Research On Hypoglycemic Brain Injury Induced By Insulin Overdose

Background More than 100 forensic identification cases related to insulin preparations have been reported since the first case of insulin homicide was reported.In the last decade,insulin related suicide and homicide cases increased slowly,whereas,insulin overdose death remains challenging in forensic practice.The detection of insulin content may indicate the passage of exogenous insulin injection,whereas,it was difficult to determine the role of insulin excess.Hypoglycemic brain damage caused by severe hypoglycemia is the underlying mechanism in insulin overdose death.The neuro necrosis in dentate gyrus of hippocampus pathologic was unique in hypoglycemic brain injury,the research on the hippocampus of hypoglycemic brain injury can provide solid basis and innovative ideas for forensic identification.Objectives 1.To sequence the hippocampus of insulin-induced hypoglycemic brain injury rats through transcriptomics technology,correlate the sequence with proteomics,and screen differentially expressed genes and proteins.2.To verify the selected oxidative stress-related proteins in animal models with the use of molecular biology techniques multidimensionally,and explore the possibility of selected oxidative stress-related proteins as biomarkers in hypoglycemic brain injury.3.To verify the selected oxidative stress-related proteins in autopsy samples and explore the possibility of selected oxidative stress-related proteins as biomarkers in hypoglycemic brain injury.4.To detect the metabolites in the hippocampus of hypoglycemic brain injury by utilizing magnetic resonance spectroscopy,and explore the mechanism of hypoglycemic brain injury.Methods 1.The rat models of hypoglycemic brain injury were successfully established,Sprague Dawley rats were randomly and equally divided into four groups: control,acute hypoglycemia,hypoglycemia resuscitation 24 h,and hypoglycemia resuscitation 7 d.2.Transcriptome sequencing was performed on the hippocampus of hypoglycemic brain injury rats.The data was first further analyzed through software by combining the bioinformatics database annotation results,and then was correlated with previous proteomics.3.Elisa was used to detect the expression of oxidative stress-related indicators in serum.Western-blot and immunohistochemical methods were utilized to conduct the validation of selected proteins in animal models.4.Immunohistochemical methods were used to conduct the validation of selected proteins autopsy samples.5.The changes of metabolites were detected by proton localized magnetic resonance spectroscopy in the right hippocampus of rats,and the hippocampal neurons were observed by transmission electron microscopy.Results 1.The cell adhesion molecule family(CAMS)and PI3K-Akt signaling pathway were significantly changed during acute hypoglycemia.The CLDN m RNA,OCLN m RNA,CDH3 m RNA,MHC1 m RNA,GLYCAM1 m RNA and MPZ m RNA in the cell adhesion molecule family,PI3K-Akt signaling pathway SGK m RNA,GF m RNA,p21 m RNA,ITGB1 m RNA,VWF m RNA,GHR m RNA and ITGA2 B m RNA are the common differentially expressed genes in acute hypoglycemia.2.The verification of the genes in the PI3K-Akt signaling pathway revealed CDKN1 A,IGF2,OSMR,SGK1,WFIKKN2 and Fn1 genes were significantly up-regulated(P<0.05)and that the SPP1 gene was significantly down-regulated(P<0.05).3.Transcriptomic and proteomic of hypoglycemic brain injury correlation showed that oxidative stress-related proteins PEX1/12 were significantly up-regulated(P<0.05),DJ-1 and NDRG1 proteins were significantly down-regulated(P<0.05).4.The serum oxidative stress indexes SOD and MDA in the acute hypoglycemia group were significantly different from those in the control group(P<0.01).The expression levels of oxidative stress-related proteins PEX1/5/12 were decreased in hypoglycemia resuscitation 24 h(P<0.05).The DJ-1 and NDRG1 of hippocampal,cortex,hypothalamus in the acute hypoglycemia group were substantially different from those in control(P<0.05).5.Elevated cortical DJ-1 and NDRG1 protein expression in insulin overdose autopsy samples were observed(P<0.05).6.The NAA,NAA+NAAG and m Ins in the acute hypoglycemia group and the hypoglycemia recovery 24 h group were higher than control(P<0.01),whereas,the Glu +Gln,t PC,and Tau showed the opposite trend.7.Damaged hippocampal neurons,swollen granules,dissolved intramembrane matrix,disappeared cristae,and decreased peroxisomes were displayed in hypoglycemic brain injury.Conclusion 1.The transcript levels in the hippocampus of hypoglycemic brain injury changed significantly and differentially expressed genes are mainly enriched in PI3K-Akt and other signaling pathways.The expression of PI3K-Akt-related genes in hypoglycemic brain injury is consistent with the transcriptome sequencing results.The verification of stress-related proteins PEX1/5/12,NDRG1 and DJ-1 protein suggests an important role of PI3K-Akt pathway and oxidative stress-related proteins in hypoglycemic brain injury.2.Oxidative stress-related proteins PEX1/5/12,DJ-1 and NDRG1 proteins demonstrate their potential value as specific molecular markers in hypoglycemic brain injury.3.The metabolites in hippocampal are dramatically altered during hypoglycemic brain injury,which includes the increase in NAA concentration possibly as a specific biomarker in hypoglycemic brain injury.Metabolic disorders in the hippocampus are associated with peroxisome and mitochondrial dysfunction,suggesting the crucial role of oxidative stress and mitochondrial dysfunction in hypoglycemic brain injury.