| Diabetes mellitus represents a common metabolic disorder, which may lead to severe microvascular complications, such as kidney failure, retinopathy, and peripheral nerve system. Recently, accumulating evidence suggests that diabetes may also affect the central nervous system and lead to memory and learning impairment accompanied by the alterations of structure, function and metabolism of the brain, referred to as diabetic encephalopathy. Recently, ample evidence suggests that uncontrolled diabetes may increase the risk of vascular dementia and Alzheimer's disease, leading to memory and learning impairment in diabetic patients. But the diagnosis diabetic encephalopathy lacks clear criteria and is therefore difficult to ascertain.The Smad family comprises receptor-regulated Smads (R-Smads, including Smad2 and Smad3), common partner Smads (CO-Smads, including Smad4) and inhibitory Smads (I-Smads, including Smad6 and Smad7). They modulate the activity of transforming growth factor beta ligands. The phosphorylation of Smads (to form pSmads) triggers the recruitment of the cofactor Smad4, and the entire Smad complex is translocated to the nucleus where it binds to DNA response elements and, together with transcription factors and various cofactors, promotes transcription of a rangeof target genes. This pathway is associated with the transcriptional activity of APP. Therefore, investigation of functional role of Smad signaling pathway in astrocyte of diabetic associated cognitive decline may offer a better understanding of pathogenesis in cognitive impairment and important targets for treatment strategies.The aim of this study is to clarify the exact role for Smad signaling pathway in the hippocampus of cognitive impairment diabetic rat and effect of advanced glycation end products on astrocytes in primary culture. This study is consisted of three main parts:the first part is to investigate the effect of streptozotocin-induced diabetes on cognitive function and hippocampal ultrastructure in the rat, the second is to see whether Smad signallingwas activated in the hippocampus of streptozotocin-induced diabetic cognitive impairment rats and investigate the expression of pSmad2,pSmad3,GFAP and TGFβ1 in hippocampus of diabetic cognitive impairment rat. Finally, the effect of AGEs and TGFβ1 on astrocytes in primary culture and observe the expression of pSmad2,pSmad3,APP,Aβ, further investigation underlying molecules mechanism of diabetic astrocyts. Part I Effect of Streptozotocin-induced diabetes on cognitive function and hippocampal ultrastructure in the ratsObjective:To evaluate the effect of streptozotocin-induced diabetes on cognitive function and hippocampal ultrastructure in the rat.Methods:Diabetes was induced by a single intraperitoneal injection of streptozotocin at a dose of 60mg·kg-1 body weight dissolved in citrate buffer. The streptozotocin-injected rats with blood glucose levels≥16.7 mmol·L-1 3 days after injection were included in the study and were randomly divided into two groups:4w DM (n=10) and 8w DM (n=10). Control rats were divided into two groups:4w CON (n=10) and 8w CON (n=10). After 8 weeks of experiment, rats were tested in Morris water maze. The animals were trained to locate the platform on 5 consecutive days, and both latency times and distances to reach the platform were recorded. On the 5th day, the rat was given a maximum of 60 s to locate the hidden platform and the time spent in the quadrant of the pool containing the hidden platform was recorded. SOD,GSH-Px and MDA were measured in plasma and hippocampal tissues, and the cellular ultrastructure of the hippocampus were observed through light microscope and transmission electronic microscope.Results:In Morris water maze test, in diabetic rats the latency (P<0.001) and distance (P<0.01) swum reach to the platform increased significantly compared to controls, and the percentage of time spent in the quadrant of the pool with platform decreased notably (P<0.01). The levels of GSH-Px and SOD decreased significantly (P<0.001) and the levels of MDA increased significantly in diabetic groups compared to controls (P<0.001). Conspicuous intracytoplasmic vacuole formation was present in neurons and astrocytes of diabetic rats'hippocampus. Obvious chromosome aberrations, dilatate, widened synaptic clefts as well as disappeared mitochondriales crista were also observed in the diabetic rats' hippocampus by TEM. However, H-E staining is not obvious pathological change by light microscope.Conclusion:In summary, we demonstrate that moderate impairment of learning and memory occurred in diabetic rats. At the same time, oxidative stress induced by hyperglycemia and ultrastructure of diabetic rats'hippocampus are abnormal. PartⅡExpression changes of phosphorylated Smad2 and Smad3 in the hippocampus of streptozotocin-induced diabetic cognitive impairment ratsObjective:Activation of the Smad signalling pathway has been implicated in the pathological process of diabetic associated complications.The current study was designed to see whether Smad signallingwas activated in the hippocampus of streptozotocin-induced diabetic cognitive impairment rats.Methods:We determined the expression level of phosphorylated Smad2 and Smad3 in the hippocampus by western blot analysis and double immunofluorescene labeling assay. At same time, we determined the expression level of TGFβ1 in the hippocampus by western blot analysis, and pSmad2,pSmad3,GFAP staining by immunohistochemistry.Results:Compared with the normal controls, immunoblot analysis showed that pSmad2 averagely increased by 0.10-fold at week 4 and 2.04-fold at week 8 post STZ induction in the hippocampal homogenates (P<0.01 at week 8), pSmad3 increased by 0.66-fold at week 4 and 1.91-fold at week 8 of diabetic duration (P<0.01). pSmad2 and pSmad3 immunoreactivity were predominantly detected in the nuclei of astrocyte in hippocampus The percentage of pSmad2+astrocyteswas signifi cantly increased in CA1 region (31.45±3.06%vs.9.77±1.87%, P<0.01), CA3 region (34.98±4.33%vs.11.06±3.97%, P< 0.01) and DG region (36.87±5.17%vs.15.01±3.10%, P<0.01) in the hippocampus of diabetic rats as compared to controls at week 8. Similarly, double immunofl uorescence labeling assay revealed a signifi cant increase in the percentage of pSmad3 immunoreactive astrocytes in CA1 region (23.33±4.53%vs.7.35±1.14%, P<0.01), CA3 region (18.32±6.34%vs.11.36±4.53%, P<0.05) and DG region (20.64±4.45% vs.14.39±4.10%, P<0.05) in the hippocampus of diabetic rats as compared with controls at week 8. Expression levels of TGFβ1 and GFAP increased in hippocampus of diabetic rats, whereas expression levels of pSmad2 and pSmad3 are not changed by IHC.Conclusion:These data indicate that Smad signalling is enhanced in hippocampal astrocytes of diabetic rats, and may thereby represent a clue to explore its exact role in the development of diabetic associated cognitive decline. PartⅢEffect of AGEs on smad signaling pathway and APP in primary cultured astrocytesObjective:To investigate the effects of AGEs on smad signaling pathway and APP in primary cultured astrocytes.Methods:Primary cultured astrocytes were treated by advanced glycation end products at different concentration of 0,50,100ug·ml-1. Cell proliferative activity was assessed by MTT assay. Immunofluorescene labelling assay was employed to investigate the celluar location of pSmad2 and pSmad3 protein in primary cultured astrocytes. A neutralizing anti-RAGE antibody and TβR I inhibitor were added into cells 30 min before AGEs stimulations. The expression levels of pSmad2,Smad2,pSmad3,Smad3,APP,Aβprotein were measured by western blot.Results:Proliferation of astrocytes was significantly increased compared with that of control at 24h (0.078±0.019 vs.0.117±0.028, P<0.05),48h (0.109±0.044 vs.0.284±0.043, P<0.01),72h (0.178±0.034 vs.0.512±0.103, P<0.01) post AGEs stimulation. By immunofluorescene, pSmad2 and pSmad3 were predominantly detected in the nuclei of astrocytes, and expression of pSmad2 and pSmad3 increased at 1h (P<0.01),24h (P<0.01), 48h (P<0.01), peaking at 24h. Western blotting showed that AGEs induced Smad2 and Smad3 phosphorylation by astrocytes in a dose-dependent manner, being at 1h, peaking at 24h and 48h after AGEs stimulation. AGEs were able to induce TGFβ1 expression by astrocytes, being significant at 24h (P<0.01) and 48h (P<0.01). At the same time, AGEs induced marked APP and Ap protein expression at 24h (P<0.01). Finally, western blot analysis showed that the neutralizing anti-RAGE antibody and TβR I inhibitor completely inhibited AGE-induced Smad2 phosphorylation and APP at 24h. This indicates that AGEs signal through RAGE to activate smad signaling via TGFβ-dependent pathways.Conclusion:Overall, our results indicate that AGEs can induce astrocytes proliferation and act by activating smad signaling to mediate diabeteic cognitive dysfunction via TGFβ-dependent pathways. Therefore, inhibit smad pathway and AGEs may represent a potentially treatment strategy against diabetic cognitive dysfunction.
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