| Objective:To study the protective effect and potential mechanism of gastrodin in early diabetic retinal nerve cytopathy (RNC) by observing and testig the effect of gastrodin in symbiotic relationship between BV2 microglia and retinal ganglion cells(RGCs) cultured by high glucose and expression of pro-nerve growth factor(proNGF) and exploring the early damage mechanisms of diabetic retinal ganglion cells to explore gastrodin protective effect and possible protection mechanisms on early.Methods:On the cellular level, RGCs cultured primary and BV2 mouse Microglia were cultured and passaged,3-5 generations were taken to experiments. These two kinds of cells were cultured together and given different concentration of gastrodin. The morphology of two kinds of cells and symbiotic relationship between the two cells were observed and taken photographs under an inverted microscope. Western blotting detected the expression of proNGF in cell supernatant from each group and statistical analysis was going on. On the animal level, eighty SD rats were divided into four groups as follow:Control group, physiological saline group, low and high concentration gastrodin group. SD rat diabetic models were established by streptozotocin intraperitoneal injection which was considered as success when the blood sugar was measured over 16.7mmol·L —1. The model rats were given 50mg · kg—1, 100mg·kg—1 gastrodin respectively by intragastric administration for 8 weeks. Physiological saline group only received physiological saline by intragastric administration for 8 weeks. All rats eyeballs were executed and made into frozen sections. The nuclear was stained by PI. RGCs in ganglion cell layer was observed and counted. THY-1 and p75 nerve growth factor receptor proNGF expression were debected by immunohistochemical staining. The expression of retinal nerve growth factor proNGF was checked by western blot.Results:On the cellular level, the results showed that hyperplasia of RGCs in high glucose group weakened and axons were reduced compared with the control group, while the BV2 cells proliferation was more active and two cells aggregation reduced compared with the control group. After gastrodin treated, proliferation of RGC in high glucose was more active, axons grew longer, and quantity of BV2 cells reduced, meanwhile both kinds of cells aggregation increased compared with high glucose group. This change was dependent on the concentration of gastrodin. Western blotting strips and semi-quantitative analysis showed that the expression of proNGF in control group was lower while the high glucose group was significantly higher, and there was no significant difference of proNGF expression in the low and moderate concentration gastrodin groups compared with high glucose group, but the expression of proNGF in the high concentration gastrodin group was significantly reduced compared with the high glucose group. On the animal level, SD rat retina RGCs number reduced significantly compared with the control group after hyperglycemic.Cell counts were 5.50±4.50 and 24.75±3.50, and the difference was statistically significant (P<0.05). Double immunofluorescence display that p75NTR diabetic rats and RGCs cell markers THY-1 co-expressed in the GCL layer, which showed a clear yellow fluorescence. The GCL cell number of gastrodin treated diabetic rats was significantly more compared with physiological saline control group. Western blot displayed that physiological retina of diabetic rats proNGF protein expression was significantly increased compared with control group (P<0.05).Conclusion:So gastrodin could improve the effects of high glucose on RGCs morphology and aggregation with microglial cells and decrease the expression of proNGF, which may be one of the protective mechanisms of gastrodin against RGCs damage in early diabetes. |
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