| BackgroundThe mechanisms of death of insulin overdose basically contain central nervous system(CNS)function failure caused by severe hypoglycemic brain damage,anaphylactic shock caused by allergy to ingredients in insulin preparations,and pulmonary embolism.CNS function failure caused by severe hypoglycemic brain damage is the main consideration.Therefore,the study of CNS after insulin overdose is helpful to dig in depth and find out the exact pathophysiological process,so as to find a relatively stable diagnostic parameter(biomarker).Insulin is an endogenous substance that can be produced physiologically in human body,and it degrades quickly after being released into blood,so the injection of insulin as a murder method is hard to distinguish sometimes.Furthermore,postmortem autolysis of blood can disrupt the structure of insulin,making the concentration of insulin decline or undetectable.Biomarkers are characteristic indicators that can objectively reflect pathophysiological or pharmacological processes,and can be used for disease screening,diagnosis,characterization,and monitoring.By the utilization of proteomics method,the molecular mechanism of brain damage caused by insulin overdose and hypoglycemia can be further elucidated,and specific biomarkers may be obtained,which has practical significance for forensic pathological diagnosis.Metabotropic glutamate a receptor 4(m Glu R4)belongs to the class Ⅲ metabotropic glutamate receptors.m Glu R4 is extensively expressed in the rats’ brain.Its expression was demonstrated in the cerebral cortex,striatum,basal ganglia,hippocampus,thalamus,cerebellum and spinal cord in rats,among them and it is strongly expressed in the cerebellar cortex,basal ganglia,some sensory delayed nuclei of thalamus and part of hippocampus.m Glu R4 is relative to cognitive activities such as learning,memory,and fear acquisition,and is associated with a variety of diseases.Proteomic research showed that m Glu R4 was elevated during acute hypoglycemic brain damage and decreased 7 days after resuscitation,showing a time correlation with damage.Therefore,as a potential biomarker of brain damage caused by insulin overdose hypoglycemia,whether m Glu R4 has a certain diagnostic significance in brain damage caused by insulin overdose hypoglycemia,and the potential pathophysiological function of m Glu R4 is of research value.Objectives1.To study the expression of mGluR4 after hypoglycemia brain damage in rats.2.To investigate the function of m Glu R4 in the occurrence and development of hypoglycemic brain damage in rats.3.The application value of m Glu R4 as a biomarker for insulin overdose death was discussed.Methods1.The experimental groups were divided into control group,acute hypoglycemic brain damage group and resuscitation group.Adult male Sprague-Dawley(SD)rats were used to establish the hypoglycemic brain damage model induced by insulin overdose according to the Auer classic model and modified appropriately.In the resuscitation group,the specific m Glu R4 positive allosteric modulators(PAMS)Lu AF21934 was used at 10mg/kg as a single intervention or for intervention in 7 consecutive days.2.Brain tissues of rats in the acute and resuscitation groups after hypoglycemic brain damage were collected,and pathological changes were evaluated by HE staining to verify the model establishment.The expression of m Glu R4 in hippocampus and cortex after hypoglycemic brain damage was detected by Western Blot.The expression of m Glu R4 in neurons in the hippocampus region was detected by double immunofluorescence.The relationship between the expression level of m Glu R4 and the time of hypoglycemic brain damage was analyzed.3.Brain tissues of rats in the Lu AF21934 intervention groups were collected.Western Blot was used to detect the changes in the expression of Caspase 3 after the single intervention and consecutive 7 days intervention group.The TUNEL staining method was used to evaluate the apoptosis level of hippocampus and to explore the function of m Glu R4 in secondary damage after hypoglycemic brain damage.Results1.After the injection of insulin,the rats in the experimental group experienced coma,dyspnoea or even apnea after their blood glucose dropped below 3.0 mmol/L.Some rats were accompanied by flexion of the limb,seizures,urinary incontinence,and other reactions.Electroencephalogram(EEG)turned into flat waves about 1h after insulin injection.2.In the acute stage of hypoglycemia,severe cerebral edema and neuronal swelling occurred in the cerebral cortex.The edema of neurons in CA1 and CA3 regions was obvious in acute hypoglycemia group.The granule cells in Dentate gyrus(DG)were compact.In the resuscitation group,some neurons in the cerebral cortex were shrunk and the neurons in the superficial cortex were necrotic.The lesions were less severe than those in the acute hypoglycemia group.In addition,we also observed delayed neuronal necrosis in CA1 and CA3 areas.Western Blot results showed that the expression level of m Glu R4 in the acute group was increased in the hippocampus.But in the resuscitation group m Glu R4 fell back to the level that had no statistical difference with the control group.There was no statistical difference between groups in the cortex.DG,CA1、CA3 in the hippocampus were observed in Neu N and MGlu R4 immunofluorescence double staining.The m Glu R4 expression was increased in DG/CA1/CA3 in the acute hypoglycemia group compared with the control group,while the resuscitation group fell back to the level of no significant difference from the control group.3.Western Blot results showed that the expression of Caspase 3 in the Lu AF21934 intervention group(Group I and Group K)was lower than that in the solvent group for single and consecutive 7 days,and there was no significant difference between Group I and Group K.Compared with the solvent group,the apoptosis of hippocampal cells in group I and K was decreased,but the apoptosis in cortex was not significantly different.Conclusions1.m Glu R4 was up-regulated in the hippocampus in the acute hypoglycemic brain damage group,and its expression was enhanced in the hippocampus DG/CA1 and CA3 regions,and then decreased in the resuscitation group,which indicated that the changes of m Glu R4 in the hypoglycemic brain damage were timely correlated with excitatory toxicity damage.2.Enhancement of the function of m Glu R4 alleviates the delayed apoptosis effect induced by hypoglycemia,and plays a certain protective role in the hippocampus after brain damage caused by hypoglycemia.3.The increased expression of m Glu R4 in acute hypoglycemic damage may be one of the protective mechanisms of neurons in the face of excitatory toxicity,and may be related to the special metabolic changes of hypoglycemic brain damage,which has the value of becoming a molecular marker of hypoglycemic brain damage caused by insulin overdose. |
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