OBJECTIVE  To investigate the role of Bai Ling Long Zao An Shen formula (BLLZ) in sleep improvement in an environmental stress-induced insomnia rat model and explore its underlying mechanisms. METHODS  (1) Component analysis: the chemical constituents of the BLLZ extract were analyzed using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). (2) Evaluation of the sedative and hypnotic effect: ① Mice: 50 ICR mice were randomly divided into normal control group, BLLZ-L group (5, 10 and 20 g·kg^-1) and diazepam group (DZP, 3 mg·kg^-1). After five days of intragastric administration, pentobarbital sodium-induced righting reflex and locomotor activity tests were performed. ② Rats: 8 SD rats were implanted with electrodes and allowed to recover for seven days before baseline EEG data was collected over 24 h. A crossover design (7 d washout period) was employed,  with rats randomly assigned to the DZP (3 mg·kg^-1) and BLLZ (20 g·kg^-1) group. After five days of treatment, 24 h EEG recordings were obtained. (3) Insomnia model and interventions: ① 8 SD rats were allowed to recover for seven days post-surgery, followed by 6 h (14:00-20:00) baseline EEG recording. A 3×3 crossover design was used to assign rats to model (environmental stress-induced insomnia), model+DZP, or model+BLLZ groups. After five days of treatment, insomnia was induced by frequent cage changes (14:00, 16:00 and 18:00), and EEG changes were monitored. (4) Mechanistic study: 32 SD rats were randomly divided into the normal control group, model group, and model+DZP group. After five days of treatment, hypothalamic tissues were collected for biochemical analysis. γ-aminobutyric acid (GABA), glutamate (Glu), and dopamine (DA) levels were measured 
using biochemical kits while γ aminobutyric acid receptor subunit alpha-1 (GABAA1), core clock proteins period circadian regulator 2 (PER2) and circadian locomotor output cycles (CLOCK) protein expressions were assessed by Western blotting. RESULTS  (1) Compared with the normal control group, the sleep latency of BLLZ 10 and 20 g·kg^-1 and DZP groups was significantly shortened, and the locomotor activity of BLLZ 20 g·kg^-1 and DZP groups was significantly reduced; BLLZ 20 g·kg^-1 significantly increased the total sleep time, slow-wave sleep time, and average duration of sleep in normal rats, and significantly reduced the wakefulness time. (2) The total sleep time and slow-wave sleep time of the model group significantly decreased and the wakefulness time significantly increased compared with baseline. (3) Compared with the model group, the total sleep time and slow-wave sleep time of the model+BLLZ group and the model+DZP group were significantly increased, and the wakefulness time significantly shortened. (4) Compared with the normal control group, the Glu/GABA ratio, DA content and CLOCK protein expression were significantly increased and GABAA1 and PER2 protein expression were significantly decreased in the model group; compared with the model group, the Glu/GABA ratio, DA content and CLOCK protein expression were significantly decreased, and the expression of GABAA1 and PER2 were significantly increased in the model+BLLLZ group and the model+DZP group. CONCLUSION  BLLZ has sedative and hypnotic effects. It can prolong the total slow-wave sleep time by increasing the average duration of slow-wave sleep episodes, thereby increasing the total sleep time and improving environmental stress-induced insomnia. The mechanism may be related to the downregulation of the Glu/GABA ratio and DA levels as well as the enhancement of GABAA1 
expressions and the regulation of hypothalamic core clock protein expressions.

OBJECTIVE  To investigate the regulatory effects of salvianolic acid C (SAC) on the level of cuproptosis and inflammatory injury in cardiomyocytes after myocardial infarction (MI). METHODS  ① C57BL/6 mice were divided into a sham group, an MI model group, and SAC (5, 10 and 20 mg·kg^-1) groups, with 10 mice in each group. Mice in the SAC groups were pretreated with oral gavage of SAC for 1 week, while those in the sham and model groups received an equal volume of saline. One week later, an MI model was established in the model and SAC groups by ligating the left anterior descending coronary artery, while the sham group underwent thoracotomy without ligation. MI size was assessed using triphenyltetrazolium chloride (TTC) staining. Cardiomyocyte apoptosis was evaluated by TUNEL staining. The ultrastructure of cardiomyocyte mitochondria was observed under a transmission electron microscope. ② Mouse cardiomyocytes HL-1 were divided into a control group, an oxygen-glucose deprivation (OGD) model group, OGD+SAC 1, 5 and 10 μmol·L^-1  groups, and a OGD+SAC (5 μmol·L^-1)+nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor ML385 (2 μmol·L^-1) group. Cells in the OGD+SAC groups were pretreated with SAC for 24 h while those in the OGD+SAC+ML385 group were pretreated with both SAC 5 μmol·L^-1 and ML385 2 μmol·L^-1 for 24 h. Except for the control group, an OGD model was established in HL-1 cells. ELISA was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in mouse serum and HL-1 cell culture supernatants. The Cu⁺ detection kit was used to measure Cu⁺ levels in myocardial tissue and HL-1 cells. Cell viability was assessed using the CCK-8 kit. Apoptosis rates of HL-1 cells were detected by flow cytometry. Reactive oxygen species (ROS) levels in HL-1 cells were measured using a ROS detection kit. Western blotting analysis was performed to detect the expression levels of Nrf2, heme oxygenase-1 (HO-1), and cuproptosis markers, ferredoxin 1 (FDX1) and solute carrier family 31 member 1 (SLC31A1) in myocardial tissue and HL-1 cells. RESLUTS  ① Compared with the sham group, the MI model group exhibited increased myocardial infarction size, elevated cardiomyocyte apoptosis rates, mitochondrial swelling, vacuolation, and cristae rupture in cardiomyocytes, increased serum levels of TNF-α, IL-6, and IL-1β, elevated Cu+ levels and expressions of FDX1 and SLC31A1 in myocardial tissue, and decreased expressions of Nrf2 and HO-1 (P<0.01). Compared with the model group, the SAC 5, 10 and 20 mg·kg^-1 groups showed 
reduced MI size, decreased cardiomyocyte apoptosis rates, alleviated mitochondrial swelling, vacuolation, and cristae rupture, lower serum levels of TNF-α, IL-6 and IL-1β, decreased Cu⁺ levels and expressions of FDX1 and SLC31A1 in myocardial tissue, and increased expressions of Nrf2 and HO-1 (P<0.05, P<0.01). ② Compared with the cell control group, the OGD model group demonstrated significantly decreased HL-1 cell viability, increased cell apoptosis rates, Cu⁺ and ROS levels, expressions of FDX1 and SLC31A1, elevated levels of TNF-α, IL-6 and IL-1β in cell culture supernatants, and 
decreased expressions of Nrf2 and HO-1 (P<0.01). Compared with the OGD model group, the SAC 1, 5 and 10 μmol·L^-1 groups showed increased HL-1 cell viability, decreased cell apoptosis rates, Cu⁺ and ROS levels, expressions of FDX1 and SLC31A1, reduced levels of TNF-α, IL-6 and IL-1β in cell culture supernatants, and increased expressions of Nrf2 and HO-1 (P<0.05, P<0.01). Compared with the SAC 5 μmol·L^-1 group, the SAC 5 μmol·L^-1+ML385 2 μmol·L^-1 group exhibited decreased cell viability, increased cell apoptosis rates, Cu⁺ and ROS levels, expressions of FDX1 and SLC31A1, elevated 
levels of TNF-α, IL-6, and IL-1β in cell culture supernatants, and decreased expressions of Nrf2 and HO-1 (P<0.01). CONSLUSION  SAC can activate the Nrf2/HO-1 signaling pathway, alleviate cuproptosis in cardiomyocytes after MI, and reduces inflammatory damage.

OBJECTIVE  To explore the effect and underlying mechanism of leucine (Leu) on metabolic dysfunction-associated fatty liver disease induced by high fat diet (HFD) in mice. METHODS  C57BL/6J male mice were randomly divided into chow diet (normal), chow diet+Leu (normal+Leu), HFD and HFD+Leu groups, with 10 mice in each group. The mice in the normal and normal+Leu groups received chow diet while those in the HFD and HFD+Leu groups received HFD. Drinking water for mice in the normal+Leu and HFD+Leu groups was supplemented with 1.5% Leu. The experiment lasted 24 weeks, total food and water intake of mice were recorded weekly to calculate energy and Leu intake respectively. Energy metabolism of mice was detected at week 20 by the Oxymas/CLAMS Animal Metabolic System heat production, CO₂ exhalation, O₂ consumption and respiratory exchange rate (RER). At the end of week 24, the mice were sacrificed and the livers were harvested, followed by the oil red O staining to reveal the fat content. Western blotting was performed to analyze the changes in the activity of the liver branched-chain α-keto acid dehydrogenase E1α  (BCKDE1A), the activation of AMP-activated protein kinase alpha subunit (AMPKα), and the protein expressions of downstream effector molecules including silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), fatty acid synthetase (FAS) and fatty acid binding protein 4 (FABP4) in the liver of mice. RESULTS  Total Leu intake of mice was significantly reduced in the HFD+Leu group, compared with the normal+Leu group. The mice fed with HFD significantly increased the energy intake body mass gain and liver mass, accompanied by fat accumulation in the liver, compared to the mice in the normal group. Simultaneously, the mice in the HFD group showed a decrease in  CO₂ exhalation both by day and by night, and in the respiratory exchange ratio by day compared to the normal group. Compared with the HFD group, the body mass gain and liver mass obviously decreased in mice of the HFD+Leu group, and the liver fat accumulation was reduced. The mice in the HFD+Leu group exhibited higher heat production and O₂ consumption, along with an increase in CO₂ exhalation by day and by night. In addition, heat production, CO2 exhalation, and O₂ consumption were significantly higher by night than by day (P<0.01). As for the respiratory exchange ratio, a night increase was seen in the mice from the normal group, normal+ Leu group, and HFD group, but not in the HFD+Leu group. The results of Western blotting showed that compared with the normal group, the BCKDE1A phosphorylation inactivation was enhanced, AMPKα phosphorylation activation alleviated, the protein expressions of SIRT1 and PGC-1α downregulated (P<0.05), and the protein expressions of FAS and FABP4 increased in the livers of mice in the HFD group. Compared with the HFD group, the BCKDE1A phosphorylation inactivation was alleviated, AMPKα phosphorylation activation enhanced, the protein expressions of SIRT1 and PGC-1α increased, and the protein expressions of FAS and FABP4 downregulated in the livers of mice in the HFD+Leu group. CONCLUSION  Leu can alleviate HFD-induced metabolic dysfunction-associated fatty liver disease in mice, which may be closely related to the promotion of energy metabolism and inhibition of fat synthesis.

OBJECTIVE  To quantify pulmonary vascular endothelial subpopulations during bleomycin-induced pulmonary fibrosis in mice. METHODS  Sixty male C57BL/6 mice were randomly divided into five groups (n=12 per group) corresponding to distinct observation timepoints: 0, 1, 2, 3, and 4 weeks. A model was established via intratracheal instillation of bleomycin (3 mg·kg^-1). Lung tissues were harvested at 0, 1, 2, 3 and 4 weeks post-bleomycin induction. Pathological staining was performed to assess lung histoarchitecture and collagen fiber deposition. Single-cell suspensions were analyzed by flow cytometry to quantify temporal changes in pulmonary vascular endothelial subpopulations, including pulmonary macrovascular endothelial cells, general capillaries, and aerocyte capillaries. Immunofluorescence staining was performed to validate the expressions of endothelial markers (CD31, APLN, APLNR, CD93). Single-cell transcriptomic data from the Tabula Muris database was analyzed to evaluate gene expression profiles of vascular endothelial subpopulations. RESULTS  Pathological staining revealed progressive destruction of lung histoarchitecture and collagen deposition during bleomycin-
induced pulmonary fibrosis. Flow cytometry demonstrated three-phase dynamics in vascular endothelial cells (CD45⁻CD31⁺CD90.2⁻): a significant decrease during the acute inflammatory phase, stabilization in the fibrotic phase, and partial recovery during the resolution phase. The proportion of von Willebrand factor-positive (VWF⁺) vascular endothelial cells significantly decreased during the resolution phase, whereas VWF⁻ vascular endothelial cells increased. Single-cell transcriptomics identified Cd93 as a specific gene for general capillary endothelial cells, with a negative correlation with "aerocyte" genes enriched in gas-exchange alveolar capillary endothelial cells. Immunofluorescence confirmed CD93 
localization to general capillary endothelial cells. A flow sorting strategy based on CD45⁻CD31⁺CD90.2⁻VWF⁻CD93⁻ effectively enriched alveolar capillary endothelial cells. This subpopulation trended upward in pulmonary vascular endothelial composition during bleomycin induction. CONCLUSION  During bleomycin-induced pulmonary fibrosis in mice, pulmonary vascular endothelial subpopulations exhibit dynamic compositional heterogeneity across fibrotic injury and repair phases.

Large inter-individual and intra-individual differences in the expression and activity of drug-metabolizing enzymes (DMEs) contribute to unpredictable drug response and toxicity, which is a challenge facing precision medicine. Nuclear receptor-mediated transcriptional regulation and epigenetic mechanisms including histone modifications, non-coding RNAs, and DNA methylation can help to explain the individual variability in DME expressions. However, several questions remain unanswered. Recently, epitranscriptomics, an emerging field, provides new insights into the regulation of gene expression. As the most abundant RNA modification in eukaryotes, N ⁶-methyladenosine (m⁶A) modification plays key roles in various physiological and pathological processes. This review summarizes the recent progress in m6A modification-mediated individual variability in DME expression in terms of the role of m⁶A modification in regulating basal expression of DMEs, crosstalk between m⁶A modification and nuclear receptors during the ontogeny of DMEs, and the contribution of m⁶A modification to xenobiotic exposure-mediated changes in DME expression. This review aims to provide data for the elucidation of individual variations in drug metabolism in clinic.

Assessment of eye irritation potential is part of the international regulatory requirements for safety testing of chemicals. Because of increasing concerns about to animal welfare in recent years, the development of alternative methods to reduce the number of animals used in eye irritation tests has become imperative. This article introduces in vitro test methods based on organotypic models, cell lines and reconstructed human cornea-like epithelium (RhCE), and analyzes strengths and weaknesses of the individual in vitro test methods. To overcome the limitations of the individual in vitro test methods, it is recommended that tiered testing strategies be adopted that combine the strengths of individual in vitro test methods to address the required ranges of irritation potential. Future applications of new technologies such as genomics, proteomics and computational models in the assessment of eye irritation potential are also predicted.

Microplastics (particles<5 μm in diameter), generated through plastic processing or natural degradation, have emerged as novel environmental contaminants with increasing threats to ecosystems and human health. Recent studies have demonstrated the accumulation of microplastics in various environmental matrices, human tissues and even placentas, highlighting the urgent need to investigate their health effects. These particles enter the human system primarily through dietary intake and water consumption, yet critical knowledge gaps remain regarding their bioaccumulation potential, toxicological profiles, and synergistic interactions with co-existing environmental contaminants. The Drosophila melanogaster (fruit fly), a well-established model organism, offers distinct advantages for contaminant toxicity assessment, including a compact life cycle, small body size, rapid reproduction, ease of laboratory maintenance, and extensive availability of transgenic strains. In particular, its genome has a high degree of homology with the human genome, which further enhances its scientific relevance for toxicological studies. In this review, we summarize the advantages of Drosophila models in microplastic toxicity studies, with particular emphasis on recent advances in understanding intestinal toxicity, neuro toxicity, genotoxicity, reproductive toxicity, developmental toxicity, and transgenerational toxicity. We point to critical research gaps that require urgent investigation, including the bioaccumulation dynamics of microplastics, metabolic transformation and elimination pathways, multi-pollutant interaction effects, molecular mechanisms of toxicity, and comprehensive health risk assessment frameworks. The review aims to provide data for elucidating the mechanisms of microplastic toxicity and formulating evidence-based prevention strategies.

The spread of antibiotic resistance genes (ARGs) poses a serious threat to global public health. Recent studies have shown that in addition to the difficulty in antibiotic selection, many non-antibiotic environmental pollutants, such as microplastics, heavy metals, nanomaterials and non-antibiotic drugs, can accelerate the spread of ARGs. This paper begins by outlining the influence of non-antibiotic environmental pollutants on the conjugative transfer of ARGs. Then, the mechanisms of ARGs are analyzed from the perspective of molecular biology, which include inducing the generation of large amounts of reactive oxygen radicals, triggering cellular oxidative stress, increasing intercellular contact, altering the permeability of cell membranes, affecting energy metabolism, triggering group sensing effects, and regulating the expression of genes related to the transfer of ARGs. Finally, the limitations of current studies are presented, and tips are given about future research. In conclusion, the effects of non-antibiotic environmental pollutants on the spread and dispersal of ARGs need to be studied more extensively.