| ObjectiveThe purpose of research is to explore the effect of Tibetan Medicine Berberis Cortex (TMBC) on spontaneous type2diabetec mice by observing the histology of retina, regulation of blood glucose level and the level of VEGF, HIF-1, PKC-β to reveal the mechanism of its prevention and treatment of DR.Methoddb/db monogenic genetic diabtic mutant mice model were select as subjects, seventy-two db/db mice of10postnatal weeks were selected while their thin db/+litters as normal contronls. To evaluate the experimental animal model of spontanneous diabetic retinopathy mice in morphology. Blood glucose and pathology were measured at the end of18weeks. The db/db mice were randomly divided into7groups according to the weight and the level of blood glucose:model control group, treated groups with high, middle, low dosage of TMBC, metformin group, calcium dobesilate group and normal control group, respectively. After two-month intragastric administration, during which all the animals were weighed and measured blood glucose every two weeks, the mice were put to death and eyeballs of the mice are taken out.Conventional HE staining was performed to observe pathologic change, retina stretched preparation was performed to observe microvascular changes and calculation of pericytes/endothelial cell ratio; Real-time quantitative PCR and Western Blot were carried out to detect the genes and proteins expression of retina VEGF, HIF-1, PKC-p. Immunohistochemistry was used to detect distributions of these three factors in the retina. Effects of TMBC was observed on spontaneously diabetic retinopathy in mice.ResultsFirstly, Evaluated by blood glucose level and retinal morphology (retinal tissue sections) for multi-angle db/db mice,18-week-old mice in model groups were significantly higher (P<0.01) in blood glucose as compared to normal group. Retinal edema, swelling and reduced number of retinal ganglion cells, inner plexiform layer swelling, capillary congestion and expansion, endothelial cell proliferation, thicken capillary wall were observed in retinal tissue sections of18-week-old spontaneous diabetic mice. We conclude that spontaneous diabetic mice (db/db mice) can be used as an animal model of human diabetic retinopathy,Secondly, In TMBC high group, blood glucose level significantly decreased (P<0.05) after two weeks treatment, whereas it took6weeks to show superior decrease of blood glucose (P<0.05) in TMBC low group. All3TMBC treatment groups showed significantly decreased blood glucose (P<0.05) after8weeks administration, the hypoglycemic effect of low dose of TMBC gradually increased with prolonged administration time.Thirdly, the results of light microscope observation:rat retinal tissue in blank control group neatly arranges on each floor. It has no obvious abnormalities. In retinal tissue of experimental model rats, the outer nuclear layer and inner nuclear layer cells disorder, the inner nuclear layer is obviously thinner, the number of retinal ganglion cells decreases significantly than the blank control group. There was significant difference. Retinal damage in experimental group rats was slighter than the experimental model group. Both inner nuclear layer cells and outer nuclear layer cells order, ganglion cells for inner and outer nuclear layer cells was relatively increased.Fourthly, retina stretched preparation:mice of model group showed varying retinal capillary diameter, incrassation of vascular basement membrane and wall, disorganization or even distortion of blood vessels alignment. Capillary lumen blood vessels were filled with leukocyte thrombosis, endothelial cell hyperplasia and perithelial cell decrease were observed. Compared with the control group, E/P value of model group mice retinas was significantly higher (P<0.05), meanwhile significantly lower E/P value (P<0.05) was obtained in high TMBC group, as compared to that of the control group.Fifthly, immunohistochemical results showed that retina of control group mice showed weakly positive HIF-la, PKC-β, VEGF immunohistochemical reaction and low expression but mice of model group showed positive PKC-β, VEGF immunohistochemical reaction and high expression, which are significantly different than the control group (P<0.01). Although no statistical differences was found in HIF-la expression of db/db mouse retina when compared with the model group, there was a trend of decrease in the treatment groups: expression of PKC-Pwas significantly reduced (P<0.01) in high TMBC group, significant decrease of VEGF was observed in both low TMBC group and berberine group (P<0.01).Sixthly, the protein expression results indicated that protein expression of HIF-la, PKC-β, VEGF in model group was significantly higher than those of control group (P<0.05). Compared with model group, high dose treatment of TMBC significantly down-regulated the protein expressions of HIF-1α and PKC-β (P<0.05). TMBC also lowered the VEGF expression as compared with the model group, but no significant difference was detected.western blot.Finally, mRNA expression results showed that there is a significant increase of HIF-la, PKC-β,(P<0.01) VEGF mRNA expression in model group compared with the control group. High dose TMBC group significantly reduced PKC-β, VEGF mRNA expression (P<0.05). HIF-1α expression also decreased, but in a non-significant way.ConclusionSpontaneously diabetic mice (db/db mice) can be considered as an animal model for human diabetic retinopathy. TMBC has a good preventive effect for DR, which is possibly through inhibiting the expression of HIF-la, PKC-β, VEGF to regulate of retinal vascular endothelial cell function, improve spontaneous retinal microvascular damage in diabetic mice, protect retinal capillaries while preventing the formation of new blood vessels, so that it could relieve symptoms and living conditions of spontaneous diabetic mice to improve their quality of life. |
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