Keming Yun

Objective: Fentanyl exposure is accompanied by myocardial and cellular alterations, but the exact mechanisms underlying cardiomyocyte injury and effective intervention strategies remain to be fully elucidated. This study aims to investigate the association between fentanyl exposure, changes in autophagy related markers, mitochondrial structural alterations, and PI3K/Akt/mTOR pathway marker changes in mouse myocardial tissue and human cardiomyocyte (AC16), while evaluating subcellular marker changes following Tetrahydropalmatine (THP) co-exposure. Methods: In vivo mouse models of fentanyl exposure and in vitro AC16 injury models were established. Myocardial histological changes were assessed using hematoxylin and eosin (H&E) staining and immunohistochemistry. Myocardial cell ultrastructure and mitochondrial morphology were evaluated using transmission electron microscopy (TEM). Mitochondrial membrane potential fluorescence, MitoTracker Green (MTG) signals, and intracellular Ca ²⁺ fluorescence were quantitatively assessed using JC-1, MTG, and Fluo-4-AM staining. The phosphorylation ratios of PI3K/Akt/mTOR markers and autophagy related markers (total ULK1, Beclin-1, LC3-II/I protein ratio,and p62 protein; Map1lc3b for transcript measurement) was analyzed by Western blot, immunohistochemistry, immunofluorescence, and RT-qPCR. Results: Fentanyl exposure was accompanied by myocardial histological shifts in mice, alongside elevated Beclin-1, LC3-II/I ratio, and total ULK1 expression and reduced p62 levels in myocardial tissue and AC16 cells (P < 0.05). THP co-exposure was accompanied by attenuation of these myocardial histological changes and autophagy related marker alterations (P < 0.05). TEM and fluorescent staining showed that fentanyl exposure was accompanied by mitochondrial structural changes, reduced MTG fluorescence intensity, increased intracellular Ca ²⁺ fluorescence signals, and altered mitochondrial membrane potential fluorescence (P < 0.05). Conversely, THP co-exposure was accompanied by lower Ca ²⁺ fluorescence signals, a higher JC-1 red/green fluorescence intensity ratio, and reduced mitochondrial structural alterations (P < 0.05). Similarly, while fentanyl exposure was accompanied by lower phosphorylation levels and mRNA expression of Pi3k, Akt, and Mtor, whereas THP co-exposure was accompanied by higher phosphorylation ratios and mRNA expression of these pathway markers (P < 0.05). Conclusion: Fentanyl exposure is accompanied by alterations in autophagy related markers, mitochondrial structural changes, changes in intracellular Ca ²⁺ fluorescence, mitochondrial membrane potential fluorescence changes, and abnormal phosphorylation ratios of PI3K/Akt/mTOR pathway markers. THP co-exposure was associated with shifts in biological markers relative to fentanyl exposure alone. These findings provide preliminary baseline data for evaluating Corydalis yanhusuo alkaloids in opioid associated cardiomyocyte models.

Introduction: Methamphetamine (METH) addiction is a chronic neuropsychiatric disorder characterized by severe abstinence symptoms and high relapse rates, yet the temporal dynamics of gut microbiota alterations across addiction and abstinence phases remain poorly understood. Methods: In this study, we performed a longitudinal microbiome analysis to characterize differences between phases and time-dependent remodeling of the gut microbiota in a mouse model. Fecal samples were collected at defined time points across a 60-day addiction phase followed by a 60-day abstinence phase and analyzed using 16S rRNA gene sequencing. Results: Distinct microbial compositions, diversity and community structure differences were observed between addiction and abstinence phases. Microbial communities during the addiction phase exhibited greater dispersion, whereas progressive stabilization was observed during abstinence. Time-series analyses revealed characteristic alterations in Verrucomicrobia and Actinobacteria during prolonged addiction and early abstinence. At the genus level, Butyricimonas, Candidatus Arthromitus, Enterococcus , and Turicibacter exhibited reproducible phase- and temporal-dependent dynamic patterns. Based on these microbial signatures, random forest models were constructed for phase classification and temporal prediction. The classification model achieved higher model performance during the addiction phase, whereas performance declined during abstinence, reflected by reduced variance explained and increased heterogeneity in gut microbiota composition. Temporal prediction models further demonstrated differential predictive performance between phases. Discussion: These findings identify reproducible temporal changes in gut microbiota across addiction and abstinence and highlight candidate microbial signatures for understanding addiction progression and distinguishing different phases.

Nitrous oxide (N₂O), commonly known as laughing gas, is widely used for its anesthetic and analgesic properties in medical settings. However, its recreational abuse has escalated, leading to severe neurological complications including cognitive impairment, yet the underlying mechanisms remain poorly understood. In this study, male C57BL/6 J mice were exposed to either normal air inhalation or nitrous oxide inhalation for 28 days to evaluate the effects of repeated nitrous oxide exposure on cognitive function. We employed hippocampal transcriptome sequencing, real-time PCR, Western blotting, and non-targeted plasma metabolomics to explore potential regulatory mechanisms. Repeated nitrous oxide exposure impaired motor skills, learning, and cognitive abilities in mice. Integrative multi-omics analysis revealed that differentially expressed metabolites and genes synergistically disrupted energy metabolism through co-regulation of the TCA cycle, MAPK signaling pathway, and pyrimidine metabolism, ultimately leading to impaired cellular signaling, nucleic acid synthesis, and cognitive dysfunction. Our study provides novel multi-omics insights into the MAPK/ERK/CREB-mediated neurotoxicity and energy metabolism collapse induced by nitrous oxide as a recreational drug of abuse, offering new directions for clinical intervention.

Background: Nitrous oxide (N₂O) is increasingly used as a recreational drug, leading to neurological and systemic toxicities. However, due to the rapid elimination and minimal alteration of nitrogen oxides, the short direct detection window complicates the assessment of N₂O exposure. Method: In this study, we investigated the effects of chronic N₂O exposure on plasma metabolites using an untargeted metabolomics approach in a mouse model. C57BL/6 mice were exposed to 90,000 ppm N₂O (1 h, twice daily for 28 days) or room air. Plasma samples were analyzed via UHPLC -Triple TOF -MS. Orthogonal partial least squares discriminant analysis (OPLS-DA) and receiver operating characteristic (ROC) curves were used to identify differential metabolites. Result: A total of 35 differential metabolites were identified. Eight metabolites with an area under the curve (AUC) > 0.90 were selected as candidate biomarkers, including up-regulated SOPC and PC(16:0/16:0) (suggesting disrupted phospholipid remodeling and membrane integrity), and down-regulated DL-tryptophan, creatine, ectoine, indole, His-Ser, and Ile-Pro. Pathway enrichment analysis revealed significant alterations in glycine, serine and threonine metabolism; phenylalanine, tyrosine and tryptophan biosynthesis; protein digestion and absorption; and tryptophan metabolism. Conclusions: Our data indicate that chronic N₂O exposure disrupts multiple amino acid-related metabolic pathways (e.g., tryptophan-kynurenine pathway) and phospholipid homeostasis. The identified metabolite changes, along with vitamin B₁₂, homocysteine, and methylmalonic acid, may constitute a specific metabolic fingerprint for N₂O exposure. These findings help reveal the intrinsic mechanistic links underlying metabolic disorders induced by N₂O exposure.

Objectives: Amantadine (AMD) is an antiviral and antiparkinsonian drug with a narrow therapeutic window and a recognized risk of severe intoxication. Interpretation of postmortem drug concentrations is complicated by postmortem redistribution (PMR), yet systematic toxicokinetic and multi-tissue PMR data for amantadine remain limited. Methods: An integrated investigation of amantadine toxicokinetics and postmortem redistribution was conducted in male rats. For toxicokinetic assessment, a single oral dose of 450 mg/kg (LD50) was administered, and concentrations were quantified in blood and nine tissues over a 96-h period. For the PMR study, rats received low (42 mg/kg), medium (LD50), and high (2 × LD50) doses, followed by controlled postmortem storage at 4 °C and 20 °C for up to 96 h. Amantadine concentrations were determined using validated HPLC–MS/MS methods and analyzed by pharmacokinetic and statistical approaches. Results: Amantadine was rapidly absorbed and widely distributed, exhibiting pronounced tissue-specific heterogeneity. The liver and kidney showed the highest exposure, whereas accumulation in the brain and testis was limited. Postmortem redistribution was substantial and tissue dependent, and was strongly influenced by dose, postmortem interval, and storage temperature. Blood concentrations were unstable over time, while solid organs, particularly the liver and spleen, exhibited higher and more sustained postmortem concentrations. Notably, selected inter-tissue concentration ratios (e.g., liver-to-lower limb muscle and spleen-to-brain) displayed consistent, dose-dependent trends across postmortem conditions. Conclusions: This study provides a comprehensive characterization of amantadine toxicokinetics and postmortem redistribution across multiple biological matrices. The findings underscore the limitations of relying solely on postmortem blood concentrations and support the complementary use of selected tissues and inter-tissue concentration ratios as comparative indicators in toxicological interpretation. These results offer mechanistic insight into postmortem drug dynamics and provide practical reference data to improve the interpretation of suspected amantadine intoxication.

Fentanyl abuse has been associated with neurological and psychological harm. However, the metabolic changes within the peripheral circulation and central nervous system involved in fentanyl addiction have not been well explored. In the present study, metabolic changes in plasma, caudate putamen (CPu), hippocampus (Hip), and prefrontal cortex (PFC) were investigated in mouse models of drug addiction based on fentanyl-induced conditioned place preference (CPP). Metabolic profiles were measured using untargeted UHPLC-Q-Exactive HFX mass spectrometry. A total of 131, 196, 104, and 52 altered metabolites were identified in plasma, CPu, Hip, and PFC, respectively. The identified metabolites mainly included lipid mediators, carbohydrate metabolites, fatty acids, amino acids (AAs) and their derivatives, and nucleotide metabolites. The disturbed metabolic pathways were primarily involved in lipid metabolism, carbohydrate metabolism, AA metabolism, nucleotide metabolism, and the metabolism of cofactors and vitamins. These findings indicate disturbances in cell membrane metabolism, energy metabolism, AA metabolism, and neurotransmitter systems caused by fentanyl addiction. Our study provides a valuable resource for future investigations aimed at defining the role of metabolites in fentanyl addiction, which may help develop new pharmacotherapies.

We report the novel HLA‐A*02:07:26 allele.