Objective To investigate the effects of Amanita caojizong on cardiomyocyte apoptosis and the expression of apoptosis-related factors Bcl-2 and Bax,thereby providing experimental evidence for the prevention and treatment of Amanita caojizong poisoning.Methods Mouse cardiomyocytes(HL-1 cells)cultured in vitro were divided into an experimental group(treated with Amanita caojizong extract)and a control group(treated with PBS).After treatment with Amanita caojizong extract,apoptosis of HL-1 cells was observed using TUNEL staining,and the protein expression levels of Bax,Bcl-2,Caspase-3,and Cleaved Caspase-3 in HL-1 cardiomyocytes were detected by Western blot.Results Compared with the control group,the TUNEL staining showed significantly increased apoptotic fluorescence intensity in the Amanita caojizong extract-treated group.The protein expressions of Bax,Caspase-3,and Cleaved Caspase-3 in HL-1 cells in the Amanita caojizong-treated group were upregulated,while the expression of Bcl-2 was downregulated.Conclusion Amanita caojizong can promote apoptosis of mouse cardiomyocytes,and its mechanism may be associated with the Bcl-2/Bax pathway.

Objective To investigate the effects of Amanita caojizong on cardiomyocyte apoptosis and the expression of apoptosis-related factors Bcl-2 and Bax,thereby providing experimental evidence for the prevention and treatment of Amanita caojizong poisoning.Methods Mouse cardiomyocytes(HL-1 cells)cultured in vitro were divided into an experimental group(treated with Amanita caojizong extract)and a control group(treated with PBS).After treatment with Amanita caojizong extract,apoptosis of HL-1 cells was observed using TUNEL staining,and the protein expression levels of Bax,Bcl-2,Caspase-3,and Cleaved Caspase-3 in HL-1 cardiomyocytes were detected by Western blot.Results Compared with the control group,the TUNEL staining showed significantly increased apoptotic fluorescence intensity in the Amanita caojizong extract-treated group.The protein expressions of Bax,Caspase-3,and Cleaved Caspase-3 in HL-1 cells in the Amanita caojizong-treated group were upregulated,while the expression of Bcl-2 was downregulated.Conclusion Amanita caojizong can promote apoptosis of mouse cardiomyocytes,and its mechanism may be associated with the Bcl-2/Bax pathway.

Objective:To explore the cause of death of 2 suspected Yunnan sudden unexplained death (YNSUD) cases in Dayao County, Yunnan Province.Methods:The field epidemiological investigation and autopsy of 2 cases of YNSUD in Dayao County from June 15 to 20, 2020 were conducted; and blood and tissue samples were collected for qualitative analysis of common poisons and drugs.Results:The areas where the two cases were located were all seriously ill villages with a history of YNSUD, and the time of death occurred in the onset season of YNSUD. There was no blood relationship between the 2 cases, no obvious abnormal symptoms before death, no special diet, no history of exposure to pesticides and other toxic chemicals, and the test results of common poisons were all negative. Autopsy pathological examination results showed that case 1 died of acute cardiac dysfunction caused by sudden acute myocardial infarction of coronary heart disease, and case 2 died of central respiratory and circulatory failure caused by spontaneous subarachnoid hemorrhage.Conclusions:The two cases are excluded from YNSUD through autopsy, and the cause of death is determined. It is suggested that emergency response should be taken as soon as possible for YNSUD cases, and autopsy should be actively carried out to clarify the cause of death from a pathological point of view.

Objective:To analyze common pathogenic gene mutations of arrhythmogenic right ventricular cardiomyopathy (ARVC) in Yunnan unexplained sudden death (hereinafter referred to as Yunnan sudden death) cases, and explore the etiological relationship between Yunnan sudden death and ARVC.Methods:Four typical Yunnan sudden death affected counties (cities) were selected as investigation sites. Cryopreserved autopsy cardiac cavity blood samples were collected from Yunnan sudden death cases ( n = 3), and peripheral venous blood samples were harvested from their relatives (first, second, third and immediate degree of kinship, n = 67) and control population ( n = 49). The DNA of blood samples was extracted for amplification and sequencing of 97 exons of 5 common ARVC desmosomal protein [desmoplakin (DSP), desmocollin-2 (DSC2), desmoglein-2 (DSG2), plakophilin-2 (PKP2) and junction plakoglobin (JUP)] genes, and genetic lineage of Yunnan sudden death cases was investigated. Results:A total of 17 gene mutation sites were discovered in Yunnan sudden death cases and their relatives, with 6, 5, 4, 1 and 1 in the DSP, DSC2, DSG2, PKP2 and JUP genes, which were not found in the control population. Among them, 9 were newly discovered mutation sites and 8 were reported mutation sites. The DSP gene exon 24 c.8472 G>C, a pure contractual sense mutation, was common in the relatives of 4 cases in the same family surveyed; and one immediate relative carried a deletion mutation at c.2368 - 2370 of exon 15 of DSC2 gene.Conclusion:Yunnan sudden death cases and their relatives carry mutations in the ARVC desmosomal protein DSP, DSC2, DSG2, PKP2, and JUP genes, and the onset of some Yunnan sudden death may be associated with mutations in the ARVC desmosomal protein genes.

Objective:To investigate the mutation of desmosomal protein gene of arrhythmogenic right ventricular cardiomyopathy (ARVC) in people from Yunnan unexplained sudden death (YUSD) area in Xiangyun County, Dali Prefecture, Yunnan Province, and to explore the etiological relationship between the mutation of ARVC desmosomal protein gene and YUSD.Methods:The autopsy cardiac blood sample of YUSD case ( n = 1) and the peripheral venous blood samples of the same time case ( n = 1) and relatives of YUSD case ( n = 16) were collected in Xiangyun County. Blood DNA was extracted for PCR amplification and sequencing of a total of 97 exons of the ARVC desmosomal protein genes [plakophilin 2 (PKP2), junction plakoglobin (JUP), desmoplakin (DSP), desmoglein 2 (DSG2) and desmocollin 2 (DSC2)] were conducted by Sanger method. At the same time, basic information and genetic family of YUSD case, the same time case and relatives of YUSD case were investigated, and gene mutations were comprehensively analyzed. Results:The YUSD case and the same time case carried JUP, DSP and DSG2 gene mutations. Among the relatives of YUSD case, 2, 14, 16, 15 and 4 cases had mutations in PKP2, JUP, DSP, DSG2 and DSC2 genes, respectively. The YUSD case, the same time case and the relatives of YUSD case carried 6 identical mutation sites: JUP gene exon 3 c.213 T>C synonymous mutation, exon 14 c.2089 A>T missense mutation; DSP gene exon 19 c.2631 G>A synonymous mutation, exon 24 c.8472 G>C synonymous mutation; DSG2 gene exon 8 c.861 C>T synonymous mutation, and exon 15 c.3321 T>C synonymous mutation.Conclusion:In Xiangyun County, six identical mutation sites (JUP gene c.213 T>C and c.2089 A>T, DSP gene c.2631 G>A and c.8472 G>C, DSG2 gene c.861 C>T and c.3321 T>C) carried by YUSD case, the same time case and the relatives of YUSD case may be related to the incidence of some YUSD cases.

Objective:To explore the relationship between arrhythmogenic right ventricular cardiomyopathy (ARVC) desmosomal protein gene mutations and Yunnan unexplained sudden death (hereinafter referred to as Yunnan sudden death) by detecting 5 common ARVC desmosomal protein gene mutations of Yunnan sudden death cases and their relatives in Heqing County, Yunnan Province.Methods:In January 2021, the autopsy heart cavity blood was collected from Yunnan sudden death cases in 8 villages in Heqing County, and peripheral venous blood samples of relatives of the cases were collected. Blood samples' DNA was extracted, after PCR amplification, 97 exons of 5 desmosomal protein genes [desmoplakin (DSP), desmoglein-2 (DSG2), plakophilin-2 (PKP2), junction plakoglobin (JUP) and desmocollin-2 (DSC2)] were sequenced by Sanger method to analyze gene mutations.Results:Three blood samples of Yunnan sudden death cases and 36 blood samples of relatives were collected. A total of 26 gene mutation sites were detected in 39 blood samples, with a total mutation rate of 26.80% (26/97). There were 13, 5, 3, 3 and 2 mutation sites in DSP, DSG2, PKP2, JUP and DSC2 genes, respectively. Among them, 19 were reported mutations and 7 were new mutations: DSP gene exon 3 c.372G>A, exon 15 c.2090A>G, exon 17 c.2371C>A, exon 24-I c.8458T>G; DSG2 gene exon 8 c.861C>T; PKP2 gene exon 3 c.892C>A, exon 8 c.1725G>T. Three Yunnan sudden death cases and 36 relatives were all carriers of compound gene mutation, and the same person carried 3 - 9 gene mutation sites at the same time.Conclusion:Mutations of ARVC desmosomal protein genes DSP, DSG2, PKP2, JUP and DSC2 exist in Yunnan sudden death cases and their relatives, which may be the genetic background factors of some Yunnan sudden death.

Objective:To analyze the mutations in desmosomal protein genes of arrhythmogenic right ventricular cardiomyopathy (ARVC) in relatives of Yunnan unexplained sudden death (YUSD) cases in Nanjian County, Dali Prefecture, Yunnan Province, and provide a basis for etiological hypothesis and control measures.Methods:The blood samples of YUSD case relatives ( n = 7) and control villagers ( n = 7) were collected, and basic situation investigation and electrocardiography (ECG) examination were performed at the same time. Blood DNA was extracted as a template for PCR amplification, and Sanger method was used to perform plakophilin 2 (PKP2), desmoglein 2 (DSG2), desmocollin 2 (DSC2), desmoplakin (DSP), and junction plakoglobin (JUP) five ARVC desmosomal protein genes sequencing of a total of 97 exons, and comprehensive analysis of gene mutations was carried out. Results:Five of YUSD case relatives carried genetic mutation sites, including DSP gene heterozygous synonymous mutations about exon 20 c.2862 C>T (p.Cys954Cys) and exon 24F c.7122 C>T (p.Thr2374Thr), DSC2 gene heterozygous missense mutation about exon 15 c.2326 A>G (p.Ile776Val), and all the five people were single heterozygous mutation carriers. Among them, two case relatives of the father-son carried the same site mutation of the DSC2 gene; the abnormal ECGs of three YUSD case relatives were ST-T change or clockwise rotation. However, the mutation sites of PKP2, DSG2, DSC2, DSP and JUP genes in control villagers were not detected.Conclusions:YUSD case relatives in Nanjian County carry ARVC desmosomal protein genes DSP and DSC2 mutations. Pathogenic mutation of DSC2 gene c.2326 A>G (p.Ile776Val) is may related to the incidence of some YUSD cases.

Objective:To expound the pathogenesis relationship between Yunnan unexplained sudden death (YUSD) and desmosomal protein gene mutations of arrhythmogenic right ventricular cardiomyopathy (ARVC).Methods:Four YUSD cases families by ARVC pathological diagnosis were selected, to collect heart blood samples of YUSD cases by ARVC pathological diagnosis( n=3), venous blood samples of immediate relatives with genetic relationship (case relatives, n=4) and control population without genetic relationship ( n=7). DNA was extracted for PCR amplification and sequencing of a total of 97 exons of the ARVC desmosomal protein genes plakophilin 2 (PKP2), desmoplakin (DSP), desmoglein 2 (DSG2), desmocollin 2 (DSC2), and junction plakoglobin (JUP), and the mutations of the 5 genes were analyzed in combination with the genetic family. Results:DSP gene mutations were found in all YUSD cases by ARVC pathological diagnosis and case relatives, and PKP2, DSG2, DSC2 and JUP genes mutations were found in 1 person each. The same person carried 1-3 genes mutations. DSP gene existed 4 exon mutation sites, and 1 of which was a newly discovered heterozygous synonymous mutation c.4014 C>A (p.A1338A). PKP2 gene existed 2 exon missense mutation sites in 1 YUSD case by ARVC pathological diagnosis, and 1 of which was a newly discovered heterozygous mutation c.739 G>C (p.G247R). One heterozygous missense mutation site c.799 G>A (p.A267T) of JUP gene was newly discovered, and the predictive value of protein function was 0.963, the possibility of abnormal changes in protein function was high. DSG2 and DSC2 genes each had one mutation site. However, no mutation was found in control population.Conclusions:Both YUSD cases by ARVC pathological diagnosis and case relatives carry ARVC desmosomal protein genes DSP, PKP2, DSG2, DSC2 and JUP mutations. There may be a certain pathogenesis relationship between YUSD and ARVC desmosomal protein gene mutations.

Objective To study the desmosomal protein plakophilin-2(PKP2)gene mutation of arrhythmogenic right ventricular cardiomyopathy (ARVC) in different populations of Yunnan unexplained sudden death (YUSD) areas,and explore the relationship between PKP2 gene mutation and YUSD.Methods Heart blood samples of YUSD cases (n =7) and venous blood samples of YUSD immediate family (n =30) and other family (n =11) members were collected.Basic situation and genetic relationship of YUSD immediate family and other family were investigated,and electrocardiography (ECG) was examined.DNA from blood samples was extracted and 15 exons of PKP2 gene were sequenced to analyze the mutation of PKP2 gene in different populations.Results A total of 10 people carried 11 PKP2 gene mutation sites with a mutation rate of 20.83％ (10/48).Two mutation sites were novel (p.G247R,p.T298N),and the new mutation sites were carried by two YUSD cases.Eight missense mutations were heterozygous mutations,two of the three synonymous mutations were heterozygous mutations,and one was homozygous synonymous mutation.The mutation sites were significantly concentrated in 4 exons,which were No.1 097 base of exon 4,No.819 and 893 bases of exon 3.2,No.739 base of exon 3.1,and No.156 base of exon 1.One YUSD case of ARVC pathological change carried exon 3.1 (p.G247R) and exon 4 (p.L366P) compound heterozygous mutations,the other YUSD case carried exon 3.2 (p.T298N) heterozygous mutation.The YUSD cases and immediate family with PKP2 gene mutations showed obvious family genetic relationships,and they were all first-degree and second-degree relatives.The abnormal ECGs of YUSD immediate family and other family mainly were conduction block,arrhythmia and premature beat.Conclusion There is a high PKP2 gene mutation rate in different populations of YUSD areas,and there may be a certain etiological connection between PKP2 gene mutations and YUSD.

Objective This study aimed to investigate the characteristics of maternal death cases, the cause of maternal death and the cause of medical dispute,and recommended the relative prevention measures. Methods We retrospectively analyzed 35 cases of maternal death identified in the College of Forensic Medicine in Kunming Medical University from January 2002 to December 2012. Results In 35 maternal death cases: (1) Most maternal deaths are over 30 years old (14 cases, 40%), followed by 17 to 19 years old (6 cases, 17.1%) . And most maternal deaths came from countryside (25 cases,71.4%) . (2) 26 cases maternal deaths happened during the perinatal period (68.5%) . (3) 26 cases happened in county hospitals, township hospitals and informal medical organizations (74.3%) . 5 maternal deaths happened on the way to the hospital or at home (14.3%) . (4) 17 cases died of obstetric hemorrhagic shock (48.6%), 11 cases died of amniotic fluid embolism (31.4%), 2 cases died of amniotic fluid embolism and obstetric hemorrhagic shock (5.7%) . (5) 30 cases of medical disputes were caused by maternal death. Among them, the clinical diagnosis was not consistent with the pathological diagnosis in 20 cases (The misdiagnosis rate was 66.7%) . (6) The causes of medical dispute:clinical diagnosis was unknown in 11 cases (36.7%),11 cases were suspected that rescue was not timely or improper (36.7%),and 8 cases were suspected of misdiagnosis (26.6%) . Conclusion Obstetric hemorrhage and pulmonary amniotic fluid embolism were the main reasons of the maternal death. The incidence of maternal death and medical disputes could be reduced by improving the ability of obstetric staff,and strengthening the diagnosis and treatment about obstetric hemorrhage and pulmonary amniotic fluid embolism. At the same time, women should improve their self-care awareness, reduce early marriage and early pregnancy,and enhance the consciousness of hospital treat and cure.