| Aims/hypothesis:The association between long-term hyperglycemia and chronic complications of diabetes mellitus has been reported in both type 1 and type 2 diabetes. Mechanisms involved including excess sorbitol-aldose reductase pathway flux, hyperactivity of protein kinase C (PKC) isoforms, increased oxidative stress and microangiopathic changes leading to ischemia. Advanced glycation end-products (AGEs) are unstable, reactive and toxic compounds that alter the extracellular matrix and exacerbate oxidative stress which has been known as the major factors that contribute to the pathogenesis of diabetic complications. AGEs interactions with the receptor for AGEs (RAGE) modify proinflammatory cytokine expression, increase free radical production via quenching of nitric oxide contributing to defective vasodilatation. induce irreversible crosslinks in extracellular matrix structural proteins and transform intracellular signaling pathways in part through nuclear factor-kappa B (NF-κB). Whereas kidney, retina, blood vessels and peripheral nerves are the primary targets of long-term diabetes, brain damage was previously considered secondary to vascular disease. With the increased understanding of diabetes, it has been disclosed that humans with long-term, particularly poorly controlled diabetes, however, develop cognitive dysfunction and an increased risk for dementia and cerebral atrophy, which is now an accepted concept as encephalopathy that still needs a defination. To prevent the development of this disease and to improve advanced brain injury, effective therapies directed toward the key molecular target are required. Grape seed proanthocyanidin extracts (GSPE), an antioxidant derived from grape seeds have been reported to possess a variety of potent properties may have therapeutic potential in the prevention and treatment of complications in patients with diabetes. In this study, we examined whether GSPE could attenuate the degeneration changes in the diabetic brain by modulating the AGEs/RAGE and NF-κB pathway.Methods:Male Wistar rats weighting 180-220 g were purchased from the Animal Centre of Shandong University. Animals were kept in individual cages on a 12-h light-dark cycle with an ambient temperature of 22±1℃, with free access to food and water. 40 randomly selected rats were divided into 2 groups: Control group 1(C1) and control group treated with GSPE (C2, administrated with GSPE with a dosage of 250mg/kg).Streptozotocin ( STZ) induced diabetic rats received a single dose of STZ (55 mg/kg, injected into tail veins) freshly dissolved in 0.1 M sodium citrate buffer (pH 4.5 ) after a 12 hours' overnight fasting. Control animals received a single tail vein injection of 0.1M citrate buffer only. Glucose concentration from fasting animals was measured daily, in a blood sample obtained from the tail by pinprick, with a glucose oxidase-impregnated test strip and a reflectance meter (One Touch II, Lifescan, USA). Hippocampus of the brains were immunohistochemically stained for RAGE and for the detection of mRNA of RAGE and NF-κB by reverse transcriptase coupled to polymerase chain reaction (RT-PCR). For morphological observations, hippocampus of the brains were stained by immunohistochemistry for neuron growth factor (NGF) and glial fibrillary acidic protein (GFAP).Results:1,At the end of this study, the weight of diabetic rats was lower than that of the controls . Mouse glycated hemoglobin, fasting plasma glucose and AGEs were increased in diabetic mice relative to controls. After treated with GSPE, the serum AGEs decreased in DM2 group than that of DM1, while there were no differences in their weights and FPG (21.35±3.59 vs 19.04±3.24mmol/L, t=1.494, P=0.153).2,It was found that the number of neurons of hippocampus in STZ induced rats decreased (t=3.672,P=0.002) and had degenerative changes: the layers of neurons were not intact, the nuclei of neurons were shrinked and in irregular orders, the cytoplasma of neurons turned into pink-colored. After treated with GSPE, the above changes were improved, and the number of neurons in DM2 group increased comparing to DM1 group (t=-2.702,p=0.015) .3,By GFAP immunochemistry, it was found that the grey value of astrocytes in CA region of hippocampus in STZ induced rats decreased comparing to the non-diabetic controls (t=3.065, p=0.007) , while the number of GFAP positive cells increased (t=-3.524, p=0.005); After treated with GSPE, the grey value of astrocytes in CA region of hippocampus in STZ induced rats increased comapring to DM1 group(t=-2.360, p=0.030) , while the number of GFAP positive cells decreased (t=2.981, p=0.013). Recent evidence suggests that the upregulation of GFAP, an astrocyte-specific intermediate filament component, is a biological marker of neurotoxicity after cerebral injury. It is suggested that there is proliferation of astrocytes in the hippocampus of diabetic rats, and GSPE can decrease the proliferation of astrocytes.4,By RT-PCR, it was found that the expression of RAGE mRNA in CA region of hippocampus of STZ induced rats increased comparing to the non-diabetic controls(t=-3.795,P=0.002); After treated with GSPE, the expression of RAGE mRNA in CA region of hippocampus decreased comapring to DM1 group(t=2.588,P=0.019).5,By RT-PCR, it was found that the expression of NF-κB mRNA in CA region of hippocampus of STZ induced rats increased comparing to the non-diabetic controls(t=-3.972,P=0.001); After treated with GSPE, the expression of NF-κB mRNA in CA region of hippocampus in STZ induced rats decreased comapring to DM1 group(t=3.480,P=0.003).6,By immunochemistry, it was found that the grey value of RAGE in CA region of hippocampus of STZ induced rats decreased comparing to the non-diabetic controls (t=2.482,P=0.027) while the number of RAGE positive cells increased (t=-5.218, p<0.001); After treated with GSPE, the grey value of RAGE in CA region of hippocampus in STZ induced rats increased comapring to DM1 group(t=-2.437, P=0.026), while the number of RAGE positive cells decreased (t=3.609, p=0.003).7,By immunochemistry, it was found that the grey value of NF-κB in CA region of hippocampus of STZ induced rats decreased comparing to the non-diabetic controls (t=2.747,P=0.019), while the number of NF-κB positive cells increased (t=-3.720, p=0.002); After treated with GSPE, the grey value of NF-κB in CA region of hippocampus in STZ induced rats increased comapring to DM1 group(t=-2.859, P=0.017), while the number of NF-κB positive cells decreased (t=4.302, p=0.001).8,By Pearson's relationship test, it was found that the expression of RAGE and NF-κB was realted to serum AGEs, respectively.Conclusions:1,Long term chronic hyperglycemia can cause degenerative changes in CA region of hippocampus in STZ induced diabetic rats, including the decrease of neuron numbers and the proliferation of glial astrocytes.2,At both mRNA and protein levels, long term chronic hyperglycemia can cause the overexpression of RAGE and NF-κB in CA region of hippocampus in STZ induced diabetic rats. The serum AGEs increased and was related to the the expression of RAGE and NF-κB, which suggusted that the signal transdcution pathway of AGEs/RAGE/NF-κB plays an important role in the pathogenesis of diabetic encepholopathy.3,Comparing with the controls, in STZ induced diabetic rats , after treated with GSPE, the number of neurons increased, the serum AGEs decreased, the degeneration in CA regions of hippocampus were improved , while the expression of RAGE and NF-κB also decreased in both mRNA and protien levels , which suggests that GSPE can improve and protect diabetic encepholopathy while modulating the AGEs/RAGE/NF-κB pathway. In summary, long term chronic hyperglycemia can cause the over-expression of AGEs/RAGE and NF-κB in CA region of hippocampus in STZ induced diabetic rats, hyperglycemia induced activation of AGEs/RAGE/NF-κB pathway takes an important role in the pathogenesis of the degenerative changes of diabetic hippocampus. GSPE improves some changes including neuron degeneration and the proliferation of astrocytes compared with non-treated diabetic rats, via decreasing the expression of AGEs/RAGE and NF-κB in the CA region of hippocampus in diabetic rats at a daily oral dosage of 250mg/kg and act as an antagonist to RAGE, which suggests that GSPE be a useful remedy in the treatment of diabetic encephalopathy.
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