Postmortem Interval Estimation Using Protein Chip Technology Combined with Multivariate Analysis Methods / 法医学杂志

OBJECTIVES@#To estimate postmortem interval (PMI) by analyzing the protein changes in skeletal muscle tissues with the protein chip technology combined with multivariate analysis methods.@*METHODS@#Rats were sacrificed for cervical dislocation and placed at 16 ℃. Water-soluble proteins in skeletal muscles were extracted at 10 time points (0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d and 9 d) after death. Protein expression profile data with relative molecular mass of 14 000-230 000 were obtained. Principal component analysis (PCA) and orthogonal partial least squares (OPLS) were used for data analysis. Fisher discriminant model and back propagation (BP) neural network model were constructed to classify and preliminarily estimate the PMI. In addition, the protein expression profiles data of human skeletal muscles at different time points after death were collected, and the relationship between them and PMI was analyzed by heat map and cluster analysis.@*RESULTS@#The protein peak of rat skeletal muscle changed with PMI. The result of PCA combined with OPLS discriminant analysis showed statistical significance in groups with different time points (P<0.05) except 6 d, 7 d and 8 d after death. By Fisher discriminant analysis, the accuracy of internal cross-validation was 71.4% and the accuracy of external validation was 66.7%. The BP neural network model classification and preliminary estimation results showed the accuracy of internal cross-validation was 98.2%, and the accuracy of external validation was 95.8%. There was a significant difference in protein expression between 4 d and 25 h after death by the cluster analysis of the human skeletal muscle samples.@*CONCLUSIONS@#The protein chip technology can quickly, accurately and repeatedly obtain water-soluble protein expression profiles in rats' and human skeletal muscles with the relative molecular mass of 14 000-230 000 at different time points postmortem. The establishment of multiple PMI estimation models based on multivariate analysis can provide a new idea and method for PMI estimation.

Application of Linear Regression Model of Gpnmb Gene in Rat Injury Time Estimation / 法医学杂志

OBJECTIVES@#To investigate the effects of injury time, postmortem interval (PMI) and postmortem storage temperature on mRNA expression of glycoprotein non-metastatic melanoma protein B (Gpnmb), and to establish a linear regression model between Gpnmb mRNA expression and injury time, to provide aimed at providing potential indexes for injury time estimation.@*METHODS@#Test group SD rats were anesthetized and subjected to blunt contusion and randomly divided into 0 h, 4 h, 8 h, 12 h, 16 h, 20 h and 24 h groups after injury, with 18 rats in each group. After cervical dislocation, 6 rats in each group were collected and stored at 0 ℃, 16 ℃ and 26 ℃, respectively. The muscle tissue samples of quadriceps femoris injury were collected at 0 h, 12 h and 24 h postmortem at the same temperature. The grouping method and treatment method of the rats in the validation group were the same as above. The expression of Gpnmb mRNA in rat skeletal muscle was detected by RT-qPCR. The Pearson correlation coefficient was used to evaluate the correlation between Gpnmb mRNA expression and injury time, PMI, and postmortem storage temperature. SPSS 25.0 software was used to construct a linear regression model, and the validation group data was used for the back-substitution test.@*RESULTS@#The expression of Gpnmb mRNA continued to increase with the prolongation of injury time, and the expression level was highly correlated with injury time (P<0.05), but had little correlation with PMI and postmortem storage temperature (P>0.05). The linear regression equation between injury time (y) and Gpnmb mRNA relative expression (x) was y=0.611 x+4.489. The back-substitution test proved that the prediction of the model was accurate.@*CONCLUSIONS@#The expression of Gpnmb mRNA is almost not affected by the PMI and postmortem storage temperature, but is mainly related to the time of injury. Therefore, a linear regression model can be established to infer the time of injury.

Estimating Postmortem Interval Using Intestinal Microbiota Diversity Based on 16S rRNA High-throughput Sequencing Technology / 法医学杂志

OBJECTIVES@#To explore the correlation between intestinal microbiota and postmortem interval(PMI) in rats by using 16S rRNA high-throughput sequencing technology.@*METHODS@#Rats were killed by anesthesia and placed at 16 ℃, and DNA was extracted in caecum at 14 time points of 0, 1, 2, 3, 5, 7, 9, 12, 15, 18, 21, 24, 27 and 30 d after death. The 16S rRNA high-throughput sequencing technology was used to detect intestinal microbiota in rat cecal contents, and the results were used to analyze the rat intestinal microbiota diversity and differences.@*RESULTS@#The total number of intestinal microbial communities did not change significantly within 30 days after death, but the diversity showed an upward trend. A total of 119 bacterial communities were significantly changed at 13 time points after death. The models for PMI estimation were established by using partial least squares (PLS) regression at all time points, before 9 days and after 12 days, reaching an R2 of 0.795, 0.767 and 0.445, respectively; and the root mean square errors (RMSEs) were 6.57, 1.96 and 5.37 d, respectively.@*CONCLUSIONS@#Using 16S rRNA high-throughput sequencing technology, the composition and structure of intestinal microbiota changed significantly within 30 d after death. In addition, the established PLS regression model suggested that the PMI was highly correlated with intestinal microbiota composition, showing a certain time series change.

Application Prospects and Prospect of Metabolomics in the Identification of Sudden Cardiac Death / 法医学杂志

In cases of sudden death, the prevention of sudden cardiac death and the analysis of the cause of death after sudden cardiac death have always been a difficult problem. Therefore, clinical research and forensic pathological identification of sudden cardiac death are of great significance. In recent years, metabolomics has gradually developed into a popular field of life science research. The detection of "metabolic fingerprints" of biological fluids can provide an important basis for early diagnosis of diseases and the discovery of potential biomarkers. This article reviews the current research status of sudden cardiac death and the research on metabolomics of cardiovascular diseases that is closely related to sudden cardiac death and analyzes the application prospects of metabolomics in the identification of the cause of sudden cardiac death.

Correlation between the Polymorphism of Coagulation-Related Genes and Lower Extremity Deep Venous Thrombosis / 法医学杂志

Objective To investigate the correlation between the polymorphism of 4 coagulation-related genes, rs1799963 （coagulation factor V gene Leiden）, rs6025 （prothrombin gene G20210A）, rs1042579 （thrombomodulin protein gene c.1418C>T） and rs1801131 （methylenetetrahydroflate reductase gene） and lower extremity deep venous thrombosis （LEDVT）. Methods The 4 genotypes mentioned above of 150 LEDVT patients and 153 healthy controls were detected by the kompetitive allele specific polymerase chain reaction （KASP）, then related blood biochemical indicators were collected, binary Logistic regression was established to screen the independent risk factors of LEDVT, and the correlation between polymorphism of 4 coagulation-related genes and LEDVT and its indicators under different genetic modes after adjusting confounding factors were analyzed. Results Five variables, D-dimer, fibrinogen degradation product, homocysteine, sex and age might be the risk factors of LEDVT. These variables were put into 4 genetic inheritance models, and adjusted in binary Logistic regression. The results suggested that the mutations of rs1042579 were correlated with LEDVT under dominant inheritance mode. Conclusion The gene polymorphism of rs1799963, rs6025 and rs1801131 has no significant correlation with the formation of LEDVT. The gene polymorphism of rs1042579 plays a role under dominant inheritance mode, and might be an independent risk factor for formation of LEDVT.

Expression of Autophagy-Associated Protein in Rat Muscle Tissues after Antemortem and Postmortem Injury / 法医学杂志

Objective To study the expression of the three autophagy-associated proteins, BECN1, LC3 and p62, after the injury of the skeletal muscle of rats and to explore its application in differentiation between antemortem and postmortem injury. Methods The 72 healthy Sprague-Dawley rats were randomly divided into the undamaged control group, the antemortem injury group （0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h and 24 h） and postmortem injury group （0.5 h, 1 h, 2 h and 4 h）. A model of the injured right hind limb of rats was constructed. The expressions of the autophagy-associated proteins, BECN1, LC3-2/LC3-1 and p62, in the control group, the antemortem injury group and postmortem injury group were detected by Western blotting method. The data were respectively centralized and standardized and the orthogonal partial least square-discrimination analysis （OPLS-DA） identification model of antemortem and postmortem injury groups was constructed. Results The expression of BECN1, p62 protein and LC3-2/LC3-1 after the injury of the skeletal muscle of the rats showed different degrees of changes, but the differences among the 3 groups had no statistical significance. Antemortem and postmortem injury groups can be distinguished by centralizing and standardizing the expression levels of autophagy protein BECN1 and the ratio of LC3-2/LC3-1. The principal components extracted from OPLS-DA model of antemortem injury and postmortem injury had a relatively good interpretation of the model （Rx2=0.563, Ry2=0.439）, but it were less predictive （Q2=0.366）. Conclusion The expression of BECN1 and the ratio of LC3-2/LC3-1 in injured local tissue of the rat skeletal muscle can be used for the differentiation of antemortem injury group and postmortem injury group.

Wound Age Estimation by Neutrophil Migration Distance / 法医学杂志

Objective To explore the application of neutrophil migration distance for wound age estimation of skeletal muscles in rats, and to provide methodological basis for follow-up study in future. Methods The skeletal muscle contusion model was established in rats, and the control group and the 2, 4, 6 h post-traumatic groups were set. The law of response of neutrophils that participated in the inflammation after injury was detected by immunohistochemical staining, and the relationship between neutrophil migration distance and injury time was detected by TissueFAXS PLUS software. Results The skeletal muscle was obviously infiltrated with neutrophils 2-6 h after injury. The positive rate of neutrophil was （28.75±0.94）% at 2 h post-traumatic, and reached the peak （45.50±3.63）% at 4 h post-traumatic, then decreased to （31.92±1.56）% at 6 h post-traumatic. The neutrophil migration distances increased with the progress of inflammation, and reached （124.80±12.32） μm, （229.03±21.45） μm and （335.04±16.75） μm at 2 h, 4 h and 6 h, respectively. Conclusion There is a relationship of neutrophil infiltrated number and migration distance and wound age within the 2-6 h after skeletal muscle injury, which could be used for the inference of skeletal muscle wound age.

Combination of mRNA of Repair-related Genes in Rat Skeletal Muscles for Wound Age Estimation / 法医学杂志

Objective To investigate the estimation of early and mid-term wound age by a combination of four mRNAs, the DNA polymerase delta-interacting protein 3 （POLDIP3） mRNA, regulator of chromosome condensation 1 like （RCC1L） mRNA, proline-rich 5 （PRR5） mRNA, and ribonucleic acid export 1 （RAE1） mRNA in rats skeletal muscles. Methods The model of rat skeletal muscle contusion was established, and then contusion area muscle tissue was extracted 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 h after injury. Histomorphological changes during the repair process after rat skeletal muscle contusion were observed. The relative expressions of Poldip3, Rcc1l, Prr5 and Rae1 mRNAs were detected by reverse transcription real-time quantitative polymerase chain reaction （RT-qPCR）. Different stages of wound age were classified by using the expression patterns of four genes at various time points after injury. The accuracy of the results was verified by Fisher discriminant analysis. Results Histomorphological results showed that the repair process after skeletal muscle contusion occurred with the prolonging of time. Through combination of the expression trends of the four kinds of mRNAs, the 48 h after injury could be divided into three periods, 4-12 h, 16-28 h and 32-48 h. The Fisher discrimination method showed that the classification accuracy rates of the three periods were 83.3%, 75.0% and 73.3%, respectively. Conclusion The classification discrimination based on the relative expression of every gene has a higher accuracy, and the accuracy of wound age estimation with combination of mRNA relative expressions is higher than that with a single indicator. By combining with Fisher discrimination method, this method can be used for early and mid-term wound age estimation.

Time-dependent Expression of Nrf2 Protein and its Effects on Wound Repair in Skeletal Muscle / 法医学杂志

Objective To detect the expression of nuclear factor erythroid 2-related factor 2(Nrf2)to explore its change rule and effects in the repair process of skeletal muscle. Methods An injury model of skeletal muscle was established by injection of cardiotoxin into the gastrocnemius muscle of SD male rats. The muscle samples were taken from control group and injury group at each time point(1 h, 4 h, 8 h, 12 h, 16 h, 1 d, 3 d, 5 d, 7 d, 9 d, 13 d, 17 d, 21 d). The morphological changes of skeletal muscle in repair process were observed by H&E staining, and the content of reactive oxygen species(ROS)was detected. The expression level of Nrf2 protein was investigated by Western blotting and immunohisto-chemistry staining. Results After the skeletal muscle injury, the injury region was infiltrated with inflam-matory cells which constituted mainly by lymphocyte neutrophils and mononuclear cells. There were large amounts of immature muscle cells appeared at the 5th day and the muscle repair was completed at the 21th day. Comparing with control group, the ROS content decreased in injury group at the 12th hour(P<0.05), while Nrf2 expression increased at the 16th hour(P<0.05). Moreover, the positive rate of Nrf2 increased after injury, and peaked at 16 h-1 d, then decreased gradually and returned to the level of control group(P<0.05). Conclusion The Nrf2 protein involves in the regulation of the repair of skeletal muscle injury. The positive rate of Nrf2 shows a time-dependent expression, which can be used to esti-mate the wound age of skeletal muscle.

Establishment of the Mathematical Model for PMI Estimation Using FTIR Spec-troscopy and Data Mining Method / 法医学杂志

Objective To analyse the relationship between Fourier transform infrared （FTIR） spectrum ofrat's spleen tissue and postmortem interval （PMI） for PMI estimation using FTIR spectroscopy combinedwith data mining method. Methods Rats were sacrificed by cervical dislocation, and the cadavers were placed at 20 ℃. The FTIR spectrum data of rats' spleen tissues were taken and measured at different time points. After pretreatment, the data was analysed by data mining method. Results The absorption peak intensity of rat's spleen tissue spectrum changed with the PMI, while the absorption peak position was unchanged. The results of principal component analysis （PCA） showed that the cumulative contribution rate of the first three principal components was 96%. There was an obvious clustering tendency for the spectrum sample at each time point. The methods of partial least squares discriminant analysis （PLS- DA） and support vector machine classification （SVMC） effectively divided the spectrum samples with different PMI into four categories （0-24 h, 48-72 h, 96-120 h and 144-168 h）. The determination coefficient （R2） of the PMI estimation model established by PLS regression analysis was 0.96, and the root mean square error of calibration （RMSEC） and root mean square error of cross validation （RMSECV） were 9.90 h and 11.39 h respectively. In prediction set, the R2 was 0.97, and the root mean square error of prediction （RMSEP） was 10.49 h. Conclusion The FTIR spectrum of the rat's spleen tissue can be effectively analyzed qualitatively and quantitatively by the combination of FTIR spectroscopy and data mining method, and the classification and PLS regression models can be established for PMI estimation.

Homogeneity of Different Functional mRNA Indicators for Wound Age Estimation / 法医学杂志

OBJECTIVES@#To explore the homogeneity level of four different functional mRNA （PUM2, TAB2, Cx45 and CHRNA1） expressions in rats with skeletal muscle contusion.@*METHODS@#The relative expressions of PUM2, TAB2, Cx45 and CHRNA1 mRNAs were detected by quantitative real-time reverse transcription-polymerase chain reaction （RT-qPCR）. The coefficient of variation （CV） of the relative expressions for different individuals in each injury group was calculated. The extreme value of CV, cumulative variability, and CVCV were compared.@*RESULTS@#A high CV of PUM2 and TAB2 mRNAs appeared on several different time points. However, the CV of Cx45 and CHRNA1 mRNAs was relatively low. The cumulative variability from high to low was PUM2, CHRNA1, TAB2 and Cx45 mRNAs. The relative expression of PUM2 mRNA was significantly higher than that of TAB2, Cx45 and CHRNA1 mRNAs （ P<0.05）. There was no statistical significance （P>0.05） in the CVCV of the relative expression of TAB2, CHRNA1 and Cx45 mRNAs.@*CONCLUSIONS@#As the mRNAs involving in biological process regulation, PUM2 and CHRNA1 mRNAs show a lowest individual homogeneity of the relative expression followed by TAB2 mRNA. As the mRNAs participating in the composition of cellular structure, Cx45 and CHRNA1 mRNAs show a high individual homogeneity of the relative expressions. The functional classification should be considered for the screening of the mRNA indicators used for wound age estimation.

Expression of SFRP5 mRNA in Rat Skeletal Muscle after Contusion / 法医学杂志

OBJECTIVE@#To investigate the relationship between the expression of secreted frizzled-related protein 5 (SFRP5) mRNA and the time interval after skeletal muscle injury in rats by real-time PCR.@*METHODS@#A total of ninety SD rats were randomly divided into the contusion groups at different times including 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, 48h after contusion, incision groups at different times including 4h and 8h after incision and the control group. The samples were taken from the contused zone at different time points. The total RNA was isolated from the samples and reversely transcribed to analyze the expression levels of SFRP5 mRNA.@*RESULTS@#Compared to the control group, the expression of SFRP5 mRNA in contusion groups were down-regulated within 48 h after contusion and reached the lowest level at 20 h, and the expression of SFRP5 mRNA gradually increased from 20 h to 48 h after contusion. The expression of SFRP5 mRNA in the incised groups were significantly lower than that of the contusion groups at 4 h after injury. At the time of 8 h, the expression levels between the contusion and incision groups showed no statistically significant difference.@*CONCLUSION@#It is suggested that SFRP5 mRNA analysis may show regular expression and can be a marker for estimation of skeletal muscle injury age.

Relation between Injury Time and the Expression of COX6C mRNA in Skeletal Muscle of Rats after Contusion / 法医学杂志

OBJECTIVE@#To investigate the relation between injury time and the expression of cytochrome c oxidase subunit VIc (COX6C) mRNA in skeletal muscle of rat after contusion.@*METHODS@#A total of fifty-four SD rats were divided into the control group and the contusion groups (0.5, 1, 6, 12, 18, 24, 30, and 36 h after contusion), randomly. The contusion model was established by free fall drop of gravity hammer. At corresponding time point after contusion, the regular histology was examined and expression level of COX6C mRNA was tested by real-time PCR after extraction of total RNA from the tissues.@*RESULTS@#The main pathological features of 6 h after injury included edema and hemorrhage in myocytes with no inflammatory cells found. After 6 hours, the findings included myocyte degeneration and necrosis, inflammatory cells infiltration, and fibrous connective tissue proliferation in the contused zone. The expression level of COX6C mRNA was higher than that of the control group within 6 h after contusion. The expression level was lower than that of the control group from 6-36 h after contusion.@*CONCLUSION@#The level of COX6C mRNA expresses in a regular way after contusion. It may be useful for estimating wound age in combination with the results of pathological features.

Relative Expression of Indicators for Wound Age Estimation in Forensic Pathology / 法医学杂志

OBJECTIVE@#In order to understand which kind of function genes play an important role for estimating wound age, the variation of difference genes' mRNA expression were compared after injury.@*METHODS@#The mRNA expression levels of seven candidate genes (ICAM-1, NF-κB, MX2, MT1, MT2, sTnI, and Cox6c) were analyzed in contused rat skeletal muscle at different time points using real-time fluorescent quantitative PCR (RT-qPCR). The raw Ct values were normalized relative to that of RPL32 mRNA, and converted to standard Ct values. At each time point after injury, the standard deviations (SD) of the standard Ct values were calculated by SPSS.@*RESULTS@#The expression trends of the seven genes were all found to be related to wound age, but there were lower variation coefficients and greater reliability of s TnI and Cox6c when compared with other genes.@*CONCLUSION@#The genes encoding structural proteins or proteins that perform basic functions can be suitable for wound age estimation.

Protective effects of intermedin preconditioning on hypoxic injury in rat's cardiac myocytes / 法医学杂志

OBJECTIVE@#To observe the effects of intermedin preconditioning on hypoxic injury in rat's cardiac myocytes and to provide the hypothetical mechanism of sudden cardiac death in the field of forensic pathology.@*METHODS@#The H9c2 cultured rat cardiac myocytes were randomly divided into control group, hypoxia group and IMD group. The myocardial cell viability, cellular ultrastructure, intracellular calcium concentration and apoptosis rate were determined by MTT assay, transmission electron microscopy, laser scanning confocal microscope and flow cytometry, respectively.@*RESULTS@#Compared with the control group, cell viability obviously decreased with inner ultrastructure injury in the hypoxia group (P<0.05), while cell viability significantly increased in the IMD group by reducing the hypoxia injury of cardiac myocytes (P<0.05). Compared with the control group, [Ca2+]i (fluorescence intensity) and apoptosis rate significantly increased in the hypoxia group, but decreased in the IMD group (P<0.05).@*CONCLUSION@#IMD increases the cell survival rate and decreases the cell apoptosis inhibited by intracellular calcium overload from hypoxia. This finding may reveal the mechanism of protective effects of myocardial hypoxia, and provide a scientific basis for the identification sudden cardiac death.

Effect and mechanism of intermedin in acute rat cardiac ischemic injury / 法医学杂志

OBJECTIVE@#To investigate the effect and potential mechanism of intermedin (IMD) in acute cardiac ischemic injury and to provide a new approach for exploring mechanism of sudden cardiac death.@*METHODS@#Seventy-two healthy male rats were randomly divided into 3 groups: control, ischemic and the IMD-treated group. The activity of lactate dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD) in heart blood were tested by enzyme chemistry method. The mRNA changes of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) in cardiac were measured by real-time PCR analysis. Myocardial cyclic adenosine monophosphate (cAMP) content was determined by enzyme linked immunosorbent assay (ELISA). Apoptosis related factors Bcl-2 and Bax were detected by immunohistochemistry.@*RESULTS@#Comparing with the control group, LDH and MDA activity of ischemic group in heart blood increased and SOD activity decreased. The concentration of cAMP increased in ventricular muscle, Bcl-2 and Bax proteins expression ratio level decreased. The intravenation of IMD decreased the level of increased activity of LDH and MDA, and lessened the level of decreased activity of SOD. The mRNA expression of CRLR and RAMPs obviously increased in ventricular muscle.@*CONCLUSION@#The protective effect of IMD against myocardial ischemic injury could be caused by decreasing the oxidative stress of ischemia and inhibiting the myocardial apoptosis.

Changes of ICAM-1 mRNA expression in rats' skin and muscle after contusion / 法医学杂志

OBJECTIVE@#To investigate the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in contused skin and muscle of rats and the relationship between the ICAM-1 expression and the wound age.@*METHODS@#The samples were taken at 0.5, 1, 6, 12, 18, 24 and 30 h after contusion of rats, respectively. Total RNA was extracted both from the skin and muscle samples of each group and reverse transcription polymerase chain reaction (RT-PCR) was conducted to synthesize the 1st strand cDNA. The amount of ICAM-1 mRNA in each sample was quantified using rp132 intrinsic fluorescent assay and compared by the 2 (-Delta Delta Ct) method with that from control samples.@*RESULTS@#After contusion the expression of ICAM-1 mRNA in skin increased rapidly and peaked at 0.5 h, at 24 h degraded to the amount that was seven times as much as the control group, then rised again. The expression of ICAM-1 mRNA in muscle increased significantly within 0.5 h and peaked at 6 h, reached the minimum at 18 h, then increased again.@*CONCLUSION@#It is suggested that ICAM-1 mRNA analysis may be useful for estimation of early wound age because of its time-related expression after contusion in skin and muscle.