ABSTRACT
Background The primary pathological basis of diabetic retinopathy (DR) is new blood formation due to anoxia and inflammation,which results in breakdown of blood-retinal barrier (BRB).Vascular endothelial growth factor (VEGF) is a key factor promoting neovascularization.Researches determined that lycium barbarum polysaccharides (LBP) can protect cells against oxidative damage.However,the study of LBP in ophthalmology is lack.Objective This study was to investigate the effects of LBP on the dynamic pathological change of retinal vessels and expression of VEGF in retina of diabetic rats.Methods One hundred and seventeen SPF male SD rats were randomly divided into the normal control group,the diabetic mellitus (DM) group and the LBP group according to random number table.Type 1 DM models were induced by intraperitoneal injection of streptozotocin (STZ,55 mg/kg) in the rats of the DM group and the LBP group,and then 250 mg/kg LBP was intragastically administered in the rats of the LBP group.The morphological change of retinal vessels was dynamically observed by retinal stretched preparation with Evans blue (EB) in 4,10 and 16 weeks after modeling.E B (45 mg/kg) was slowly injection via jugular vein,and 1％ polyoxymethylene was infused into the left ventricule.The eyeballs were extracted and retina were isolated.EB content in the retinas (mg/g) was calculated using retinal stretched preparation method at the time points mentioned above.Expressions of VEGF protein and mRNA in the retinas were detected by immunohistochemistry and real-time quantitative PCR at various time points,respectively.Results Retinal stretched preparation with EB exhibited that the abnormal degree in the shape,diameter of vessels and leakage of the retinal blood vessels were significantly slighter in the LBP group than those of the DM group in 4,10,16 weeks after modeling.At 4,10,16 weeks,EB content in the retinas was (12.17±1.55),(16.46±1.60) and (19.55±1.49) mg/g,which was significantly lower than (15.76± 1.90),(21.61 ±2.05) and (26.30±2.28) mg/g of the DM group (P＜0.05).Immunochemistry showed that the expression of VEGF protein primarily located at retinal ganglion cells (RGCs) layer.The staining intensity for VEGF protein was weaker in the LBP group than that of the DM group.The expression levels of VEGF protein (A value) in the LBP group were 0.234±0.011,0.331±0.023 and 0.536±0.031at various time points,with significant decline in comparison with 0.281±0.018,0.533±0.055 and 0.765±0.075 of the DM group (all at P＜0.05).Real-time quantitative PCR revealed that the expression levels of VEGF mRNA were 0.157±0.013,0.505 ±0.114 and 1.577±0.074 in the LBP group at various time points,which were significantly lower than 0.235±0.209,1.043±0.084 and 2.446±0.061 of the DM group (all at P＜0.05).Conclusions LBP can alleviate the DM-induced retinal vasculopathy,lessen the leakage of vessels well,and further protect the BRB.
ABSTRACT
Background Research demonstrated that mitochondrial pathway plays a key role in cell apoptosis.Purendan supermicropowder(PRD),a traditional Chinese medicine,may be a potentially effective therapy for neuron apoptosis in diabetic retina.Objective This study was carried out to investigate the effects of PRD on aldose reductase(AR)activity,neuron apoptosis and mitochondrial pathway in retina of diabetic rat.Methods Thirty-six clean male Wistar rats were randomly divided into normal control group,diabetes model group,PRD treatment group randomly and 12 rats for each group.The diabetes models were established by intraperitoneal injection of 25 mg/(kg · d)streptozotocin(STZ)for 3 consecutive days,and blood glucose ≥ 16.7 mmol/L was taken as the standard.PRD solution of 1.8 g/(kg · d)was lavaged in 12 models for 3 months.The eyeballs were enucleated for the preparation of retinal tissue homogenate and slice.AR activity in the retina was detected by ultraviolet spectrophotometry,and neuron apoptosis in retina was assayed by TUNEL staining.Western blot was used to assess the expressions of bcl-2,bax,cyt-c and caspase-3 protein in the retina.The use of animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee(Version 1988).Results Statistically significant differences were found in AR activity and AI among the normal control group,diabetic group and PRD groups(F=90.115,165.540,P＜0.01),and those of diabetic group were evidently higher than the normal control group and PRD group(P＜0.01,P＜0.01).The positive TUNEL cells mainly located in inner nuclear layer and retinal ganglion cell layer.The expressions of bax,cyt-c,caspase-3,bcl-2 and bcl-2/bax in retina were obviously different among these three groups(F =51.332,41.262,25.888,38.564,47.870,P＜0.01),and the expression of bax,cyt-c and caspase-3 protein in diabetic group evidently elevated in comparison with the normal control group and PRD group(t ＝ 10.32,11.04,6.91,P ＜ 0.01)and the expressions of bcl-2 protein and bcl-2/bax value were significantly lower in diabetic rats than in the normal control rats(t =18.05,12.23,P＜0.01).AR activity by AI of retina,the expressions of bax,cyt-c and caspase-3 proteins in retina were obviously lower in PRD group than in diabetes model rats(P ＜ 0.01),and the expression of bcl-2 protein and bcl-2/bax value were significantly higher in PRD group than in diabetes group(P＜0.01).Conclusions PRD can protect retina against the damage caused by high glucose by suppressing AR activity by downregulating the expressions of bax,cyt-c,caspase-3 proteins,increasing the expressions of bcl-2 protein in retina of diabetic rats and further inhibiting the mitochondrial pathway and reducing cell apoptosis in retina of diabetic rats.