OBJECTIVE  To explore the applicablity of ′BlueScreen HC′(BSHC), a high throughput genotoxicity screening system based on human growth arrest and DNA damage inducible 45α(GADD45α) gene, in detecting the genotoxicity of migrants mixtures from food contact materials (FCM). METHODS  The 2000 bp sequence upstream of the open reading frame of human GADD45α gene was used as the promoter to construct the lentiviral plasmid pEZX-LvPG04, which was double labeled by purinamycin and Gausluciferase (Gluc), and the lentiviral plasmid was infected with human lymphoblastocyte TK6 to obtain a stable transmutation cell line TK6-Gluc.  Methyl methylate (MMS) at concentrations of 0, 1.56, 3.13, 6.25, 12.5, 25.0 and 50.0 mg·L^-1 was selected as the genotoxin without liver S9, cyclophosphamide (CTX) 0, 0.78, 1.56, 3.13, 6.25, 12.5, 25.0 mg·L^-1 was selected as the pre-genotoxin with liver S9, and dimethyl sulfoxide (DMSO) 0, 0.35, 0.69, 1.38, 2.75, 5.5 and 11.0 g·L^-1 was selected as the non-genotoxin. The constructed BSHC was verified with the above known genetic positive and negative substance respectively. Polybutyleneadipate-co-terephthalate (MS/PBAT) was tested using 4% (V/V) acetic acid, and 10%, 20%, 50% and 95% (V/V) ethanol as food simulants at 40 ℃ for 24 hours to obtain 5 multi-component migrants of MS/PBAT that were obtained by using DMSO as a solvent. TK6-Gluc cells were treated with 5 multi-component migrants of MS/PBAT at concentrations of 0, 0.38, 0.76, 1.53, 3.05, 6.10 and 12.20 g·L^-1 with or without liver S9. Cells were treated without liver S9 for 48 h. Cells treated with liver S9-mix were incubated for 3 h at a final concentration of 1% (V/V) liver S9 before being washed and re-suspended in fresh recovery media for another 45 h. After exposure, the cell viability was detected using the CCK-8 cell activity kit, and the Gluc Luminescence in the medium was detected with Secrete-PairTM Gaussia Luciferase Assay Kit. In addition, the mutagenicity on Salmonella typhimurium TA98 and TA100 was detected by micro-fluctuation Ames test with 5 multi-component migrants of MS/PBAT at concentrations of 3.05 and 12.20 g·L^-1. The in vitro mammalian cell chromosome aberration test was performed on CHL cells with 5 multi-component migrants of MS/PBAT at concentrations of 3.05 and 12.20 g·L^-1 to detect the chromosomal aberration. The results of genotoxicity were compared with those of BSHC. RESULTS  The lowest effect centration (LEC; <80% relative cell viability) and the coytotoxicity (<30% relative cell viability) was defined. A positive genotoxicity result threshold was determined at 1.8-fold relative induction. For the liver S9 protocol, the same process was followed, and the decision threshold derived was 1.5-fold relative Gluc induction. It is considered as genetic substance only when a positive genotoxicity result was reached and there was no cytotoxicity. Compared with the vehicle control group, no genotoxicity was observed at all concentration of DMSO by BSHC. MMS 12.5, 25.0 and 50.0 mg·L^-1 produced genotoxicity without liver S9 while CTX 6.25, 12.5 and 25.0 mg·L^-1 produced genotoxicity with liver S9. Significant cell growth inhibition was observed in 95% ethanol migrants of MS/PBAT at concentrations of 6.10 and 12.20 g·L^-1, and in 50% ethanol migrants of MS/PBAT at a concentration of 12.20 g·L^-1 without liver S9. No cytotoxicity with a relative cell viability below 30% was observed in any of the treatment groups, and no high expression of Gluc was observed. Therefore, none of the 5 multi-component migrants produced genotoxicity without liver S9. Significant cell growth inhibition was observed in 95% ethanol migrants of MS/PBAT at a concentration of 12.20 g·L^-1, and in 4% acetic acid migrants of MS/PBAT at concentrations of 6.10 and 12.20 g·L^-1 with liver S9. No cytotoxicity with a relative cell viability below 30% was observed in any of the treatment groups. No high expression of Gluc was observed. Therefore, none of the 5 multi-component migrants produced genotoxicity with liver S9. In the micro fluctuation Ames test, when 5 multi-component migrants of MS/PBAT were treated with concentrations of 3.05 and 12.20 g·L^-1 on TA98 and TA100 strains, there was no significant difference in the number of mutagenic positive wells compared with DMSO control group with or without liver S9,  indicating that no mutagenic effect was produced.  When CHL cells were treated with 5 multi-component migrants of MS/PBAT at concentration of 3.05 and 12.20 g·L^-1, compared with DMSO control group, there was no significant difference in chromosome aberration rate of CHL cells with or without liver S9. CONCLUSION  BSHC based on GADD45α gene has been established, which can be used for in vitro genotoxicity evaluation of migrants mixtures of FCM, but further exploration of its minimum effective concentrations is still needed, and more types of mixtures need to be applied for further validation.

OBJECTIVE  To optimize hematopoietic stem cell transplantation therapy and provide support for drug research by investigating the dynamic process of hematopoietic and immune system reconstitution after bone marrow transplantation (BMT) in mice. METHODS  CD45.2+ C57BL/6 mice were used as recipient mice and randomly divided into the normal control group and transplantation group, with 30 mice in each. The transplantation group was irradiated by a lethal dose of cobalt-60 rays. Bone marrow cells were prepared from CD45.1⁺ C57BL/6 mice and transfused into recipient mice through the tail vein. Peripheral blood, spleens, lymph nodes, thymuses and bone marrow were collected at 1, 2, 4, 8 and 16 weeks after transplantation. Blood routine examination was performed with peripheral blood and total cell numbers in suspensions of other organs were counted by an automated cell counter. Cell classification analysis of white blood cells in peripheral blood, cell suspensions of other organs was performed by flow cytometry. RESULTS  Four weeks after BMT, the numbers of white blood cells and red blood cells in peripheral blood of recipient mice returned to the same level of or higher level than normal control (P<0.05). Although the number of platelets recovered significantly, it was still markedly lower than that of normal control until 16 weeks post BMT (P<0.05). In addition, the percentages of myeloid leukocytes and B cells in peripheral blood, spleens, lymph nodes, and bone marrow, as well as megakaryocytes and erythrocyte progenitor cells in bone marrow also returned to normal, and the majority of myeloid leukocytes and B cells were CD45.1⁺ cells from the donors. Eight weeks after BMT, T cells in peripheral blood, spleens, lymph nodes, thymuses, and bone marrow of recipient mice returned to normal, and CD45.1⁺ T cells were dominating. CONCLUSION  The hematopoietic and immune reconstitution of recipient mice is nearly completed eight weeks after BMT. However, the reconstruction speed of different kinds of cells and the reconstruction status of same kind of cell in different organs vary widely.

OBJECTIVE  To study the pharmacodynamics and pharmacokinetics of semaglutide (Sem) capsules in type 2 diabetic model rats. METHODS  Male SD rats were divided into the normal control group, type 2 diabetic model group and model+Sem capsules (0.839, 1.678 and 2.517 mg·kg^-1) groups. A type 2 diabetic rat model was induced by high sugar and high fat diet feeding combined with ip given streptozotocin (STZ) injection. Seven days after modeling, the model+Sem capsules group was  ig given Sem capsules  at the corresponding dose in a fasting state, once a day, for 14 d. Body mass, fasting blood glucose (FBG), and glycosylated hemoglobin (HbA1c) levels were regularly measured in each group of rats. Plasma from rats in the model+Sem capsules 0.839, 1.678 and 2.517 mg·kg^-1 groups at different time points was collected at the end of the continuous administration of Sem capsules, and the content of Sem in the plasma of rats was determined by liquid chromatography-tandem mass spectrometry. Concentration-time curves were plotted, and the main pharmacokinetic 
parameters were fitted by the WinNonlin non-atrial model method. RESULTS  Compared with the model group, the body mass of rats in model+Sem capsules dosing groups decreased significantly after 7 and 14 d of Sem capsules intervention (P<0.05, P<0.01), so did FBG (P<0.01) and the HbA1c level (P<0.01). Meanwhile, FBG and HbA1c levels of rats in model+Sem capsules 1.678 and 2.517 mg·kg-1 groups were not significantly different from those of the normal control group after 14 d of Sem capsules intervention, suggesting that FBG and HbA1c levels were basically restored to normal. Pharmacokinetic results showed that the elimination half-life (t_1/2) of Sem in plasma after ig administration of Sem capsules 0.839, 1.678, and 2.517 mg·kg^-1 for 14 d in rats was 7.40±1.34, 7.48±0.33 and (8.23±0.90) h, respectively, the peak concentration (C_max) was 18±9, 81±23 and (256±53) μg·L^-1, time to peak (T_max) was 0.06±0.13, 1.56±0.88, (1.50±1.00) h, respectively, the area under the curve (AUC_0-t) was 158±76 μg·h·L^-1, 858±310 and (3795±1539) μg·h·L^-1, and the accumulation index was 1.12±0.05, 1.12±0.01 and 1.15±0.04, respectively. CONCLUSION  Sem capsules ig administrated can effectively reduce body mass, FBG and HbA1c levels in type 2 diabetic model rats, and lead to glucose reduction and by mass loss. After 14 d of continuous administration of Sem capsules, there is no accumulation of semaglutide in rats in the dose range of 0.839-2.517 mg·kg^-1, and the exposure increases with the dose.

Ganoderma lucidum is one of the widely-used traditional Chinese medicines. Ganoderma lucidum extract and Ganoderma spore powder are also widely used in foods, health care and cosmetics, and are highly favored by consumers. Ganoderic acid A is one of the main effective components of Ganoderma lucidum triterpenoids. In recent years, a large number of studies have proved that ganoderic acid A has anti-tumor, anti-inflammatory, hepatoprotection,hypolipidemic, renoprotective properties, and has therapeutic potential in psychiatric and neurological disorders., which has high medicinal value and good prospects for development. Based on review of domestic and foreign literatures in recent years, the new research progress in pharmacology of ganoderic acid A is summarized in this paper in order to provide references for its further development and clinical applications.

Opioid analgesics play an important role in pain management. While opioid analgesics have been used to control various types of acute and chronic pain, they bring about the risks of non-medical use and addiction, which results in a worldwide opioid crisis. Opioid receptors are widely distributed in the central nervous system and mediate complex biological effects. The side effects of opioid analgesics, such as respiratory depression, dependence and addiction, have severely limited their clinical applications. The main strategies for developing new opioid analgesics with low addiction potential involve abuse-deterrent formulations, agonists targeting peripheral opioid receptors, G protein signaling-biased agonists of opioid receptors, agonists targeting opioid receptor heteromers and multi-functional ligands of opioid receptors. Most of them are undergoing clinical or pre-clinical research, and some new formulation medicines have been approved for marketing. Despite the analgesic or other therapeutic benefits, these strategies retain to some extent the potential risk of abuse and other 
adverse effects like to traditional opioid analgesics. How to fully separate the analgesic action from addiction potential remains challenging. Therefore, more efforts are needed to develop new opioid analgesics with low addiction potential.

Human beings are inevitably exposed to toxic substances as a result of influences of potential contamination factors in the environment, food and medicines, which poses a threat to human health. In order to effectively screen and prevent the exposure or intake of such substances, it is necessary to develop in vitro assays for the detection and toxicity evaluation of toxic substances. High content screening (HCS) has been recognized as an important tool for toxicity testing and risk assessment of compounds due to its high throughput and automation advantages, and has been widely used in in vitro toxicology research. In this review, we described the system components of HCS and its workflow in toxicity screening and toxicity evaluation by focusing on cases of their application in toxicity detection and evaluation studies, including the cytotoxicity, hepatotoxicity, nephrotoxicity, genotoxicity, neurotoxicity, cardiotoxicity, and developmental toxicity. In addition, the applications and developments of 
machine learning in HCS were explored, especially to the advantages of supervised and unsupervised machine learning strategies for high throughput image screening and data analysis. Finally, the future applications of HCS in toxicity screening and evaluation are outlined, especially in terms of binding new models and gene editing technology.

The Hippo/YAP signaling pathway is an evolutionarily conserved protein kinase cascade that plays an important role in a variety of biological processes, such as cell proliferation and differentiation, organ growth and tissue regeneration. Fibrosis is a continuous and highly dynamic process characterized by excessive deposition of extracellular matrix, resulting in irreversible pathological changes that eventually lead to the failure of multiple tissues and organs. Targeted therapeutic strategies to ameliorate or reverse fibrosis are lacking. Studies have shown that the aberrantly activated Hippo/YAP signaling pathway may play a role in the development of fibrosis by regulating collagen deposition, fibroblast overproliferation, and epithelial cell differentiation, but the specific mechanism of action has not been fully elucidated. Targeting the Hippo/YAP signaling pathway involves two mechanisms: one is to target the upstream molecules of the Hippo/YAP signaling pathway, which is mainly achieved by inhibiting the activity of the core kinase or blocking the interaction with other molecules; the other is to target the downstream activities of YAP/TAZ and YAP/TAZ-TEAD in the Hippo/YAP signaling pathway. Studies have shown that the phosphorylation and subcellular localization of YAP/TAZ are significantly altered when tissue and organ damage occurs. This article is intended to review the current research on Hippo/YAP signaling pathway and its mediation of fibrosis in the lung, heart, liver, kidney, pancreas and skin in hopes of providing new ideas for studies on the pathogenesis of fibrosis and targeted therapeutic drugs.

Metabolism-related diseases are chronic diseases caused by genetic and environmental factors. The symptoms include insulin resistance, abnormal blood glucose and lipid levels, and elevated blood pressure. This type of illness has become a major threat to human health, and there is an urgent need to find effective treatments. Advanced glycation end products (AGE) are a group of complex and heterogeneous compounds that result from reduced interactions between the carbonyl groups of  sugar and the free amino groups of proteins, lipids, and nucleic acids. Increasing evidence shows that AGE and its receptor (RAGE) are involved in the occurrence and development of such metabolism-related diseases as hypertension, diabetes, and atherosclerosis. AGE can have adverse effects on tissues through non-receptor and receptor-mediated mechanisms. In the receptor-mediated mechanism, AGE interacts with RAGE to increase the production of oxygen free radicals and activate NF-κB so that more pro-inflammatory cells are expressed and released, leading to cell damage. This article reviews the research progress in interventions with AGE and RAGE in the treatment of hypertension, diabetes, and atherosclerosis from a metabolic perspective in the hope of exploring the potential of AGE and RAGE as therapeutic targets for metabolism-related diseases.

Mitophagy is a process by which cells selectively eliminate damaged or dysfunctional mitochondria through the autophagy-lysosomal pathway under the regulation of mitophagy-related proteins to maintain mitochondrial quality and cellular homeostasis, and it is closely related to the occurrence and development of a variety of tumors. Increasing studies have shown that mitophagy plays a dual role in breast cancer progression. ① Mitophagy promotes breast cancer cell survival and invasion by triggering energy metabolic reprogramming, reducing ROS accumulation and maintaining mitochondrial homeostasis. ② Mitophagy leads to death or apoptosis of breast cancer cells by attenuating inflammatory responses and triggering mitochondrial damage. The relevant mechanisms include ① PTEN induced kinase 1，PINK1 (PINK1) /Parkin pathway; ② mitophagy receptor-associated protein pathway (including BNIP3, BNIP3L and FUNDC1); ③ mitochondrial division pathway. This review summarizes the role and mechanism of mitophagy in the hopes of providing reference for research and treatment of breast cancer.

Research into human drug metabolism, liver disease modeling as well as drug screening heavily depend on cell cultures for in vitro validation and experimental animal models for in vivo analysis. However, these methods are not without limitations. Cell stability is often compromised in vitro, and the species-specific differences between experimental animals and humans restrict the accuracy of these models in replicating in vivo drug metabolism and the pathophysiological features of human liver diseases. Humanized liver chimeric mouse models, developed through the transplantation and expansion of human liver cells into the livers of live mice, replicate the specific functionalities of the human liver. This replication is instrumental in fulfilling a variety of research objectives, including the replication of hepatitis viruses within mice, the modeling of liver metabolic disorders, the screening of liver cell-targeted therapeutic agents, and the evaluation of drug-induced hepatotoxicity. As such, these models are regarded as optimal for the preclinical validation of drug efficacy and safety assessment. This paper presents a comprehensive review and a vision of three representative humanized liver chimeric mouse models in terms of classification, construction principles, limitations, and current applications, thereby offering reference for the selection of appropriate models in research.

OBJECTIVE  To investigate the damage effect and mechanisms of cyclophosphamide (CTX) and its active metabolite derivative 4-hydroperoxycyclophosphamide (4-HC) to human neuroblastoma SH-SY5Y cells. METHODS  SH-SY5Y cells were treated with CTX [0 (cell control), 0.01, 0.1, 1, 5, 10, 20, 40 and 80 mmol·L^-1] and 4-HC [0 (cell control), 0.01, 0.1, 1, 5, 10, 20, 40 and 80 μmol·L^-1 ] for 48 h. Cell  confluence and morphology were observed by the IncuCyte ZOOM system. Cell viability was assessed by CCK-8 assay. Lactate dehydrogenase (LDH) release was measured by  LDH assay kit. SH-SY5Y cells were treated with CTX (0, 1, 5, 10 and 20 mmol·L^-1) and 4-HC (0, 1, 5, 10 and 20 μmol·L^-1) for 48 h before cell proliferation was analyzed by 5-ethynyl-2′-deoxyuridine (EdU) staining assay. Immunofluorescence was employed to assess the levels of the DNA double-strand break marker γ-H2AX and to evaluate changes in mitochondrial membrane potential. SH-SY5Y cells were treated with CTX (0, 1, 5 and 10 mmol·L^-1) and 4-HC (0, 1, 5 and 10 μmol·L^-1) for 48 h, and the alterations in glycolysis and oxidative phosphorylation levels were analyzed using the Seahorse XFe96 Analyzer. RESULTS  Compared with the cell control group, cell confluence and cell viability were significantly reduced in the CTX and 4-HC groups (P<0.01), and the half-maximal inhibitory concentrations (IC₅₀) for CTX and 4-HC were 4.44 mmol·L^-1 and 4.78 μmol·L^-1, respectively. The release rate of LDH was significantly increased while the percentage of EdU+ cells was significantly reduced in the CTX and 4-HC groups (P<0.01). The percentage of γ-H2AX+ cells was significantly increased and mitochondrial membrane potential significantly decreased in the CTX and 4-HC group (P<0.05). Treatment with CTX and 4-HC resulted in reduced levels of maximum glycolytic capacity, glycolytic reserve, maximal respiration, and ATP production (P<0.05). CONCLUSION  CTX and 4-HC exert significant cytotoxic effects on SH-SY5Y cells by disrupting cell membrane structure, impeding cell proliferation, and reducing cell viability. The mechanisms underlying these effects may involve intracellular DNA damage, disturbance of energy metabolism and mitochondrial dysfunction.

With the emergence of high-throughput technology and massive toxicology data, toxicology research has entered the era of big data. How to efficiently integrate existingtoxicological data, clarify the toxic effects of chemicals, and use these patterns to providenew information, in order to achieve efficient prediction of the toxicity of new chemicalsubstances, is one of the cutting-edge issues in toxicology. In view of the high cost, low throughput and difficulty in revealing the mechanism information of traditional chemical toxicity testing methods, high throughput prediction models are urgently needed. Machine learning methods have been applied to toxicity testing, such as supervised learning models, unsupervised learning models, deep learning models, reinforcement learning models, and transfer learning models. Chemical characteristic data commonly used in machine learning models include chemical structure data, text data, toxicological genome data and image data. There is huge potential for applying machine learning to toxicity testing and machine learning methods have made some progress. However, current research focuses on the processing of data and development of  models, which has failed to produce a widely used and accepted method. In addition, the prediction accuracy of machine learning models is not only dependent on algorithms, but also affected by data quality, and the mutual promotion and development of algorithms and data quality remains a big challenge. In short, data 
processing and model construction in the field of toxicology require interdisciplinary cooperation and technological innovation. With the increasing perfection of toxicology databases and the continuous optimization of various model algorithms, the toxicity prediction of new chemicals based on machine learning models will become increasingly efficient and accurate, playing an important role in ensuring human health and environmental safety.

The relationship between glycolysis and viral infectious diseases is close, and the interaction between viral and host glycolysis is a common mechanism found in a wide range of viruses. Therefore, the regulation of glycolysis may be an important antiviral strategy. The coronavirus diseases 2019 pandemic has brought about a tremendous disaster to humanity, compelling us to seek effective solutions from various perspectives. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can induce an increase in host glycolysis, the level of which plays an important role in virus replication and infection, associated with the progression of the disease and a variety of clinical symptoms and complications. The study on the interaction between host glycolysis and SARS-CoV-2 infection can shed light on the pathogenic mechanism of SARS-CoV-2 and promote the research on related drugs. This article reviews the interactions between SARS-CoV-2 infection and host glycolysis in the hopes of providing a new perspective to understand the relationships between viral infections and disease in terms of metabolic regulation and formulate countermeasures.

Integrated Approaches to Testing and Assessment (IATA) is a toxicological assessment decision-making method that integrates existing information from various sources, including physical and chemical properties, animal testing, and non-animal testing. Through the evaluation and analysis of a series of iterative strategies, it ultimately obtains risk assessment conclusions, thereby providing a basis for risk management decisions regarding chemical substances. The use of IATA is becoming increasingly prevalent in such areas as ocular irritation and genotoxicity. This paper introduces the conceptual connotation of IATA, sorts out the framework elements and sequential processes, explains the commonly used framework construction methods, shares cases of application in various exposure scenarios, and finally envisions future research directions in order to provide better methodological support for the risk assessment of chemical substances.

OBJECTIVE  To set up normal ranges for indexes in embryo-fetal development toxicity studies in Sprague-Dawley (SD) rats and to establish a background database to provide reference for the embryo-fetal development toxicity evaluation of drugs. METHODS  The data on embryonic development and fetal growth from embryo-fetal development toxicity studies (11 items) conducted by our center between 2013 and 2022 was statistically analyzed, involving 205 pregnant rats and 3037 fetuses in total, with the mean and standard deviation, coefficient of variation and 95% confidence interval calculated. The indexes included body mass, body mass gain and food consumption during pregnancy, pregnancy outcomes (pregnancy rate, average corpora lutea, average Implant sites, average live conceptuses, live conceptuse rate, resorption rate and dead conceptuse rate), fetal growth and development (fetal mass, placental mass and sex ratio), appearance abnormality rate, visceral abnormality rate, and skeletal abnormality rate. RESULTS The mass of pregnant rats trended up during gestation, with significant increases in the late period. Food consumption increased along with gestation. Caesarean section was conducted on gestation day 20, and the pregnancy rate was 93.2%. The average corpora lutea, Implant sites and live conceptuses were 18.0±3.2, 15.9±2.8 and 14.8±3.0, respectively. The live conceptuse rate was 93.4% while the total dead embryo rate was 6.6%. The average mass of fetuses and placenta were respectively 3.6±0.3 and (0.6±0.3) g, and the fetal sex ratio (male/female) was 0.94. The incidence of fetal appearance abnormalities was about 0.2%, and that of soft tissue abnormalities was approximately 0.8%. The rate of skeletal abnormalities was about 1.2%, with higher incidence of non-ossification and incomplete ossification mostly identified on sternum and hyoid bone. The numbers of ossifications of metacarpal bones, metatarsal bones and sacrococcygeal vertebrae were 7.0±0.7, 8.0±0.1 and 7.4±0.5, respectively. The rate of ossification of sternumⅠ to Ⅳ was higher, with an average of about 98.6%-99.9%. The ossification rates of sternum Ⅴ and Ⅵ were (68.0±28.4)% and (82.8±23.9)%. CONCLUSION The background database of indexes in the embryo-fetal development toxicity study on SD rats is established for our GLP laboratory, which provides reference for reproductive toxicity studies.

Kidney organoids are induced and differentiated from human pluripotent stem cells (PSCs) or adult stem cells (ASCs) derived from tissue sources, primarily composed of nephron structures. However, due to the absence of a supporting vascular network, kidney organoids often exhibit immature tissue structures and limited growth. Therefore, vascularization in kidney organoids remains a pressing challenge in this field. Currently, the methods such as transplantation into immunodeficient animals, alterations in induced differentiation protocols, utilization of microfluidic chips, and manipulation of extracellular matrix and oxygen concentrations may facilitate vascularization of kidney organoids, which  provides a new perspective for the scientific study and clinical application of kidney organoids.

With the rapid development of industry and economy, the emergence of a large number of chemicals has made of risk management more difficult. Traditional risk assessment relies on animal experiments for toxicity testing. However, animal experiments are time-consuming, costly, and unable to meet the practical needs of risk assessment. The increasing maturity of toxicity testing alternative technologies signifies the possibility of rapid, sensitive, and accurate identification of chemical toxicity. This article focuses on the research and applications of alternative toxicity testing by reviewing the background, developments, and current research at home and abroad. It also discusses the progress in alternative testing methods in such areas as cosmetics and food safety risk assessment and explores the problems with the development of alternative testing technologies and risk assessment in China. This review aims to provide a reference for the system construction of cosmetics health risk assessment in China.

Animal models are powerful tools for studying the mechanism of depressive disorders and screening antidepressants, but so far there is no model which can stimulate the clinical status of patients ideally. Here, we briefly introduced the research advances in classic animal models of depressive disorders, and focused on stress-related animal models, especially those induced by physical and social psychological stressors. The tests for evaluating animal depression behavior were reviewed. In this article, the strengths and weaknesses of each model were analyzed, and the precautions in its application were recommended. Finally, given the high heterogeneity of depressive disorders, this article elaborated on the research progress in models for subtypes of depressive disorders, such as treatment resistant depression, bipolar disorder, peripartum depression, and premenstrual syndrome.

OBJECTIVE  To investigate the preventive effect and mechanism of simvastatin on cerebral ischemia-reperfusion injury (CIRI) in hyperlipidemic mice. METHODS  Sixty C57BL/6J mice were randomly divided into Sham group, CIRI group (CIRI model was prepared by middle cerebral artery occlusion and reperfusion (MCAO/R)), hyperlipemia group (i.p poloxamer 407), hyperlipemia+CIRI group (i.p poloxamer 407, followed by MCAO/R operation after 24 h), and hyperlipemia+CIRI+
simvastatin 5 and 10 mg·kg^-1 groups (i.g simvastatin for 7 d, and then treated as the hyperlipemia+CIRI group). After reperfusion for 24 h, the neurological deficit score (NDS) was evaluated; the Rotarod experiment was used to determine the first drop latency; autonomous activity was used to test the horizontal and vertical movement frequency of mice; TTC staining was used to measure the volume of cerebral infarction; laser speckle flow imaging was used to detect cerebral blood flow in mice; the kits were used to detect total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), malondialdehyde (MDA) levels, and glutathione peroxidase (GSH-PX) levels in serum; qPCR and Western blotting were used to detect the mRNA and protein expression levels of clock genes Rev-erbα and Bmal1 in the right cerebral cortex of mice, respectively. RESULTS  Compared with the Sham group, the NDS of the CIRI group was significantly increased (P<0.01), the latency period was shortened (P<0.01), and the number of activities was reduced (P<0.01); Compared with the CIRI group, the hyperlipemia+CIRI group showed aggravated neurological damage (P<0.01); the volume of cerebral infarction was significantly increased (P<0.01); the cerebral blood flow was significantly decreased (P<0.01); the levels of TC, TG, LDL-C, and MDA in serum were significantly increased (P<0.01), while the level of GSH-PX was significantly decreased (P<0.01); the mRNA and protein expression levels of Rev-erbα in the cerebral cortex were significantly downregulated (P<0.01); the mRNA and protein expression levels of Bmal1 were significantly upregulated (P<0.01). Compared with the hyperlipemia+CIRI group, the hyperlipemia+CIRI+simvastatin 5 and 10 mg·kg^-1 groups showed reduced neurological damage (P<0.01); the volume of cerebral infarction was significantly decreased (P<0.01); the cerebral blood flow was significantly increased (P<0.01); the levels of TC, TG, LDL-C, and MDA in serum were significantly reduced (P<0.01), while the level of GSH-PX was significantly increased (P<0.01); the mRNA and protein expression levels of Rev-erbα in the cerebral cortex were significantly upregulated (P<0.01);  the mRNA and protein expression levels Bmal1 were significantly downregulated (P<0.01). CONCLUSION  Prophylactic administration of simvastatin can effectively alleviate hyperlipidemia combined with cerebral ischemic injury in mice, and the mechanism is related to the regulation of blood lipid and clock gene expression.

Food toxicology plays a crucial role in supporting scientific and technical aspects of food safety risk assessment. However, traditional methods relying on animal testing are becoming increasingly inadequate for identifying and evaluating emerging foods and unknown risks. There is a strong push worldwide towards the development of new approach methodologies (NAMs) based on non-animal testing methods. Policies and regulations related to NAMs are being standardized gradually in the European Union, the United States, and China. Some progress has also been made in applying these methodologies in food toxicology research in China. For instance, within the "Food Toxicology Program" at the National Center for Food Safety Risk Assessment, high-content and high-throughput in vitro hazard identification models employing model organisms like human macrophages, hepatocytes, adipocytes, embryonic stem cells, and zebrafish, as well as Toxicological Thresholds of Concern and quantitative in vitro to in vivo extrapolation based on physiologically-based toxicokinetic models have been established and applied. Nonetheless, new toxicological hazard identification technologies still face challenges such as inadequate elucidation of toxic mechanisms, insufficient collaborative research efforts, and inefficient translation of these findings into practical applications.

Antibody-based therapies are one of the crucial tumor-targeted therapies, enabling precise elimination of tumor cells by specifically binding to antigens on the tumor cell surface. However, their wide applications in solid tumor therapy are often limited by on-target toxicity. Recent advancements in antibody engineering have led to the development of novel tumor-targeted masking antibodies, which are specifically designed to address these limitations. Masking antibodies typically consist of an antibody domain, a masking domain and a linker. These antibodies are characterized by selective activation and other functional properties. Currently, various masking antibody technologies with distinct characteristics have been developed and have demonstrated favorable safety profiles in animal studies. This review summarizes the structure and characteristics of tumor-targeted masking antibodies outlines common masking technologies and their drug development in order to offer new lines of thought  for the design and development of next-generation tumor-targeted therapeutics.

OBJECTIVE To explore the ameliorative effect of ursolic acid on carbon tetrachlorideinduced acute liver injury in mice, and the feasibility of multispectral optoacoustic tomography (MSOT) for characteristic structural and functional imaging of liver tissues. METHODS Kunming mice were randomly divided into the normal control group, model group, model+ursolic acid 30, 60 mg·kg^-1 groups and model+bifendate 5.625 mg·kg^-1 group, with 14 mice in each. Each group was given the corresponding drug once daily for 7 days. An acute liver injury model was established in mice by intraperitoneal injection of 0.2% carbon tetrachloride in olive oil solution after the last administration. Blood was collected, liver tissues were taken 24 h after modeling, and the liver index was calculated, 8 mice from each group and the levels of serum glutamic pyruciv transaminase (GPT) and glutamic oxaloacetic transaminase (GOT), as well as superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in liver tissues were measured. The enzyme-linked immunosorbent assay (ELISA) method was used to detect the level of α - glutathione S-transferase (α -GST) in serum. The histopathological changes of the liver were observed under a light microscope. The remaining 6 mice in each group underwent MSOT technique was used for characteristic structural and functional imaging of liver tissue. Levels of oxygenated hemoglobin (HbO₂) and deoxygenated hemoglobin (Hb) were analyzed, oxygen saturation was calculated, and the extent of liver injury was assessed by examining the intrahepatic distribution of indocyanine green (ICG). RESULTS Compared with the normal group, the levels of GPT, GOT and α-GST in serum, content of MDA in liver tissues and the liver index in the model control group were significantly increased while the activity of SOD in liver tissues were significantly decreased. Compared with the model group, ursolic acid in each dose group significantly reduced the liver index of mice, lowered the serum levels of GPT and GOT as well as the level of α- GST, decreased the content of MDA in liver tissues, and elevated the activity of SOD in liver - injured mice. Hematoxylin-eosin staining showed that significant steatosis and hepatocyte necrosis and inflammatory cell infiltration in hepatocytes of mice in the model group. Ursolic acid significantly attenuated the degree of hepatocellular lesions and markedly reduced steatosis in mice. MSOT imaging showed that the HbO2 level and oxygen saturation were significantly lower while the Hb level was remarkably higher in the liver of mice in the model group. In each administration group, the level of HbO₂ significantly increased, the level of Hb was significantly decreased, oxygen saturation was significantly increased in the liver of model mice and the accumulation of ICG dye probe was atten⁃ uated in the body after hepatocyte injury. CONCLUSION Ursolic acid can elevate the hepatic oxygen saturation, improve the metabolism of ICG, reduce the degree of hepatic necrosis in mice, and help protect against carbon tetrachloride-induced hepatic injury in mice. The mechanism is probably related to the inhibition of oxidative stress.

OBJECTIVE  To explore the role and mechanism of dental pulp stem cells (DPSCs) in repairing hypoxic injury to rats pulmonary microvascular endothelial cells (PMVECs). METHODS  ① PMVECs were treated with cobalt chloride at 0, 10, 25, 50 and 100 μmol·L^-1 for 72 h. CCK-8 was used to detect the cell viability, and the protein levels of hypoxia-inducible factor 1α (HIF-1α), zona occludens small-band protein 1 (ZO-1), and occludin (OCLN) were detected by Western blotting. ② There was a cell control group, model group, and model+DPSCs group, and the levels of reactive oxygen species (ROS) was detected by immunofluorescence staining after at 24 and 48 h of action. The levels of ZO-1 and OCLN proteins were detected by Western blotting. ③ A cell control group, model group, model+DPSC group and model+DPSC cell knockdown superoxide dismutase 1 (SOD1) group were set up. The mRNA level of SOD1 was detected by real-time fluorescence quantitative PCR 24 and 48 h later, while the protein levels of ZO-1 and OCLN were detected by Western blotting. RESULTS  ① Compared with the cell control group, 72 h of cobalt chloride 100 μmol·L^-1 treatment of PMVECs resulted in a cell survival rate above 80%, a significant increase in the level of HIF-1α protein (P<0.05), a significant decrease in the levels of ZO-1 and OCLN proteins (P<0.01), and establishment of a  model of hypoxic injury in PMVECs. ② Compared with the cell control group, the ROS level was significantly higher in the model group (P<0.01). Compared with the model group, the ROS level was significantly lower in the model+DPSCs group (P<0.01), while the levels of ZO-1 and OCLN proteins were significantly higher in the model+DPSCs group (P<0.05). ③ Compared with the DPSC group, ZO-1 and OCLN expressions were significantly decreased after knockdown of SOD1 in DPSCs (P<0.05, P<0.01). CONCLUSIONS DPSCs can repair hypoxic injury to PMVECs, and the anti-oxidative stress capacity of DPSCs plays an important role in hypoxic injury repair of PMVECs.

Special populations such as the elderly, pregnant women, children, and patients with impaired liver and kidney function have different physiological characteristics and drug processes from other patients. As a result during medication, these populations are vulnerable to drug-drug interactions in case of combined medications, which makes it more difficult of optimize drug treatment and develop new drugs. The physiologically based pharmacokinetic (PBPK) model can predict drug-drug interaction in special populations by altering metabolism-related physiological parameters such as metabolic 
enzymes, transporter activity and clearance. Gene polymorphisms, intestinal metabolism and other 
factors also affect the accuracy of model results. This review aims to to optimize clinical regimens for special populations and provide references for new drug development.

As antibiotic resistance becomes increasingly concerning, antimicrobial peptides, as a new type of antibiotic alternative, have attracted more attention. However, the low enzymatic stability of antimicrobial peptides severely limits their clinical applications. To address this issue, researchers have developed various structural modification strategies, including the introduction of unnatural amino acids, peptide chain cyclization and chemical group modification. This article reviews the basic principles and cases of the above modification strategies analyzes the advantages and disadvantages of different modification strategies and recommends ways these strategies can be optimized. In addition, this article predicts the developments of and potential challenges to strategies for enhancing enzymatic stability of antimicrobial peptides in the hope of providing references for subsequent research and development of antimicrobial peptides.

OBJECTIVE  To investigate the effect of N-methyl-D-aspartic acid (NMDA) receptor in the secondary visual cortex (V2) on methamphetamine-associated contextual learning and memory. METHODS  With male C57BL/6J mice as subjects and using the mouse conditioned place preference (CPP) experiment, the scores of CPP were observed after microinjection of NMDA receptor selective antagonist D-AP5 (0.5 μg per side) into the bilateral V2 during the formation phase, single microinjection of D-AP5 (0.5 μg per side) into the bilateral V2 prior to the expression test, and methamphetamine (0.5 mg∙kg^-1, ip)-induced reactivation test and methamphetamine-associated contextual-induced reactivation test, to evaluate the effect of NMDA receptors on the formation, expression and reinstatement of methamphetamine-induced CPP. RESULTS  Compared with the control group, microinjection of D-AP5 (0.5 μg per side) into the bilateral V2 during the formation phase did not have significant inhibitory effect on CPP scores, nor did single microinjection of D-AP5 (0.5 μg per side) into the bilateral V2 prior to the expression test, single microinjection of D-AP5 (0.5 μg per side) into the bilateral V2 prior to methamphetamine (0.5 mg∙kg-1, ip)-induced reactivation test or single microinjection of D-AP5 (0.5 μg per side) into the bilateral V2 prior to methamphetamine-associated contextual-induced reactivation test. CONCLUSION  The NMDA receptor in the V2 is not involved in the formation, expression and reactivation of methamphetamine-associated contextual learning and memory.

The quality of toxicological data is critical to toxicity assessment and risk prediction. Integrating multiple streams of evidence and enhancing the quality of toxicological data are indispensable for more efficient and precise health risk assessment. There are several commonly used toxicology data quality evaluation systems abroad, which include Klimisch rating system, ToxRTool evaluation tool, the SciRAP evaluation tool and the integrated risk information system tool. The TRAM reliability evaluation tool is developed in China for toxicology data in food safety risk assessment.  These evaluation tools have different backgrounds and applicability of evaluation standards. Each evaluation system also has its advantages and disadvantages.

OBJECTIVE  To investigate the mechanism by which salvianolic acid A (Sal A) reduces the inflammatory response and oxidative stress of BV2 cells injured by oxygen and glucose deprivation/reperfusion (OGD/R). METHODS  An OGD/R injury model of BV2 cells was established with sugar free Earle solution containing Na₂S₂O₄ 10 mmol·L^-1. Na₂S₂O₄ sugar free Earle solution was added and cultured in an incubator (37 ℃, 5%CO₂) for 1.5 h (oxygen glucose deprivation) before a normal medium was used for 24 h (reperfusion). Then, the cells were divided into the cell control group, OGD/R group, OGD/R+Sal A 1, 5 and 10 μmol·L^-1 group, OGD/R+ML385 group, OGD/R+ML385+Sal A 1, 5 and 10 μmol·L^-1 group and OGD/R+edaravone (Eda, 50 μmol·L^-1) group. After twenty-four hours of culture, the cell survival rate was measured by CCK8 kit. The contents of Interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), IL-10, IL-4 and transforming growth factor-β (TGF-β) in the cell supernatant were detected by ELISA. Reactive oxygen species (ROS) in cells was detected using the chemical fluorescence method. The contents of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and chloramphenicol acetyltransferase (CAT) in cells were determined with the colorimetric method. Protein expressions of Kelch like ECH-associated protein 1 (Keap1), nuclear factor erythroid-2 related factor 2 (Nrf2),Heme oxygenase-1 (HO-1), NADPH: quinone oxidoreductase-1 (NQO1) and p-nuclear factor kappa-B p65 (p-NF-κB p65) were detected by Western blotting. RESULTS  ① Compared with the cell control group, the cell survival rate of the OGD/R group was significantly decreased (P<0.01). Compared with the OGD/R group, the survival rates of OGD/R+Sal A 1, 5 and 10 μmol·L-1 groups were significantly increased (P<0.05, P<0.01). ② Compared with the cell control group, the contents of IL-1β, IL-6 and TNF-α were significantly increased, the contents of IL-10, IL-4 and TGF-β were significantly decreased, the contents of ROS and MDA were significantly increased, and the activities of SOD, CAT and GSH-Px were significantly decreased in the OGD/R group (P<0.01). Compared with the OGD/R group, the content of IL-6 was significantly decreased, the contents of IL-10, IL-4 and TGF-β were significantly increased, the contents of ROS and MDA were significantly decreased, and the activities of SOD, CAT and GSH-Px were significantly increased in OGD/R+Sal A 1, 5 and 10 μmol·L-1 and OGD/R+Eda groups (P<0.05, P<0.01). ③ Compared with the cell control group, the protein expression of p-NF-κB P65 in the OGD/R group was significantly increased (P<0.01). Compared with the OGD/R group, the protein expressions of Keap1 and cytoplasmic Nrf2 were significantly decreased, the expressions of nuclear Nrf2, HO-1 and NQO1 proteins were significantly increased, and the expression of p-NF-κB p65 protein was significantly decreased in OGD/R+Sal A 5 and 10 μmol·L^-1 and OGD/R+Eda groups (P<0.05, P<0.01). In OGD/R+ML385, OGD/R+ML385+Sal A 1, 5 and 10 μmol·L^-1 groups, the protein expression of Keap1 was significantly increased, the protein expressions of cytoplasmic Nrf2, nuclear Nrf2, HO-1 and NQO1 protein were significantly decreased, and the protein expression of p-NF-κB P65 was significantly increased (P<0.01). CONCLUSION  Sal A reduces the inflammatory response and oxidative stress of OGD/R injured BV2 cells possibly by activating the Keap1/Nrf2 pathway and inhibiting the NF-κB pathway.

OBJECTIVE  To investigate whether aldo-keto reductases (AKRs) can act as a nitroreductase (NR) and bioactivate aristolochic acid Ⅰ (AA-Ⅰ) to produce AA-Ⅰ-DNA adducts. METHODS  ① Human-induced hepatocytes (hiHeps) and human bladder RT4 cells were used as tool cells and treated with AA-Ⅰ 0, 0.5, 1.0 and 2 μmol·L^-1 for 24 h. Cell viability was detected using the CCK-8 method, and the half maximal inhibition concentration (IC₅₀) was calculated using the CCK-8 method and the level of DNA adduct production was calculated. ② hiHeps and RT4 cells were treated with AKR inhibitor luteotin (0, 5, 10 and 25 μmol·L^-1)+AA-Ⅰ 0.2 and 1.0 μmol·L^-1 for 24 h, respectively, and the levels of DNA adducts were detected by a liquid chromatography-tandem mass spectrometer (LC-MS/MS). ③ hiHeps cells were incubated with 80 nmol·L^-1 small interfering RNAs (si-AKRs) for 48 h and treated with AA-Ⅰ1.0 μmol·L^-1 for 24 h. Real-time qualitative PCR (RT-qPCR) method was used to detect the mRNA expression of AKRs gene and LC-MS/MS technology was used to investigate the effect of specific AKR gene knockdown on DNA adduct levels. ④ 500 nmol·L^-1 human AKR recombinant proteins AKR1A1 and AA-Ⅰ were incubated in vitro under anaerobic conditions and the formation of AA-Ⅰ-DNA adducts was detected. RESULTS  ① The IC₅₀ of AA-Ⅰ to hiHeps and RT4 cells was 1.9 and 0.42 μmol·L^-1, respectively. The level of DNA adduct production of the two cell lines was significantly different (P<0.01). ② Luteolin≥5 μmol·L^-1 significantly inhibited the production of AA-Ⅰ-DNA adducts in both cells (P<0.05), and there was a concentration-dependent effect in hiHeps cells (P<0.01, R=0.84). ③ In the AKR family, the knockdown of AKR1A1 gene up to 80% inhibited the generation of AA-Ⅰ-DNA adducts by 30%-40%. ④ The AA-Ⅰ-DNA adducts were detected in the incubation of recombinant protein AKR1A1 and AA-Ⅰ under anaerobic conditions in vitro, approximately 1 adduct per 107 nucleotides. CONCLUSION  AKR1A1 is involved in AA-Ⅰ bioactivation, providing a reference for elucidation of the carcinogenic mechanism of AA-Ⅰ.

OBJECTIVE  The inhibitory effect of active ingredients of Tripterygium wilfordii Hook. F. (TWHF) (celastrol, triptolide, triptonide, wilforlide A, wilforgine and wilforine) on human carboxylesterase 1 (CES1) and CES2 was detected to investigate the herb-drug interactions (HDIs) of TWHF. METHODS  Human liver microsomes catalysed hydrolysis of 2-(2-benzoyl-3-methoxyphenyl) benzothiazole (BMBT) and fluorescein diacetate (FD) were used as the probe reaction to phenotype the activity of CES1 and CES2, respectively. The residual activities of CES1 and CES2 were detected by ultra-high performance liquid chromatography (UPLC) after intervention with celastrol, triptolide, triptonide, wilforlide A, wilforgine and wilforine (100 μmol·L^-1). Kinetics analysis, involving half inhibitory concentration (IC₅₀), inhibition type and kinetic parameter (K_i), and in vitro-in vivo extrapolation (IVIVE), was carried out to predict the HDIs between these compounds and CES-metabolizing drugs. Molecular docking was performed to analyze the ligand-enzyme interaction. RESULTS  Out of the six main constituents of TWHF, only celastrol exhibited strong inhibition towards both CES1 and CES2, with the inhibitory rates of 97.45% (P<0.05) and 95.62% (P<0.05) , respectively. The IC₅₀ was 9.95 and 4.02 mol·L^-1, respectively, and the types of inhibition were all non-competitive inhibition. Based on the kinetics analysis, the K_i  values were calculated to be 5.10 and 10.55 μmol·L^-1 for the inhibition of celastrol on CES1 and CES2, respectively. IVIVE indicated that celastrol might disturb the metabolic hydrolysis of clinical drugs in vivo by inhibiting CES1. Molecular docking results showed that hydrogen bonds and hydrophobic contacts contributed to the interaction of celastrol and CESs. CONCLUSION  The inhibitory effect of celastrol on CES1 and CES2 might cause HDIs with clinical drugs hydrolysed by CESs.

Macrophage-capping protein (CapG) is a member of the gelsolin superfamily. It is a 
universal multifunctional actin binding protein in the body and highly expressed in breast cancer, bladder cancer, prostate cancer and other types of cancer, which can promote the metastasis and invasion of cancer cells. This article reviews the structure, function, related signal pathways and roles of CapG in tumor invasiveness.

Over the past 30 years, nano-drug delivery systems (NDDS) have become a promising field of drug research. However, a poor knowledge of the in vivo process of NDDS, the limited methods of pharmacokinetic correlation, and the inability to effectively provide strong support for the construction of upstream drug as well as the evaluation of downstream pharmacology and toxicology have become the technical bottleneck for their clinical transformation. Lipid nanodrug (LND) is the most successful NDDS for industrial transformation with great biocompatibility. Taking LND as an example, this paper reviewed the delivery process and influencing factors in vivo, and summarized the regulatory mechanism of biological environments on drug release in vivo. Based on advanced spectroscopy and mass spectrometry techniques, the spatial and temporal distribution of the dynamic carrier particle/depolymerized molecule ratio and dynamic free/encapsulated drug ratio of LND in biological matrix were analyzed. Finally, the existing problems and future developments in this field were summarized to provide references for the analysis of NDDS in vivo, and stimulate readers' interest in nanomedical research and development. 

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.

Peptide nanomedicines have been rapidly developed in the field of tumor treatment and the discovery of new drugs due to their strong stability, precise targeting and high bioavailability. The administration methods of peptide nanomedicines include intravenous injection, oral administration, respiratory administration and transdermal administration, among which intravenous administration is the most commonly used. Once inside the body, peptide nanomedicines can be transported everywhere via the blood circulation, accumulating in large quantities at the target site. Effective drug tracking analysis methods can help to obtain accurate pharmacokinetic data, while a comprehensive understanding of the in vivo distribution and pharmacokinetic characteristics of peptide nanomedicines can contribute to the development of new drugs and promote the progress of cancer therapy. This paper reviews the advantages and disadvantages of analytical methods for peptide nanomedicines and the research progress in their metabolism and transport in order to provide references for subsequent research on peptide nanomedicines.

OBJECTIVE  To investigate the transcriptomal charactersistics of the striatum in the chronic social defeat stress (CSDS) model mice by using spatial transcriptome analysis and to address the underlying mechanism of the striatum in regulating depressive states. METHODS  The CSDS paradigm was employed to establish a depression-like mouse model. The depressive indicators of behavioral despair, anhedonia, and social disorders were assessed through a battery of tests, including the tail suspension test, forced swim test, sucrose preference test, and social interaction experiments. The control mice and the mice exhibiting CSDS-sensitive depression-like behaviors were selected for spatial transcriptome sequencing of the striatal region. This sequencing aimed to identify highly expressed genes, followed by KEGG and GO enrichment analyses using the DAVID database. RESULTS  The CSDS mouse model effectively induced behavioral despair, anhedonia and social avoidance (P<0.05, P<0.01). Spatial transcriptome analysis revealed 193 differentially expressed genes in the striatum of normal mice. KEGG and GO analyses indicated that these genes were primarily associated with striatal development, locomotor behaviors, and drug addiction. They were strongly implicated in signaling pathways such as cyclic adenosine monophosphate, cyclic guanosine monophosphate-protein kinase G, calcium signaling, Ras-related protein 1, and mitogen-activated protein kinase, and synaptic linked to GABAergic and dopaminergic neurons. In contrast, CSDS modeling mice led to the identification of 298 differentially expressed genes in the striatum compared with the normal control mice. These genes were significantly enriched in pathways related to neurodegenerative diseases, including Huntington disease, Alzheimer disease, and Parkinson disease. CONCLUSION  Depressive states induced by CSDS are associated with the pathological processes underlying neurodegenerative diseases in the striatum.

Sleep disorders are becoming a social problem that poses a danger to human health and an obstacle to economic development. As an endogenous hormone, melatonin plays an important role in regulating circadian rhythm. Research has shown that exogenous melatonin holds promising prospects in the treatment of insomnia disorders and thus merits in-depth exploration and development. Given the close relationship between pharmacokinetic properties and drug efficacy as well as toxicity, this paper reviews the quantitative analysis methods for melatonin and its major metabolites, especially the  pretreatment methods for biological samples and the related LC-MS/MS analysis methods. Studies related to the absorption, distribution, metabolism, excretion and drug-drug interactions of melatonin both in vivo and in vitro, as well as the level and species differences of endogenous melatonin are also outlined. Meanwhile, the druggability of melatonin and corresponding solutions are explored in the hope of providing data for the development of exogenous melatonin into novel sleep-regulating drugs.

OBJECTIVE  To investigate the distribution and regulation of G-quadruplex (G4) in enzymes related to glycolysis. METHODS  The sequences of the transcription start site (TSS) region upstream of 1500 bp to the 5′-Untranslated region in 200 enzymes and their subtypes in glycolysis were selected for bioinformatics analysis. Related enzymes in glycolysis containing putative G-quadruplex-forming sequences (PQSs) were identified. Circular Dichroism and Native polyacrylamide gel electrophoresis were used to verify the formation of G4. The ExonucleaseⅠhydrolysis assay was used to validate the stability of the formed G4 under 0, 0.5, 2, 8, 16, and 32 min. A reporter gene plasmid was constructed by inserting specific fragments of the related enzymes before the luciferase expression sequence. The dual-luciferase reporter assay system validate the expression level of luciferase to assess the impact of G4 on promoter activity. Real-time quantitative PCR was performed to validate the transcriptional regulatory role of G4 by detecting the mRNA levels of luciferase. RESULTS  ① Bioinformatics analysis showed that out of the 200 glycolysis-related enzymes, 12 contained PQSs. Based on the analysis of the length and structure of PQSs, aldolase A (ALDOA) and phosphoglycerate mutase 2 (PGAM2) proved to be able to form stable G4. ② ALDOA and PGAM2 had the maximum positive absorption peak at 260 nm and maximum negative absorption peak at 240 nm. Both of them could form a G4 at the same time. ③ After digestion with ExonucleaseⅠ, ALDOA and PGAM2 showed no significant hydrolysis and demonstrated the stability of G4 structures. However, both of them could be gradually hydrolyzed after mutations in their PQSs. ④ After PQS mutation of ALDOA and PGAM2, the mRNA levels and expression of downstream luciferase of ALDOA were significantly increased (P<0.05, P<0.01), while PGAM2 was significantly decreased (P<0.05, P<0.01). CONCLUSION The gene sequences of glycolysis-related enzymes and their subtypes contain a large number of PQSs. ALDOA and PGAM2 can form stable G4 and perform transcriptional regulatory functions.

OBJECTIVE  To study the way in which hypidone hydrochloride (code: YL-0919) improves motor function after ischemic stroke (IS) and explore the related mechanism. METHODS  Adult male SD rats were used to establish a middle cerebral artery occlusion (MCAO) model that simulated acute IS. All animals were randomly divided into four groups: sham group, MCAO group, MCAO+YL-0919 group, and MCAO+YL-0919+erastin (Era, ferroptosis inducer) group. The drug administration groups received the first ip injection 6 h after operation, followed by continuous ip injection once per day. After 7-10 d of drug administration, the effect of YL-0919 on motor function after IS were evaluated via neurological function test, adhesive-removal test, rotarod test, balance beam test and open field test. After 7 d of drug administration, TTC staining was used to detect the cerebral infarction area while the colorimetry method was used to measure the contents of glutathione (GSH), malondialdehyde (MDA), and ferrous ions (Fe²⁺) in the penumbra of the cerebral cortex. Western blotting was used to detect the expression levels of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (xCT), acyl-CoA synthetase long-chain family member 4 (ACSL4), and transferrin receptor 1 (TFR1) in the cortical penumbra. RESULTS  Compared with the sham group, the MCAO group showed higher neurological function scores (P<0.01), with notably prolonged time for tape removal and first contact with the right forepaw (P<0.01), spent significantly more time crossing the balance beam (P<0.01) but endured a notably shorter duration on the rotarod (P<0.01), reduced the movement distance in the open field (P<0.01), had a remarkably increased infarct area (P<0.01) but significantly level of GSH in the cortical penumbra region decreased (P<0.01), while MDA and Fe²⁺ levels were markedly increased (P<0.01). Protein expression levels of GPX4 and xCT were reduced (P<0.05), while those of ACSL4 and TFR1 were elevated (P<0.05). Compared with the MCAO group, these changes were significantly reversed after YL-0919 administration. However, when Era and YL-0919 were administered simultaneously, the reversal effect of YL-0919 was significantly weakened. CONCLUSION  YL-0919 can improve motor function impairment and reduce cerebral infarction areas in rats after IS, and the mechanism may be related to the inhibition of ferroptosis.