Markers Of Mechanical Asphyxia Death Based On Postmortem Biochemical Detection And Proteomics Technique

[Background]Mechanical asphyxia is asphyxia resulting from impaired breathing or respiratory failure caused by external mechanical violence.It is a rapid process of death and accounts for a high proportion of causes of death in forensic pathology,with an incidence second only to that caused by mechanical injury.Common types of mechanical asphyxia include constriction,strangulation,suffocation,drowning,and death by compression of the chest and abdomen.The main features of the body include bruising and swelling of the face,significant and extensive post-mortem staining,dental maceration,and petechial haemorrhaging of internal organs.However,a significant number of homicide cases such as death by mouth and nose,smothering,death by compression of the chest and abdomen,and post-mortem division of the body do not show obvious signs of mechanical asphyxiation.The rapidity of the death process and the lack of appropriate eyewitnesses make it difficult to solve the case.Therefore,it is important to investigate the mechanism of mechanical asphyxiation in forensic medicine and to screen for the corresponding biological markers to assist in the diagnosis of mechanical asphyxiation leading to death.[Objective]In this experiment,an animal model of mechanical asphyxia was developed to simulate the mechanism of death by asphyxia in humans and to screen for characteristic biological markers.The post-mortem arterial blood biochemical assay and the tissue organ proteomics assay were used to study the biochemical and molecular biological parameters of mechanical asphyxia death from two aspects simultaneously,which can provide some auxiliary diagnostic indicators for the identification of mechanical asphyxia death in forensic medicine.[Methods]1.Establish a mouse model of mechanical asphyxia（rabbit modeling method is the same as that of mice）,and set up control group,hanging group and smother group respectively.After successful modeling,the corpse surface and anatomical signs of the mice were observed.HE staining was used to observe the morphological characteristics of the myocardial and brain tissue of the three groups of mice.TUNEL staining was used to evaluate the apoptosis of the myocardial and brain tissue of the mice.2.The biochemical indexes of the arterial blood in the control group,the hanging group and the smother group were determined within 12 hours（0h、2h、4h、6h、8h、10h、12h）after death.The measurement items mainly include blood gas value（p H,Pa O₂,Pa CO₂）,blood oxygen value（Sa O₂,Hct,t Hb）,electrolyte value(K⁺,Na⁺,Cl^-,Ca²⁺)and metabolite value（Lac,Glu）.We observed the above indicators of postmortem changes and differences between the three groups of models.3.Label-free proteomics technology was used to detect the differences in the proteome levels of the myocardial and brain tissue of the control group,the hanged group and the smother group.Combined with bioinformatics methods and technologies,the differential protein enrichment pathways were analyzed,and the differential proteins related to mechanical asphyxia were screened out.Finally,PRM and immunohistochemical methods were used to verify the accuracy.[Results]1.In this experiment,two animal models of mechanical asphyxia were successfully established,mouse and rabbit.In the mouse model of asphyxia,typical asphyxia characteristics can be observed in necropsy and anatomical examination.Protuberance of the eyes,exposed tongue tip,incontinence was observed in the hanged mice.After dissection,the neck muscle hemorrhage,internal organ congestion,and petechial hemorrhage under the organ capsule were observed.HE staining showed scattered hemorrhage in the myocardial interstitium of mice,congestion of small blood vessels in the myocardial interstitium,congestion and edema of brain tissue,and congestion,edema and hemorrhage of lung tissue.TUNEL staining showed that a small number of apoptotic signals appeared in the outer layer of the myocardium and the superficial layer of the cerebral cortex.Mice in the smother group showed symptoms of incontinence and mania during the moribund period.HE staining showed that the myocardial interstitial small blood vessels were dilated and congested,and scattered red blood cells were seen in local cardiac chambers,brain tissue congestion,edema,and lung tissue congestion,edema,and hemorrhage.TUNEL staining showed that a small number of apoptotic signals appeared in the outer layer of the myocardium and the superficial layer of the cerebral cortex.2.Postmortem arterial blood gas analysis of rabbits in control group,hanging group and suffocation group showed that the contents of Pa O₂,Sa O₂,p H,Ca²⁺and Glu decreased gradually with the prolongation of PMI within 12 hours after death.The overall change trends of the above-mentioned measurement indicators among the three groups were all statistically different.Among them,the measurement results of the four indicators Pa O₂,Sa O₂,p H and Ca²⁺were as follows:control group>smother group>hanged group,and the measurement results of Glu were hanged group>smother group>control group.The contents of Pa CO₂,K⁺and Lac increased gradually with the prolongation of PMI within 12 hours after death.The overall change trend of Pa CO₂ was statistically different among the three groups,which was the hanging group>the smother group>the control group.The contents of K⁺and Lac both reached the maximum threshold determined by this experimental instrument within 12hours after death,and the overall statistical analysis could not be carried out.The four biochemical indexes of Hct,t Hb,Na+and Cl-were relatively stable within 12 hours after death,and there was no obvious change law.3.The results of the proteomics of mouse myocardium and brain tissue showed that the differential protein functions of the myocardium of the hang group vs the control group were enriched in the regulation of ribosome function,myocardial contractile function and protein degradation,mitochondrial oxidative respiration and protein degradation.folding,etc.Based on the above results,we screened out that the up-regulated differential proteins were mainly TITIN,PSA1,PSA6 and PSB4,and the down-regulated differential proteins were mainly FIBA,FIBB,FIBG,CY1and C560.The differential protein functions of the myocardial tissue of the smother group vs the control group were enriched in the regulation of ribosome function,myocardial contraction and calcium regulation,protein synthesis and metabolism,regulation of coagulation function,mitochondrial oxidative respiration and so on.We screened up-regulated differential proteins mainly including CASQ2,TITIN,PSA6,PSB4 and RAB7A,and down-regulated differential proteins mainly included FIBA,FIBB and FIBG.Different protein functions in brain tissue of hang group vs control group were enriched in ribosome function regulation,coagulation function regulation,glycogen metabolism,protein degradation process,apoptosis regulation and so on.We screened out that the up-regulated differential proteins were mainly PP2AA,2A5G、RYR2 and CO3,and the down-regulated differential proteins were mainly UBA3.The differential protein functions of the brain tissue of the smother group vs the control group were enriched in protein synthesis and metabolism,ribosome function regulation,mitochondrial oxidative respiration,fatty acid oxidation,and glycogen synthesis regulation.The up-regulated differential proteins were mainly TRA2B,ACSL1 and CPT2,and the down-regulated differential proteins were mainly COX6A1.PRM technology and immunohistochemistry were used to re-verify some of the above differential proteins,and the results were consistent with the protein expression results screened by Label-free proteomics,which further proved the accuracy of this protein screening method.[Conclusion]In this experiment,two technical methods of postmortem biochemical detection and proteomics were used to systematically study the problems related to mechanical asphyxia in forensic medicine.We established two animal models of hanging and smother in mechanical asphyxia,and used the above-mentioned technical means to study the postmortem biochemical and histological differences in mechanical asphyxia.Through a series of methods such as PRM and immunohistochemistry,the screened differential indicators were further verified,and some indicators valuable for the diagnosis of mechanical asphyxia were preliminarily summarized.Biochemical indicators such as Pa O₂,Sa O₂,p H,Ca²⁺,Glu,p H,etc.,molecular indicators such as FIBA,FIBB,FIBG,CASQ2,TRA2B,RAB7A,etc.This study fills the gap in forensic research on mechanical asphyxia at the molecular level and lays the foundation for further research on mechanical asphyxia in the future.