BackgroundThe differentiation of naïve CD8+ T cells into effector cells upon activation is essential for eliminating intracellular pathogens and cancerous cells, although the underlying epigenetic mechanisms remain incompletely characterized.MethodsPeripheral blood mononuclear cells (PBMCs) were obtained from healthy donors. naïve CD8+ T cells were purified and activated with α-CD3/CD28-conjugated microbeads for 0, 24, or 72 h in vitro. Flow cytometry was used to assess cytokine production and activation markers at each time point. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed to identify differentially accessible chromatin regions (DARs). RNA sequencing (RNA-seq) was performed to measure gene expression. Data from ATAC-seq and RNA-seq were integrated to examine the relationship between chromatin accessibility and gene expression. Enriched pathways for DARs and differentially expressed genes (DEGs) were determined by KEGG pathway and gene ontology (GO) enrichment analysis, and transcription factor (TF) binding patterns around these genes were visualized by footprint analysis.ResultsUpon activation, naïve CD8+ T cells showed increased production of IFN-γ, TNF, and IL-2, and elevated expression of CD69 and CD95. Integrated ATAC-seq and RNA-seq analysis identified 568 and 541 dual-upregulated genes (showing both increased chromatin accessibility and expression) at 24 and 72 h post-activation, respectively. These early-response genes were enriched in pathways including pyruvate metabolism and the DNA damage response. Footprint analysis predicted the ETS and bZIP TF families as key regulators driving this coordinated chromatin and transcriptional reprogramming. Furthermore, distinct chromatin remodeling patterns were observed in gene sets associated with memory, effector function, exhaustion, and metabolism, revealing that accessibility changes did not always directly correlate with transcriptional outcomes.ConclusionThis study defines a core set of genes and TFs that critically regulate the initial activation of human naïve CD8+ T cells. These results provide a molecular roadmap for future efforts to engineer more potent and durable CD8+ T cell responses for adoptive cell therapy.

Background. Mycoplasma pneumoniae ( MP ) is a common causative pathogen of community-acquired pneumonia in children, with clinical presentations ranging in severity. Early stratification and timely intervention are essential for improving patient outcomes. However, a major clinical challenge lies in the limited ability to accurately distinguish between mild and severe cases based solely on early clinical indicators. Methods. This prospective real-world study investigated the differences in microbiome and metabolomics between mild and severe MP pneumonia (MPP) in children. Bronchoalveolar lavage fluid samples were collected from 153 children and subjected to metagenomic sequencing and non-targeted metabolomic analysis. Meanwhile, to enhance early diagnostic accuracy, this study developed a machine learning classification model and validated it using a third-party validation set. Results. The results revealed significant alterations in the abundance of specific bacterial communities in the severe group, most notably the coexistence of MP and Alphainfluenzavirus influenzae , which may contribute to disease exacerbation through synergistic pathogenic mechanisms. Furthermore, the macrolide resistant rate of MP in the severe group exceeded 80%, emphasizing the importance of appropriate antibiotic selection. Metabolomic analysis showed a significant enrichment of metabolites related to cellular energy metabolism and immune regulation in severe cases. The model demonstrated exceptional predictive performance, achieving an area under the curve ranging from 0.909 to 0.991, which significantly outperformed conventional clinical stratification methods. Conclusions. These findings elucidate the distinct pathophysiological mechanisms underlying both mild and severe MP infections and provide a promising framework for improving early diagnosis and personalized treatment strategies in paediatric MPP.

Neuroblastoma (NB) is a pediatric solid tumor with poor outcomes in high-risk cases, largely driven by the tumor microenvironment (TME). Tumor-associated neutrophils (TANs) exert potent regulatory effects within the TME, but the mechanisms orchestrating their activation and functional polarization in NB remain elusive. Here, utilizing proteomic and single-cell transcriptomic analyses, we identify a distinct tumor-enriched SCART1+ T cell subset within the NB TME. This population prominently secretes IL-17A, GM-CSF, IL-22, and TNF-α. Moreover, the abundance of SCART1+ T cells correlates positively with TAN infiltration. Further functional assays reveal that IL-17A and GM-CSF promote neutrophil migration, while GM-CSF and TNF-α induce neutrophil activation and immunosuppression. Collectively, our data demonstrate that the specific cytokines are capable of driving the recruitment and immunosuppressive polarization of neutrophils in NB TME. These findings suggest SCART1+ T cells as potential architects of neutrophil suppressive polarization via a cytokine-mediated network, representing a promising target for immunotherapy.

ABSTRACT Background Papillary thyroid carcinoma (PTC) is the most commonly diagnosed subtype of thyroid cancer and represents a highly prevalent form of endocrine malignancy. Aims This study aimed to investigate the role and molecular mechanism of CHI3L1 in PTC progression. Methods and Results CHI3L1 expression in PTC was analyzed using public datasets. Cell proliferation was assessed using the CCK‐8 assay and colony formation assay. Tumor growth was evaluated using nude mouse xenograft models. Cell invasion was evaluated using the transwell assay, while cell migration was assessed with the wound healing assay. Transcriptomic analysis was conducted to examine the molecular mechanism, and real‐time quantitative PCR was performed for gene expression validation. The findings revealed that CHI3L1 expression was upregulated in various cancers, mainly in PTC. Both in vitro and in vivo assays demonstrated that cell proliferation was suppressed when CHI3L1 was knocked down. Transcriptome sequencing indicated that CHI3L1 knockdown was associated with migration‐related pathways and the TP53 signaling pathway. Transwell assays showed reduced cell invasion upon CHI3L1 suppression, while wound healing assays demonstrated decreased cell migration. Following CHI3L1 silencing, real‐time quantitative PCR verified the overexpression of TP53‐related genes. Survival analysis further indicated a correlation between elevated CHI3L1 expression and reduced survival rates. Conclusion This study identified that CHI3L1 was an oncogene in PTC and promotes tumor cell proliferation associated with downregulating the TP53 pathway. It provides new evidence supporting CHI3L1 as a potential molecular target for future therapeutic investigation in PTC.

Herpes zoster (HZ) is the clinically apparent manifestation of latent varicella-zoster virus (VZV) reactivation. Aging and immunosuppression are established risk contexts. SARS-CoV-2 infection has prompted renewed attention to whether acute or post-acute immune changes can reduce the reserve needed to maintain VZV latency. Large observational studies report a modest increase in HZ after COVID-19, most consistently after severe disease or hospitalization and within early post-infection windows. These associations do not establish direct causation or a population-wide shift of HZ toward younger adults. A threshold-lowering interpretation is more consistent with the available evidence: SARS-CoV-2 infection may narrow latency-control reserve through cellular immune disruption, interferon dysregulation, and inflammation, particularly in hosts already affected by comorbidity or treatment-related immunosuppression. Long COVID provides a setting in which persistent immune dysregulation can be studied, but it is not yet a proven causal framework for VZV disease. Post-COVID HZ may therefore represent a clinically visible manifestation of disrupted host-virus homeostasis in susceptible individuals. Prospective studies that combine clinical phenotyping with VZV-specific cellular immune measurements are needed to test this model.

BACKGROUND:Tissue-nonspecific alkaline phosphatase (TNAP) expression increases after liver injury, but its role in liver fibrosis remains unclear. This study investigated the effect of TNAP on liver fibrosis and its mechanism in regulating TGF-β1 signaling. METHODS:Human liver samples and a CCl4-induced liver fibrosis mouse model with adv-TNAP and a TNAP inhibitor (tetramisole, Tetra) were used to study the function of TNAP in liver fibrosis. Primary HSCs were used to study the mechanism of TNAP in regulating the TGF-β1 signal. RESULTS:Elevated TNAP expression was observed in human and murine fibrotic liver tissues, correlating with increased fibrotic markers. In vivo experiments using TNAP overexpression and inhibition in a CCl4-induced liver fibrosis mouse model demonstrated that TNAP exacerbated, while its inhibition alleviated, liver fibrosis. In vitro studies revealed that TNAP regulated TGF-β1 conversion and HSCs activation through the TGF-β1/SMAD pathway. TNAP facilitated TGF-β1 conversion by promoting the interaction between CD47 and thrombospondin-1 (TSP1). Membrane expression of CD47 modulated by TNAP might contribute to the binding effect of CD47 and TSP1. CONCLUSIONS:TNAP plays a critical regulatory role in TGF-β1-mediated liver fibrosis, probably by promoting the binding of CD47/TSP1. Targeting TNAP-mediated pathways may offer new therapeutic strategies for liver fibrosis.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the four plots shown for the flow cytometric (FCM) data in Fig. 2 on p. 5728 contained overlapping areas, where differently performed experiments were intended to have been represented. Moreover, there were also indications that certain of the data were potentially overlapping with Figs. 4 and 5 (the other pair of figures containing FCM data in this paper). Owing to the fact that the abovementioned data bore notable similarities comparing across different quadrants among these three figures, the Editor of Molecular Medicine Reports has decided that this paper should be retracted from the Journal on the grounds of an overall lack of confidence in the presented data. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 18: 5726‑5732, 2018; DOI: 10.3892/mmr.2018.9604].

Wilms tumor (WT), the most common pediatric renal malignancy, exhibits a relatively low mutational burden compared to adult cancers, which hinders the development of targeted therapies. To elucidate the molecular landscape of WT, we perform integrative proteomic, phosphoproteomic, transcriptomic, and whole-exome sequencing analyses of WT and normal kidney tissue adjacent to tumor. Our multi-omics approach uncovers prognostic genetic alterations, distinct molecular subgroups, immune microenvironment features, and potential biomarkers and therapeutic targets. Proteome- and transcriptome-based stratification identifies three molecular subgroups with unique signatures, correlating with different histopathological subtypes and putative cellular origins at different stages of embryonic kidney development. Notably, we identify EHMT2 as a promising prognostic biomarker and therapeutic target associated with epigenetic regulation and Wnt/β-catenin pathway. In this work, we provide a comprehensive molecular characterization of WT, offering valuable insights into its pathogenesis and a foundational resource for future therapeutic development.

BackgroundEpstein–Barr virus (EBV) infects more than 95% of the global population, and EBV reactivation is associated with the development of various diseases. The aims of this study were (1) to investigate the epidemiology of EBV, CMV, HSV, and Toxoplasma gondii infections and their associations with serum antibody profiles and (2) to explore the relationships between EBV infection and reactivation and the antibody status of Toxoplasma gondii, CMV, and HSV.MethodsThis retrospective study detected EBV-specific antibody profiles or plasma EBV-DNA, as well as antibodies against Toxoplasma gondii, cytomegalovirus (CMV), and herpes simplex virus (HSV). Basic demographic information, including age and sex, was collected to assess the EBV infection status and the prevalence of antibodies against Toxoplasma gondii, CMV, and HSV in different populations.ResultsA total of 3,046 hospitalized patients (1,524 male, 1,522 female) who underwent antibody testing for Toxoplasma gondii, CMV, and HSV were included in the study. The overall serum positivity rates for Toxoplasma gondii IgG, CMV IgG, and HSV IgG increased with age, with overall rates of 16.84, 97.50, and 91.20%, respectively. Among the 1,079 patients who underwent EBV-DNA testing, the lowest virus detection rate (9.97%) was found in the 21–40-year-old age group, with a progressively increasing rate with age. Additionally, compared with patients who were negative for Toxoplasma gondii IgG, those with Toxoplasma gondii IgG positivity had significantly higher rates of EBV-EA-IgG and EBV-VCA-IgA antibody positivity (p = 0.032; p < 0.001). Furthermore, patients with EBV reactivation had the highest CMV IgM antibody positivity rate (60.53%), followed by those with primary EBV infection (45.45%), whereas patients without EBV infection had the lowest rate (25.75%), with statistically significant differences between the groups.ConclusionEBV antibody profiles positivity rates were higher in patients with Toxoplasma gondii IgG positivity than in those with Toxoplasma gondii IgG negativity. The CMV IgM antibody positivity rate was significantly higher in EBV reactivation group than in other groups. These results highlight potential diagnostic relevance of co-testing for EBV and CMV in suspected reactivation cases.

Autosomal dominant non-syndromic hearing loss (ADNSHL) caused by MYO6 gene variation typically manifests as progressive post-lingual deafness. We identified an 11-member Chinese family across four generations with ADNSHL. We investigated the genetic patterns and mechanisms and employed whole-exome sequencing on genomic DNA derived from peripheral blood to screen and identify pathogenic genes. We verified the potential pathogenicity of the mutation site through minigene assays. Our investigation revealed a novel splice variant, c.554-4A > G (NM_004999.4), located in intron 7 of MYO6, which exhibited clear co-segregation with the hearing impairment phenotype among the family members. Subsequent minigene splicing assays demonstrated that the c.554-4A > G variation resulted in an insertion of three bp in intron 7 of MYO6 (c.554-1_554-3 insTAG, p.184_185insV). The novel splice variant: c.554-4A > G was associated with ADNSHL. Our study expands the spectrum of pathogenic genes and variant sites associated with ADNSHL.

As immunotherapy gains increasing attention and clinical application, the immune modulation therapy has been widely used in the treatment of infectious and critical diseases. Clinical evidence has been accumulated for application of thymosin alpha 1 (T alpha 1), a classical immune modulator, in related domains. The National Clinical Research Center for Infectious Diseases, National Medical Center for Infectious Diseases and other institutions invited multidisciplinary experts to develop this expert consensus on clinical application of T alpha 1 in infectious diseases and critical care medicine. Based on the latest domestic and international research findings and considering relevant factors, including economics, patient preferences and values, tradeoffs, accessibility, fairness and acceptability, the consensus assesses the quality levels of current evidence and forms 10 recommendations on the application of T alpha 1 in treatment of liver diseases, viral infections, bacterial infections and critical illnesses. This consensus aims to enhance understanding of T alpha 1 and improving its standardized application for clinicians.

IntroductionDecreased stability of coronary atherosclerotic plaques correlates with a heightened risk of acute coronary syndrome (ACS). Thus, early diagnosis and treatment of unstable plaques are imperative in averting adverse cardiovascular events. This study aims to identify diagnostic biomarkers for unstable coronary atherosclerotic plaques and investigate the role of immune cell infiltration in their formation.MethodsThe datasets GSE163154 and GSE111782, obtained from the gene expression omnibus (GEO) database, were amalgamated for bioinformatics analysis, using the dataset GSE43292 as a test set. Sequentially, we performed principal component analysis (PCA), differential gene expression analysis, enrichment analysis, weighted gene co-expression network analysis (WGCNA), utilized a machine learning algorithm to screen key genes, conducted receiver operating characteristic (ROC) curve analysis and nomogram model to assess biomarker diagnostic efficacy, validated the biomarkers, and analyzed immune cell infiltration.ResultsIn conclusion, enrichment analyses demonstrate that genes are significantly enriched in inflammatory and immune-related pathways. We identified HSPA2 and GEM as key genes and validated them experimentally. Significant differences existed in immune cell infiltration between subgroups. Additionally, HSPA2 and GEM showed significant associations with a wide range of immune cells.DiscussionHSPA2 and GEM can function as diagnostic biomarkers for unstable coronary atherosclerotic plaques. In combination with immune cell infiltration analyses, our study provides new insights into the future study of unstable plaque occurrence and molecular mechanisms.

Epstein-Barr virus (EBV) is a carcinogenic γ-herpesvirus that remains latent in more than 95% of adults. The virus can undergo lytic activation when immune function is suppressed or when stimulated by drugs or pathogens. EBV reactivation poses a significant threat to human health and is closely associated with various cancers, such as Burkitt's lymphoma and nasopharyngeal carcinoma. Inhibiting EBV reactivation is a current clinical challenge. Tumour necrosis factor-α (TNF-α), an important cytokine, has different effects on various viruses. It also exerts varying effects on the same virus depending on the type of infected cell. This study aimed to investigate the impact of TNF-α on EBV reactivation and its underlying mechanisms. Our experimental research revealed that TNF-α significantly inhibits EBV reactivation and that this inhibitory effect is mediated primarily through its receptor TNFR1. Furthermore, TNF-α affects the expression of the GPX4 protein and regulates the potential ferroptosis state of cells. Using transmission electron microscopy and other methods, we observed typical characteristics of ferroptosis, such as changes in mitochondrial morphology and Fe2 + accumulation. Additionally, we established stable GPX4-knockdown cell lines, which demonstrated the crucial role of GPX4 in the process of TNF-α-mediated inhibition of EBV reactivation. Overall, TNF-α acts on the TNFR1 receptor, thereby affecting the GPX4 protein and the ferroptosis pathway to achieve its inhibitory effect on EBV reactivation. These findings provide new insights into the mechanisms of EBV reactivation and may offer new perspectives for the early treatment of EBV-related diseases.

Doxorubicin (DOX) is one of the most widely used antineoplastic drugs with known cardiotoxicity while other organ toxicity, such as hepatotoxicity is not well defined. This study was to explore the role of nicotinamide adenine dinucleotide (NAD+) in DOX-induced hepatotoxicity. DOX (20 mg/kg) induced acute liver injury and oxidative stress in C57BL/6 J mice at 48 h. Notably, the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and NAD(P)H dehydrogenase quinone 1 (NQO1) were downregulated. NAD+ deficiency was confirmed due to DOX exposure. Mechanistically, the downregulation of nicotinamide mononucleotide adenylyl transferase 1 (NMNAT1), NMNAT2 and NMNAT3, while no alteration of nicotinamide phosphoribosyl transferase was proved. As a consequence of NAD+ deficiency, the expression of poly-ADP-ribose polymerase1 (PARP1), CD38 and Sirtuin1 (SIRT1) were reduced. Furthermore, supplementation of NAD+ (200 mg/kg/day) or its precursor nicotinamide mononucleotide (NMN) (500 mg/kg/day) alleviated liver injury, attenuated oxidative stress, and elevated the downregulation of Nrf2 and NQO1. More importantly, compromised expression of NMNAT1-3, PARP1, CD38 and SIRT1 were improved by NAD+ and NMN. In conclusion, NAD+ deficiency due to NMNATs expression inhibition may attribute to the pathogenesis of DOX-induced hepatotoxicity, thus providing new insights for mitigating DOX side effects.

Abstract Metabolic dysfunction-associated steatotic liver disease (MASLD) has emerged as a predominant liver disease worldwide, lacking approved drugs for clinical intervention at present. The composite dietary antioxidant index (CDAI) is used to assess the anti-inflammatory properties of diets, with higher CDAI indicating greater exposure to antioxidants. Therefore, our study aimed to explore the relationship between CDAI and MASLD in order to identify potential therapeutic approaches. We collected data from 12,286 participants in the National Health and Nutrition Examination Survey (NHANES) database from 2017 to 2020 for analysis. The correlation between CDAI and MASLD status, controlled attenuation parameter (CAP), and liver stiffness measurement (LSM) was evaluated by adjusting for confounding variables using weighted binary logistic regression model, linear regression model, and restricted cubic spline (RCS) regression. The median CDAI in this study was − 0.3055 (interquartile range [IQR], − 2.299 to 2.290). The CDAI was higher in the population characterized by being young, female, higher income, absence of diabetes, and non-MASLD. After multivariable adjustment, the results of the weighted linear regression model suggested that higher CDAI may be associated with a decrease in CAP values; the results of the RCS regression model indicated significant non-linear relationships between MASLD status, CAP, LSM, and CDAI. The CDAI corresponding to the inflection points of the relationship curves between MASLD status, CAP, LSM, and CDAI were 0.349, 0.699, and 0.174, respectively. After further stratification by gender, we found that the relationship between MASLD status, CAP, and CDAI was significantly linear for females, whereas for males, it was non-linear, and the CDAI values corresponding to the inflection points in the curves for males were 1.325 and 0.985, respectively. We found that higher CDAI may be associated with decreased CAP values, particularly significant in females, suggesting that the intake of complex dietary antioxidants may ameliorate hepatic steatosis and reduce the occurrence of MASLD. Therefore, promoting dietary patterns rich in antioxidants may be an appropriate strategy to reduce the incidence of MASLD.

Background Epigenetic remodeling at effector gene loci has been reported to be critical in regulating T cell differentiation and function. However, efforts to investigate underlying epigenetic mechanisms that control T cell behaviors have been largely hindered by very limited experimental tools, especially in humans. Results In this study, we employed a flow cytometric assay to analyze histone acetylation at single-cell level in human T cells. The data showed that histone acetylation was increased during T cell activation. Among T cell subsets, terminally differentiated effector memory T (T EMRA ) cells robustly producing effector cytokines were hyper-acetylated. Conversely, these T EMRA cells had lower expression levels of TCF-1, a key transcription factor for maintaining stem cell features. Pharmaceutical inhibition of histone acetylation using a small molecule C646 restrained the production of effector molecules, but retained stem cell-like properties in T cells after expansion. Conclusions Per-cell histone acetylation is associated with terminal differentiation and poor stemness in human T cells. These observations suggest a new approach to enhance the stem cell-like properties of T cells and improve the efficacy of immunotherapy.

In this study, by pooling the clinical data of patients who died with a history of long-term clozapine use and by examining their hearts, it was found that long-term clozapine use can lead to cardiomyopathy and that its presentation resembles arrhythmogenic cardiomyopathy (ACM), i.e., it exhibits a predominantly right ventricular fatty infiltration with mild left ventricular damage. The transcriptomic data of rat cardiomyocytes after clozapine intervention were analyzed by transcriptomic approach to explore the causes of clozapine cardiomyopathy. The cause of clozapine cardiomyopathy was then explored by a transcriptomic approach, which revealed that its clozapine action on cardiomyocytes enriched cardiomyocyte-related differential genes in biological processes such as muscle development and response to hypoxia, as well as pathways such as fatty acid metabolism and cellular autophagy. Transcriptomic analysis showed that Egr1, Egr2, ler2, Jun, Mapk9, Nr1d2, Atf3, Bhlhe40, Crem, Cry1, Cry2, Dbp were hub genes for clozapine injury to the myocardium, and that these genes may play an important role in the myocardial ACM-like changes caused by clozapine. Combined with the results of pathological examination and transcriptomic analysis, it can be concluded that the long-term action of clozapine on cardiomyocytes leads to cellular autophagy and subsequent structural remodeling of the heart, and in the remodeling affects fatty acid metabolism, which eventually leads to ACM-like changes.

Background:The mechanical properties of the aorta are particularly important in clinical medicine and forensic science, serving as basic data for further exploration of aortic disease or injury mechanisms.Objective:To study the influence of various factors (age, gender, test direction, anatomical location, and pathological characteristics) on the mechanical properties and thickness of the aorta.Methods:In this study, a total of 24 aortas (age range: 54–88 years old) were collected, one hundred and seventy-four dog-bone-shaped samples were made, and then the uniaxial tensile test was run, finally, pathological grouping was performed through histological staining.Results:Atherosclerotic plaques were mainly distributed near the openings of blood vessel branches. The distribution was most severe in the abdominal aorta, followed by the aortic arch. Aortic atherosclerosis was a more severe trend in the male group. In the comparison of thickness, there were no significant differences in age (over 50 years) and test direction, the average thickness of the aorta was greater in the male group than the female group and decreased progressively from the ascending aorta to the abdominal aorta. Comparing the mechanical parameters, various parameters are mainly negatively correlated with age, especially in the circumferential ascending aorta (εp “Y = −0.01402*X + 1.762, R2 = 0.6882”, εt “Y = −0.01062*X + 1.250, R2 = 0.6772”); the parameters of males in the healthy group were larger, while the parameters of females were larger in atherosclerosis group; the aorta has anisotropy, the parameters in the circumferential direction were greater than those in the axial direction; the parameters of the ascending aorta were the largest in the circumferential direction, the ultimate stress [σp “1.69 (1.08,2.32)”] and ultimate elastic modulus [E2“8.28 (6.67,10.25)”] of the abdominal aorta were significantly larger in the axial direction; In the circumferential direction, the stress [σp “2.2 (1.31,3.98)”, σt “0.13 (0.09,0.31)”] and ultimate elastic modulus (E2 “14.10 ± 7.21”) of adaptive intimal thickening were greater than those of other groups, the strain (εp “0.82 ± 0.17”, εt “0.53 ± 0.14”) of pathological intimal thickening was the largest in the pathological group.Conclusion:The present study systematically analyzed the influence of age, sex, test direction, anatomical site, and pathological characteristics on the biomechanical properties of the aorta, described the distribution of aortic atherosclerosis, and illustrated the characteristics of aortic thickness changes. At the same time, new insights into the grouping of pathological features were presented.

Due to nonspecific pathological changes and the rapid degradation of insulin in postmortem blood samples, the identification of the cause of death during insulin overdose has always been a difficulty in forensic medicine. At present, there is a lack of studies on the toxicological changes and related mechanisms of an insulin overdose, and the specific molecular markers of insulin overdose are still unclear. In this study, an animal model of insulin overdose was established, and 24 SD rats were randomly divided into a control group, insulin overdose group, and a recovery group (n = 8). We detected the biochemical changes and analyzed the toxicological mechanism of an insulin overdose. The results showed that after insulin overdose, the rats developed irregular convulsions, Eclampsia, Opisthotonos, and other symptoms. The levels of glucose, glycogen, and C-peptide in the body decreased significantly, while the levels of lactate, insulin, and glucagon increased significantly. The decrease in plasma K+ was accompanied by the increase in skeletal muscle K+. The PI3K-AKT signaling pathway was significantly activated in skeletal muscle, and the translocation of GLUT4/Na+-K+-ATPase to sarcolemma was significantly increased. Rare glycogenic hepatopathy occurred in the recovery group after insulin overdose. Our study showed that insulin overdose also plays a role in skeletal muscle cells, mainly through the PI3K-Akt signaling pathway. Therefore, the detection of signaling pathway proteins of the skeletal muscle cell membrane GLUT4 and Na+-K+-ATPase has a certain auxiliary diagnostic value for forensic insulin overdose identification. Glycogen detection in the liver and skeletal muscle is important for the diagnosis of insulin overdose, but it still needs to be differentiated from other causes of death. Skeletal muscle has great potential for insulin detection, and the ratio of insulin to the C-peptide (I:C) can determine whether an exogenous insulin overdose is present.