( DR) is a common microvascular complication of diabetes mellitus( DM), it is also a neural vascular diseases,oxidative stress is one of the main pathogenesis theory of DR. Hyperglycemia or DM will increase oxidative stress and cause the damage of retinal tissue. Nrf2/ARE signal pathway is an important endogenous antioxidant stress pathway, When the reactive oxygen species( ROS) or electrophilic reagent stimulate nuclear factor EF-E2 related factor( Nrf2), Nrf2 transfer into the nucleus, recognize and bind to antioxidant response element( ARE), and start the transcription of heme oxygenase-1( HO-1) protein and other downstream antioxidant gene, enhance cell antioxidant capacity, reduce the oxidative stress injury, so protecting the retina. In the study, we use high fat and sugar diet with intraperitoneal injection of a small dose of streptozotocin( STZ) to preparing the type 2 diabetic rat model, and study the effect of tert-butyl hydroquinone( t BHQ) on the retina of type 2 diabetic rats at different time of Nrf2/ARE signal pathway, to exploring whether t BHQ has protective effects on the retina and its mechanism, so provide a theoretical basis for the prevention and treatment of DR. Method Selecting 60, 4 week old male SD rats, weighing 180-200 g, after one week Adaptive feeding, We randomly divide into three groups: normal control group( NC group, n = 20), Model group( n = 40). The NC group were fed with basic diet, but Model group were given high fat and sugar diet,for one month later, Model group inject the STZ of 30mg/kg into intraperitoneal to establishing the rat model of type 2 DM after fasting 12h( 7 days after the tail of fasting blood glucose( FBG) greater than 16.7mmol/L illustrate the modeling is success), NC group was injected the same volume ofbuffer into intraperitoneal as control. Successful modeled, were randomly divided into diabetic group( DM group) and drug intervention group( t BHQ group).one week after modeling, the t BHQ group fed with high fat and sugar diet, added 1% t BHQ, but the NC and DM group feeding methods unchanged.The first 4 weeks and 12 weeks of t BHQ intervention, each group rats determined their weight, then Cardiac puncture to determinate FBG and blood lipid. The rats eyes use HE staining to observe the morphological changes and immunohistochemical to detect the distribution of Nrf2 and HO-1 in retinal tissues, the remaining rat eye retina use quantitative PCR to detect the expression of Nrf2 and HO-1 m RNA in retinal tissues. Result 1. The Molded type 2 diabetic rats was 87.5%( 40 rats 35 molded). 2. The general condition of rats: DM, t BHQ group rats began to emerge weight loss, listlessness, unresponsive, and more food, polyuria, polydipsia and other symptoms, and the t BHQ group was less than that in DM group, close to the NC group. NC group were normal weight, good spirit, normal food and water. 3. Biochemical indicators: Compared with the NC group, 4 weeks and 12 weeks DM group, FBG increased significantly, the difference is statistically significant( t=3.015, 2.977, P=0.000, 0.000, P<0.01); Compared with the NC group, 4 weeks and 12 weeks t BHQ group, FBG increased significantly, the difference is statistically significant( t=5.915, 5.018, P=0.000, 0.000, P<0.01); Compared with 12 weeks DM group, 12 weeks t BHQ group FBG decreased, the difference is statistically significant( t=3.415, P=0.002, P<0.01); Comparedwith the NC group, 4 weeks and 12 weeks DM group, the total cholesterol( TC), triglyceride( TG), low density lipoprotein cholesterol( LDL) increased, the difference is statistically significant(FTC=65.641, FTG=63.415, FLDL=61.241, P=0.01, 0.01, 0.01, P<0.05), High-density lipoprotein cholesterol( HDL) lower( FHDL=58.317, P=0.02, P <0.05); Compared with the NC group,4 weeks and 12 weeks t BHQ group, the total cholesterol( TC), triglyceride( TG), low density lipoprotein cholesterol( LDL) increased, the difference is statistically significant( FTC=65.641, FTG=63.415, FLDL=61.241, P=0.03, 0.02, 0.02, P<0.05), High-density lipoprotein cholesterol( HDL) lower( FHDL = 58.317, P=0.03, P <0.05); 4. Histological changes: 4 week HE staining showed that the NC group retinal tissue structure is clear, no significant pathological changes; DM group rat retinal cells arranged in a little disorder, there are different degrees of cell edema phenomenon; t BHQ rat retinal cells arranged in neat, little integration of internal and external nuclear layer, individual cells can be seen edema. 12 week HE staining showed that the NC group retinal tissue layers of the structure are normal, basic neatly arranged, no obviously pathological changes; DM group rat retinal structure loosely arranged, the cell layer disordered, cell edema obviously, the inner and outer nuclear layer disordered; t BHQ retina tissue structure is more clearly, some cells edema, no obvious abnormalities. 5. Immunohistochemical results: Compared with the NC group, the expression of Nrf2 and HO-1 protein was higher than that of 4 weeks DM group at rat retina tissue(tNrf2=3.115,PNrf2=0.002, PNrf2<0.01; tHO-1=3.485, PHO-1=0.000, PHO-1<0.01); 4 week t BHQ group was higher than that of 4 weeks DM group( tNrf2=2.473, PNrf2=0.031, PNrf2<0.05; tHO-1=2.785, PHO-1=0.024, PHO-1<0.05). Compared with the NC group, the expression of Nrf2 and HO-1 protein was higher than that of 12 weeks DM group( tNrf2=3.781, PNrf2=0.000, PNrf2< 0.01; tHO-1=3.785, PHO-1=0.000, PHO-1<0.01); 12 weeks t BHQ group was higher than that of 12 weeks DM group( tNrf2=2.576, PNrf2=0.038, PNrf2< 0.05; tHO-1=2.879, PHO-1=0.018, PHO-1<0.05); 12 weeks t BHQ group was higher than that of 4 weeks t BHQ group( tNrf2=2.516, PNrf2=0.042, PNrf2< 0.05; tHO-1=2.776, PHO-1=0.037, PHO-1<0.05);12 weeks DM group the expression of Nrf2 protein was higher than that of 4 weeks DM group( t=0.276, P=0.040, P<0.05); 6. Quantitative PCR: Compared with the NC group,the expression of Nrf2 and HO-1 m RNA was higher than that of 4 weeks DM group( tNrf2=4.758, PNrf2=0.031, PNrf2<0.05; tHO-1=5.114, PHO-1=0.029, PHO-1<0.05); 4 weeks t BHQ group was higher than that of 4 weeks DM group( tNrf2=5.133, PNrf2=0.023, PNrf2<0.05; tHO-1=4.758, PHO-1=0.033, PHO-1<0.05); Compared with the NC group, the expression of Nrf2 and HO-1 m RNA was higher than that of 12 weeks DM group( tNrf2=4.285, PNrf2=0.041, PNrf2<0.05; tHO-1 =4.514, PHO-1 =0.037, PHO-1 <0.05); 12 weeks t BHQ group was higher than that of 12 weeks DM group( tNrf2=4.976, PNrf2=0.028, PNrf2< 0.05; tHO-1=4.251, PHO-1=0.039, PHO-1<0.05); 12 weeks t BHQ group was higher than that of 4 weeks t BHQ group( tNrf2=4.292, PNrf2=0.040, PNrf2< 0.05; tHO-1=4.974,PHO-1=0.028, PHO-1<0.05);12 weeks DM group the expression of Nrf2 m RNA was higher than that of 4 week DM group( t=5.114, P=0.022, P<0.05). Conclusion: 1. t BHQ intervention can increased the expression of Nrf2, HO-1 significantly, and the retinal tissue antioxidant capacity, thereby reduced oxidative stress damage, delayed the progression of DR. 2. t BHQ intervention can lower blood sugar and reduce the pathological changes in retina, May become a new retina protective agent. |
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