| Diabetes mellitus can cause central nervous system complications characterized by mild and moderate cognitive impairment and decreased learning and memory ability,known as diabetic encephalopathy.Elderly diabetics are about twice as likely to develop dementia as their normal peers.At present,the prevalence rate of type 2 diabetes is more than 10%in the elderly population,and more than half of them are accompanied by the decrease of learning,memory and cognitive ability.Diabetic encephalopathy has both cerebrovascular disease-and Alzheimer’s disease-like changes,it’s pathogenesis is complex and pathological mechanism is still unclear.It is believed that the damage of central nervous system neurons and support cells by diabetic brain microenvironment,such as high glucose,high cholesterol,high triglyceride,high fatty acid and disturbance of insulin signals,is an important factor in inducing diabetic encephalopathy.Astrocytes are one of the most abundant and versatile nerve cells in the central nervous system.It plays an important role in the homeostasis of brain ions,the release and uptake of neurotransmitters,the growth and development of neurons as well as the cellular signal transductions.Therefore,with the deepening of research,the cytopathic mechanism of neuron and its support cells has gradually become the focus of diabetic encephalopathy.The aim of this study was to investigate the effects of hyperlipidemia or hyperglycemia on primary cultured neurons or astrocytes in order to find out the key pathological factors in the pathogenesis of diabetic encephalopathy,and also the potential mechanisms.This work is divided into the following three parts:Part I Effects of high glucose and free fatty acid on cell viability of neurons and astrocytesIn order to find out the key pathological factors in the pathogenesis of diabetic encephalopathy.The effects of high free fatty acid and high glucose on neurons and astrocytes in vivo were simulated by glucose or palmitic acid injury model of primary cultured neurons and astrocytes in vitro.Primary neurons and astrocytes were isolated and cultured from hippocampal or cortical tissues of neonatal Wistar rats within 12 hours after birth.Immunofluorescence staining of specific labeled proteins showed that,the purity of primary cultured astrocytes was greater than 98%and that of neurons was greater than 95%.For hyperglycemic injury experiment,the viability of neurons or astrocytes was detected by MTT assay after 24h,48h and 72h cultured in medium containing 35mmol·L-1 G,17.5mmol·L-1 G,5.5mmol·L-1 G or 5.5mmol·L-1 G plus 17.5mmol·L-1 mannitol respectively.For hyperlipidemia damage experiment,the viability of neurons or astrocytes was detected by MTT assay after 24h,48h and 72h cultured in medium containing 0.1,0.05,0.01 or 0 mmol·L-1 PA respectively.The results showed that high glucose significantly injured astrocytes while had no obvious effect on neurons,the cell viability of astrocytes was decreased by 20%after cultured in medium containing 35mmol·L-1 G for 72h.Similarly,high free acid significantly damaged astrocyte while also had no obvious effect on neurons,the cell viability of astrocytes was decreased by 51%after cultured in medium containing 0.1mmol·L-1 PA for 72h.It is concluded that injury of astrocytes by free fatty acids may be an important pathological mechanism of diabetic encephalopathy.Part Ⅱ Mechanism research on palmitate-induced astrocyte damageThe first part of the study found that free fatty acid damage to astrocytes may play an important role in the development of diabetic encephalopathy.In this part,the potential mechanisms were explored.TUNEL staining was used to detect apoptosis and it was found that palmitic acid could induce apoptosis of astrocytes in a concentration-dependent manner.The uptake of BODIPY FL C16 by astrocytes was measured by flow cytometry.The results showed that CD36 plays a very important role in the process of palmitate uptake by astrocytes.The changes of intracellular calcium concentration were detected by FLIPR real-time fluorescence recording system.It was found that mediated by IP3R palmitate could induce significant intracellular calcium release and finally cause endoplasmic reticulum calcium depletion.The intracellular ROS level was detected by CM-H2DCFDA fluorescence staining.The results showed that palmitate could obviously increase the ROS level in astrocytes.CD36 inhibitor SSO can reduce palmitate uptake,thereby inhibiting the production of reactive oxygen species,calcium overload,mitochondrial damage,cell apoptosis,decreased glutamate uptake and BDNF synthesis and secretion.Palmitate-induced calcium overload and ROS increasement could also be prevented by IP3R inhibitor APB.CD36 inhibitor SSO,IP3R inhibitor APB and antioxidant NAC all had significant inhibitory effects on palmitate-induced astrocyte cell viability decrease.In conclusion,CD36 mediates the apoptosis of astrocytes induced by palmitate via calcium overload and oxidative stress.Part Ⅲ Mechanism research on different sensitivity of neurons and astrocytes to palmitate-induced cell damageThe first part of the study found that high free fatty acids significantly damaged astrocytes but had no obvious effect on neurons.The second part revealed that CD36 mediated free fatty acids induced astrocytes apoptosis via calcium overload and oxidative stress.Based on the above results,this part primarily focused on the molecular mechanism of the different reactivity of astrocytes and neurons to free fatty acid injury.The expression of fatty acid translocation enzyme CD36 in primary cultured astrocytes and neurons was detected by Western-blot and flow cytometry.The results showed that the expression of CD36 in astrocytes was about 2.1-fold that of neurons.The uptake of BODIPY FL C16,a fluorescent labeled free fatty acid analogue,was detected by flow cytometry.The results showed that the uptake rate of free fatty acid by astrocytes was about 6.4 times that of neurons.Free fatty acids could strongly promote the release of calcium from the endoplasmic reticulum of astrocytes while had weaker effect in neurons.Under the same condition,the calcium content of endoplasmic reticulum of astrocytes decreased by about 80%after being subjected to free fatty acids and that of neurons decreased by about 25%.Down-regulation of CD36 by siRNA could attenuate PA-induced astrocyte damage while overexpression of CD36 could aggravate PA-induced neuron damage... |
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