| Diabetic retinopathy(DR) is the most common and most serious eye complication of diabetic mellitus, which is recognized as a significant cause of irreversible blindness. As the change of the life-style and the prolongation of the average life span, the morbidity rate of DR shows rising trend. The quality of patients’life are influenced badly because of the damage of eyesight caused by microangiopathy and the lesion of nervous tissue in retina. Therefore, the prophylaxis and treatment of DR become to a momentous subject in the research field of diabetic mellitus and its complications. Chinese materia medica(CMM) formula which has multi-targets and multi-pathways treatment effect, could fully produce the best effect as for its integral regulation. Purendan superfine powder(PRD) has the functions of boosting the energy, nourishing yin, clearing away heat, activating blood, dissolving stasis and removing obstruction in collaterals, which aims at the key of the nosogeny of diabetic mellitus, heat, deficiency and stasis. In this study, diabetic rats models built up by intraperitoneal injection of Streptozotocin(STZ), are used to investigate the protective effects of PRD on retinopathy of diabetic rats and provide experimental and theoretical basis of the treatment of DR by CMM formula.Part I Effects of Purendan superfine powder on expression of retinal VEGF and PEDF in diabetic ratsObjective:To investigate the protective effects of Purendan superfine powder(PRD) on microangiopathy in retina of diabetic rats by observing expression of retinal vascular endothelial growth factor(VEGF) and pigment epithelium-derived factor(PEDF) expression in diabetic rats. Methods:136male Wistar rats were randomly divided into3groups:normal control group, diabetes model group and PRD treatment group. The rats in normal control group were bred as normal. The rats in diabetes model group and PRD treatment group were made diabetes model by injecting2%STZ(25mg/kg,3d) into the abdominal cavity continuously and blood glucose (BG)≥16.7mmol/L was taken as standard. After the diabetes model was successfully established, the rats in model group had no more treatment, the rats in PRD treatment group were lavaged with PRD (1.8g/kg/d,3m).2ONE TOUCH blood glucose meter was used to detect the BG of rats in each group.3HE staining was used to observe histopathological change in retina of rats in each group.4SP immunohistochemical staining, Western blotting were used to assess the expression of VEGF and PEDF protein in retina of rats in each group.5RT-PCR was employed to examine the expression of VEGF and PEDF mRNA in retina of rats in each group.Results:1BGThe BG of rats in model group was (21.63±1.01)mmol/L, which increased obviously compared with rats in normal control group (P<0.01). The BG of rats in PRD treatment group was (14.60±0.95) mmol/L, which was obviously lower than that of model rats (P<0.01).2Change of retinal histomorphologyNormal rats showed integrated configuration in each retinal layer, the layers were clear, the surface of inner limiting membrane was smooth; For the neurocyte in different levels, the size was uniformity and the arrangement is orderly. In diabetic model group, inner limiting membrane became edema and irregular, the retinal vascular endothelial cells broke out the inner limiting membrane; the layers are unclear, the arrangement of the ganglion cells was disorderly and sparse, the quantity of the neurocyte reduced, vacuole-like transformed, karyopycnosis, partial hypochromatosis. In PRD treatment group, inner limiting membrane became dema and irregular lightly, the arrangement of the ganglion cells was disorderly lightly, the quantity of the neurocyte reduced lesser.3VEGF expression in retina of ratsVEGF immunopositive products were buffy granules and located in cytoplasm, VEGF immunopositive cells were seen in retinal ganglion cell layer and inner nuclear layer. The expression of VEGF protein and mRNA in retina were obviously higher in diabetic rats than in normal control rats (P<0.01), those were obviously lower in PRD treatment group than in diabetes model rats (P<0.01).4PEDF expression in retina of ratsPEDF immunopositive products were buffy granules and located in cytoplasm, PEDF immunopositive cells were seen in retinal ganglion cell layer and inner nuclear layer. The expression of PEDF protein and mRNA in retina were obviously lower in diabetic rats than in normal control rats (P<0.01), those were obviously higher in PRD treatment group than in diabetes model rats (P<0.01).Conclusions:PRD can reduce vascular leakage and inhibit pathologic neovascularization by up-regulating PEDF expression and down-regulating VEGF expression in retina of diabetic rats, so has protective effects on retinal microangiopathy in diabetic rats.Part II Effects of Purendan superfine powder on retinal neuron apoptosis and expression of apoptosis-associated genes in diabetic ratsObjective:To investigate the protective effects of PRD on nervous tissue damage in retina of diabetic rats by observing retinal neuron apoptosis and expression of apoptosis-associated genes in diabetic rats.Methods: 136male Wistar rats were randomly divided into3groups:normal control group, diabetes model group and PRD treatment group. The rats in normal control group were bred as normal. The rats in diabetes model group and PRD treatment group were made diabetes model by injecting2%STZ(25mg/kg,3d) into the abdominal cavity continuously and blood glucose (BG)≥16.7mmol/L was taken as standard. After the diabetes model was successfully established, the rats in model group had no more treatment, the rats in PRD treatment group were lavaged with PRD (1.8g/kg/d,3m).2TUNEL technique was used to detect neuron apoptosis in retina of rats in each group.3SP immunohistochemical staining, Western blotting were used to assess the expression of bcl-2, bax and caspase-3protein in retina of rats in each group.4RT-PCR was employed to examine the expression of bcl-2, bax and caspase-3mRNA in retina of rats in each group.Results:1Neuron apoptosis in retinaTUNEL positive products were brown granules and located in nucleus. TUNEL positive cells were seen in retinal ganglion cell layer and inner nuclear layer. AI of retina was obviously higher in diabetic rats than in normal control rats(P<0.01), AI of retina was obviously lower in PRD treatment group than in diabetes model rats(P<0.01).2Bcl-2and bax expression in retina of ratsBcl-2and bax immunopositive products were buffy granules and located in cytoplasm, bcl-2and bax immunopositive cells were seen in retinal ganglion cell layer and inner nuclear layer. The expression of bcl-2protein and mRNA in retina, bcl-2/bax ratio were obviously lower in diabetic rats than in normal control rats(P<0.01), the expression of bax protein and mRNA in retina were obviously higher in diabetic rats than in normal control rats (P<0.01). The expression of bcl-2protein and mRNA in retina, bcl-2/bax ratio were obviously higher in PRD treatment group than in diabetes model rats (P<0.01), the expression of bax protein and mRNA in retina were obviously lower in PRD treatment group than in diabetes model rats (P<0.01).3Caspase-3expression in retina of ratsCaspase-3immunopositive products were buffy granules and located in nucleus, caspase-3immunopositive cells were seen in retinal ganglion cell layer and inner nuclear layer. The expression of caspase-3protein and mRNA in retina were obviously higher in diabetic rats than in normal control rats (P<0.01), those were obviously lower in PRD treatment group than in diabetes model rats (P<0.01).Conclusions:1The neuron apoptosis and expression of apoptosis-associated genes in retina of diabetic rats were disorder, manifested as, elevated AI of retina, decreased bcl-2expression and increased bax, caspase-3expression.2PRD can reduce the apoptosis cells by up-regulating bcl-2expression and down-regulating bax, caspase-3expression in retina of diabetic rats, so has protective effects on retinal nervous tissue damage in diabetic rats. |
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