| Diabetic encephalopathy(DE)is the damage of central nervous system caused by diabetes mellitus(DM),which leads to the functional and structural changes of cerebral tissues.It is characterized by acquired cognitive impairment and behavioral defects.The main manifestations were memory loss,delayed response,and attention loss.It is one of the most difficult complications to prevent and cure,which brings heavy burden to the family and society globally.With the aging of society and the increasing prevalence of diabetes,the prevention and treatment of diabetic encephalopathy has become the main focus of researchers.It has been reported that diabetic encephalopathy rats present Alzheimer's disease(AD)-like pathological changes,such as neuronal degeneration,hippocampal atrophy,? deposition and neurofibrillary tangles.Epidemiological studies have also suggested that diabetes will increase the risk of AD.diabetes is one of the independent risk factors of AD.We speculate that diabetic encephalopathy and AD have similar pathological changes and pathogenesis.Oxidative stress is considered to be an pivotal mechanism in the pathogenesis of diabetes and AD.As the central nervous system(CNS)cell membrane is composed of a large number of highly unsaturated fatty acids,it is more sensitive to oxidative stress injury.Therefore,oxidative stress is an early pathopgysiology event of diabetic encephalopathy and AD,and runs through the course of the disease.Studies have shown that oxidative stress can induce a series of gene expression imbalance,participate in the body's stress response,and play a pivotal role in the pathogenesis of nervous and mental system diseases.Micro RNAs(miRNAs)are non coding small RNAs with a length of about 22 bases,which are negative regulators in biological function regulation.miR-132 is one of the most abundant miRNAs in the brain,which plays a key role in neurogenesis and plasticity of neurons.It was demonstrated that miR-132 was significantly downregulated in the brain of AD patients after death,accompanied by neuronal death,amyloid deposition,and other pathological manifestations,suggesting that miR-132 may be involved in the molecular mechanism of AD.In the early stage of our project,we used H2O2 to stimulate primary hippocampal neurons of mice to prepare oxidative stress cell model.In combination with SAMP8 and SAMP10,and in the peripheral serum of AD patients,we used microarray and q PCR technology to screen and verify a series of miRNAs related to the pathogenesis of AD,in which the expression of mir-132 was significantly downregulated,suggesting that mir-132 may play a role in AD.Glycogen synthase kinase-3?(Glycogen synthase kinase-3?,GSK-3?).It is one of the enzymes involved in the regulation of Tau abnormal hyperphosphorylation.Studies have confirmed that miR-132 mimics play an significant role in the pathogenesis of AD through GSK3?.It can reduce the phosphorylation of Tau protein at Ser 396 and Ser 404 sites,thus playing a therapeutic role in AD.Is miR-132 involved in the pathogenesis of diabetic encephalopathy? In this section,we will explore whether miR-132 mediates GSK-3 ?/Tau pathway is involved in the pathogenesis of diabetic encephalopathy.Part One Expression of miR-132 in hippocampus of DE modelObjective: To explore the expression of miR-132 in hippocampus of diabetic encephalopathy rats.Methods: 1.We selected 40 SD male rats of 200 g and 8 weeks approximately and treated them with high-fat diet and STZ.They were random Ly divided into Con group(n = 10)and model group(n = 30).Rats with fasted blood glucose ? 16.7 mmol/L was regarded as diabetes model.After 28 weeks of model building,Morris water maze was managed for the neurological function and recognition function of rats.The results of Morris water maze is referenced for the division of Con,DM,and DE groups,respectively.2.SD neonatal rats within 24 h of birth should be dissected in order to obtain intact hippocampal tissue for isolating and cultivating primary hippocampal neurons.Primary hippocampal neural cells injury model was established by incubating primary hippocampal neuron cells at different concentrations of H2O2,high glucose,and mannitol.3.The miRNA was extracted by instruction book of RNA extraction,then they were undergone reverse transcription experiments and q RT-PCR for the levels of miR-132.Results: 1.The levels of miRNA-132 was decreased in Con,DM,and DE groups,progressively(P <0.01).2.Expression level of miR-132 in primary hippocampal neurons of the mannitol,high glucose,and H2O2 intervention group was lower than that of the control group.Expression of miR-132 in the high glucose and H2O2 intervention group was significantly lower than that of the control group,which was statistically significant(P <0.01).Conclusion: The expression of miR-132 decreased in the oxidative stress injury model of hippocampus and primary hippocampal neurons of diabetic encephalopathy rats,suggesting that miR-132 might be a protective molecule in the pathogenesis of diabetic encephalopathy.Part Two miR-132 regulates GSK-3?/Tau signaling pathway and participates in the pathogenesis of diabetic encephalopathyObjective The purpose of this part is to observe the changes in the levels of GSK-3?,p-GSK-3?(Tyr216),p-Tau(Ser404),p-Tau(Ser396)in the hippocampus of DE rats and HT-22 cells damaged by oxidative stress.Methods:1.To stimulate the early pathological changes of DE,the oxidative stress cell damage model was established by culturing the HT-22 hippocampal neuron cell line and stimulating HT-22 cells with different concentrations of H2O2 and AGEs.2.The expression levels of GSK3 ?,p-GSK-3 ?(T216),p-Tau(Ser404),and p-Tau(Ser396)in hippocampus of DE rats and HT-22 cells injured by oxidative stress were detected by Western blot;the expression of GSK-3? in HT-22 cells injured by oxidative stress and influenced by miR-132 was verified by Western blot.3.Real time quantitative PCR was used to detect the successful transfection(overexpression)of miR-132 in HT-22 cells 4.The expression and co-localization of GSK-3? and p-Tau(Ser404)in Con,DM and DE groups were detected by immunofluorescence.5.Immunoprecipitation was used to detect the binding relationship between GSK-3 ? and p-Tau(Ser404)protein.6.Immunohistochemistry was used to detect the expression of p-Tau(Ser404),p-Tau(Ser396)and GSK-3? in Con,DM and DE groups.Results: 1.Western blot suggested that the protein levels of GSK-3?,p-GSK-3 ?(T216),p-Tau(Ser404),and p-Tau(Ser396)were increased in Con,DM,and DE groups,progressively(P < 0.05).2.The expression of GSK-3? in HT-22 cells stimulated by ages,Glu and H2O2 was higher than that in Con group(P < 0.05).3.GSK-3? and p-Tau(Ser404)were upregulated in the hippocampus of diabetic rats.4.The results of immunoprecipitation showed that there was a binding relationship between GSK-3? and p-Tau(Ser404)protein,and the binding degree in DE group was significantly higher than that in DM and Con groups.5.Immunohistochemical results showed that the expression levels of GSK-3 ? and p-Tau(Ser404)protein increased between Con,DM and DE groups.6.miR-132 downregulates the expression of GSK-3? in HT-22 injured cells.Conclusion: 1.miR-132-mediated GSK-3?/Tau signaling pathway is involved in the pathophysiology of diabetic encephalopathy in rats,which is one of the molecular mechanisms of diabetic encephalopathy.2.miR-132-mediated GSK-3? signaling pathway is involved in oxidative stress injury of HT-22 cells,which is one of the molecular mechanisms of diabetic encephalopathy.Part Three Clinical implications of miR-132 and GSK-3? in peripheral blood of patients with diabetic encephalopathyObjective: To verify the expression levels of miR-132 and GSK-3? in peripheral blood of patients with diabetic encephalopathy,for a purpose of prevention and treatment of T2DM-related DE.Methods: 1.Sixty DE patients were enrolled in the Department of Endocrinology,The First Hospital,School of Graduate,Hebei Medical University,Shijiazhuang,China from Dec 2018 to Dec 2020,as well as 60 healthy adult controls matched with general demography information.2.5 ml venous blood were collected from patients in DE group and healthy control group,and serum and plasma were separated respectively.3.The relative expression of miR-132 in the peripheral blood serum of DE patients was detected by ELISA.4.Enzyme linked immunosorbent assay was used to detect GSK-3? in peripheral blood plasma of DE group.Result: 1.Downregulation of miR-132 expression in peripheral blood serum of patients with diabetic encephalopathy.2.The level of GSK3? in peripheral blood plasma of patients with diabetic encephalopathy is up-regulated.Conclusions: Clinical samples suggest that miR-132 expression in peripheral blood of diabetic encephalopathy patients is down regulated and GSK-3? level is up regulated.It further proves that miR-132 may be a protective molecule hypothesis in the pathogenesis of diabetic encephalopathy. |
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