| Objectives:1.To observe the effect of Modifield Gegenqinlian Decoction in preventing and treating cognitive dysfunction in diabetic rats.2.To Analysis of diabetic cognitive dysfunction rats hippocampal neurovascular unit, microvasculer tight junction protein, endothelial progenitor cells, hippocampal neurons morphology and PI3K/AKT/CREB signaling pathways, explore Modifield Gegenqinlian Decoction protection mechanism of neurovascular unit.3.To explict new targets of Modifield Gegenqinlian Decoction in treating diabetic cognitive dysfunction.Methods:120 SD rats were selected, and divided into blank group (15 rats) and high fat diet group (105 rats) after adaptive feeding. The blank group was given ordinary feed, while the high fat group fed with high fat diet for 4 weeks. The latter ones were injected the STZ into the abdomen to replicate the diabetic model, meanwhile the equal 0.1M sodium citrate were given to the blank control group. The model would be regarded as proper if the random blood glucose was more than 16.7mmol/L after 3 days. Proceedingly, the successful model rats were randomly divided into model group, and the modified Gegen Qinlian Decoction in large, medium and small dose group as well as western medicine control group. The blank group and the model group were given purified water through intrasgatric administration, and the treatment groups were resepectively given modified Gegen Qinlian decoction, cilostazol intragastric through intrasgatric administration in duration of 8 weeks. Ultimately, the body weight, blood glucose, and insulin were measured. Simultaneously, the number of endothelial progenitor cells in the peripheral blood was detected through FCM (Flow Cytomrtry). To observe the change in permeability of BBB, hippocampal neurons and microvascular morphology, HE, electron microscope were used.Evans blue(EB)in the brain tissue was surveyed by the means of EB fluorescent quantitation. Afterwards, RT-PCR was adopted to detect PI3K, AKT, CREB, HIF-1 alpha and VEGF, while Westen-blot to detect four kinds of tight connected protein, which were occluding, claudins, JAM-1 and ZO-1.Results:1. Morris water maze test shows the learning and memory ability of the diabetic rats decreased significantly at 8 weeks, illustrating the diabetic cognitive dysfunction model were successfully reproduced.2. After injection of Evans blue, the vessels of the diabetic rats diffused outward obviously, under the light microscope, we could see the surrounding vessels blue dyed, meanwhile the content of the Evans blue is significantly higher than the blank group(P<0.05). Under the electron microscope,we could see the extravascular space had a marked edema, vascular deformation and lumens stenosis. The immunohistochemical CD34 staining indicated the positive cells were less than the blank group, demonstrating the microvascular of the diabetic rats were damaged, blood-brain barrier was impaired, decreasing of the number of microvessels and the dysfunction of the repairment and neogenesis of the microvessels.3. Under the light microscope, we can see the number of neurons in hippocampal CA1 region of the model group reduced, with irregular arrangement, necrotic cells and glial cells, which illustrates hippocampal neuron of the diabetic rats appeared was damaged. Immunohistochemistry showed the expression of TGF-1, CREB expression decreased.4. After intervention of modified Gegen Qinlian Decoction, the cells and vesseles in the treatment group improved significantly, and the content of Evans blue decreased significantly compared with model group, while the expression of occluding, claudins and ZO-1 increased markedly under the electron microscopic. The number of EPCs increased significantly than that in model group, and the CD34 positive cells increased. Meanwhile the expression of PI3K, mRNA, AKTmRNA, CREBmRNA, HIF-1 alphamRNA, VEGFmRNA increased.Conclusion:1. Modified Gegen Qinlian Decoction can obviously improve learning and memory ability in diabetic rats.2. The Modified Gegen Qinlian decoction can protect diabetic rat blood brain barrier, which is related to improving the expression of tight junction proteins,such as occludin, claudins and ZO-13. The Modified Gegen Qinlian Decoction can promote diabetic rats hippocampus angiogenesis, improve the status of ischemia hypoxia of hippocampal and reduce injury of hippocampal neurons.4.The Modified Gegen Qinlian Decoction protect hippocampal neurons in diabetic rats with cognitive dysfunction,by adjusting the hippocampal neurons of IGF-1 and PI3K/AKT/CREB signaling pathway. |
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