DFO Alleviates Cognitive Impairment In T1DM Model Rats By Reducing Iron Deposition In Hippocampus And Improving Synaptic Morphological Plasticity

Objective:Diabetes has seriously threatened human health.Cognitive dysfunction in diabetes is one of the complications of central nervous system in diabetes.It has similar clinical manifestations with Alzheimer’s disease（AD）,including thinning of cerebral cortex,damage of brain neurons,and Alzheimer’s disease specificβAmyloid protein（Aβ）,abnormal deposition and tau protein hyperphosphorylation,and in serious cases,these symptoms may develop into diabetes encephalopathy（DE）.Previous studies have shown that neuroinflammation,oxidative stress damage and mitochondrial dysfunction in the brain of diabetic patients are related to the occurrence and development of neurodegenerative diseases,thus affecting the cognitive ability of patients.However,there are few effective methods to prevent and treat cognitive dysfunction caused by diabetes.Iron is a powerful oxidant,which can catalyze various cell reactions to produce reactive oxygen.When the concentration of iron in cells reaches a high level,it can form a Fenton chemical reaction with H₂O₂ to produce a large number of hydroxyl free radicals,thus affecting cell activity.In addition,excessive intracellular iron can also lead to iron-dependent oxidative stress and cell death caused by a large amount of lipid peroxidation,eventually leading to ferroptosis.It is reported that the abnormal increase of ferritin（Ft）in diabetes indicates iron overload in the body,and the serum ferritin level is negatively correlated with insulin sensitivity,which can infer that excessive iron in the body may aggravate the occurrence and development of diabetes and its complications.So,how does iron deposit in diabetes?The pathogenesis of iron overload affecting cognitive dysfunction in diabetes is still unclear.Methods:In this experiment,we used a single intraperitoneal injection of 65 mg/kg Streptozotocin（STZ）to make the type I diabetes model（T1DM）.After 21 days,the intra-cerebroventricular was injected with a small dose of deferoxamine（DFO,5μg/kg/d）for 28 consecutive days.Morris water maze test was used to evaluate the learning and memory ability of animals to spatial location;At the same time,open field test and elevated plus maze test were used to observe the autonomous exploration and anxiety behavior of rats;The expression of ferritin（FTH/FTL）,iron metabolism related protein（Tf Rc/DMT1/FPN1）,ferroptosis related protein（GPX4/SLC7A11/ACSL4）and synaptic plasticity related protein（SYN/PSD95/VAMP2）in hippocampus and cerebral cortex were detected by Western blot;The microstructure changes of neurons in CA1,CA3 and cerebral cortex regions of the hippocampus were studied by Nissl staining and immunofluorescence chemistry;Finally,the ultrastructural changes of hippocampal CA1 neurons were observed by transmission electron microscopy in order to obtain the main mechanism of cognitive dysfunction in T1DM rats.Results:1.Morris water maze results showed that T1DM rats had cognitive dysfunction,and DFO treatment had a positive impact on learning and cognitive behavior of T1DM rats.At the same time,T1DM rats showed no anxiety-like behavior with or without DFO treatment.2.Biochemical examination of serum SOD and MDA levels in T1DM rats showed that the level of oxidative stress in T1DM rats increased;The total iron content in the hippocampus of T1DM rats increased by ICP-MS;DFO treatment has obvious improvement;At the same time,Western blot and immunofluorescence results showed that the expression of FTH and FTL protein in hippocampus and cortex increased significantly,indicating iron overload in hippocampus and cortex;DFO can alleviate brain iron overload in T1DM rats and reduce the level of oxidative stress in rats.3.The expression of Neu N protein in hippocampus and cortex of T1DM rats decreased,and immunofluorescence showed that Neu N-positive neurons were damaged.DFO intervention could improve the neuronal damage.4.The results of protein and mRNA levels showed that the expression of iron uptake protein DMT1 increased in hippocampus and cortex of T1DM rats,and the expression of iron exporting protein FPN1 decreased.The expression of DMT1 and FPN1 protein was recovered after DFO treatment,but DFO had no effect on mRNA level.Hippocampal Tf Rc protein expression increased,cortical Tf Rc protein expression decreased,and DFO reversed this result,but had no effect on mRNA levels.5.Under the electron microscope,the mitochondria of T1DM rats shrunk and the membrane density increased.The morphology of mitochondria was improved to some extent after DFO treatment;The results of Nissl staining showed that DFO could protect neurons from oxidative stress damage after inhibiting ferroptosis.Compared with Ctrl group,the expression of GPX4 and SLC7A11 protein in hippocampus and cortex of rats in T1DM group decreased significantly,while the expression of ACSL4 protein increased;After DFO intervention,the levels of GPX4,SLC7A11 and ACSL4 were recovered to different degrees.6.Western blot results showed that the expression of synaptic function-related proteins SYN,PSD95 and VAMP2 in hippocampus and cortex of rats in T1DM group decreased significantly,and the levels of these three proteins recovered significantly after DFO intervention.Under the transmission electron microscope,the length of synaptic active area and the volume of synaptic vesicles in T1DM rats treated with DFO were significantly improved,and DFO also had some effect on the formation of nerve fiber myelin sheath.Conclusion:1.DFO can alleviate the impaired learning and memory ability of T1DM rats;2.DFO can reduce iron deposition and improve the body’s oxidative stress state;3.DFO protects hippocampal neurons from injury by inhibiting ferroptosis;4.DFO can improve synaptic morphological plasticity and expression of synaptic function-related proteins.