Alzheimer disease (AD) is a degenerative disease of the central nervous system, which is characterized by progressive cognitive decline and behavioral impairment, and has no effective prevention and treatment because of its complex pathogenesis. In recent years, targeted therapies based on the pathogenesis and pathology of AD have come to be the focus of new drug development. The targeted drugs amyloid monoclonal antibody aducanumab and lecanemab were approved by the US FDA for the treatment of AD, and GV-971 was approved in China. Innovative drugs such as 
β-secretase inhibitors, anti-Aβ vaccines, tau protein aggregation inhibitors, and γ-secretase inhibitors have also shown therapeutic potential for AD and entered clinical trials. This paper is intended to summerize the recent research progress in innovative drugs for the treatment of AD and analyze the strategies of innovative drug development in order to provide reference and ideas for the treatment of AD and the development of related new drugs.

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.

OBJECTIVE  To investigate the inhibitory effect and mechanism of 5，6-dimethylxanthenone-4-acetic acid (DMXAA) on metastasis of Lewis lung cancer (LLC) in mice. METHODS  The inhibitory effect of DMXAA on tumor metastasis was analyzed via an LLC xenograft tumor model and LLC metastatic tumor model. The mice of LLC xenograft tumor model were randomly divided into three groups: model group (physiological saline containing 1% DMSO, ip, once every two days), model+sunitinib group (30 mg·kg-1, ip, once every two days), and model+DMXAA group (25 mg·kg-1, ip, once). Tumor volume and body mass were measured once every two days after administration. Two and five days after administration, tumor mass was measured by sacrificing the mice, followed by immunofluorescence staining of tumor tissues. Platelet/endothelial cell adhesion molecule-1 (CD31) and α-smooth muscle actin (α-SMA) were used to analyze the vascular structure of tumor tissues. The tumor hypoxia level was detected using the hypoxia probe pimonidazole staining. The mice of LLC metastatic tumor model were randomly divided into three groups: model group (physiological saline containing 1% DMSO, ip, twice a week), model+sunitinib group (60 mg·kg-1,ip, twice a week), and model+DMXAA group (25 mg·kg-1, ip, once). At the Two and five weeks after administration, the in vivo tumor growth and metastasis were observed and quantified using a small animal live imaging system. RESULTS  Compared with the model group, the tumor volume and mass of the model+sunitinib group and model+DMXAA group were significantly reduced (P<0.05, P<0.01), and DMXAA took effect faster and more significantly than sunitinib. At the same time, compared with the model group, the body mass in the model+sunitinib group decreased significantly (P<0.05), but there was no significant difference in body mass the model+DMXAA group. Compared with the model group, model+sunitinib had no effect on tumor metastasis, but model+DMXAA significantly reduced tumor metastasis two weeks after administration (P<0.01). Compared with the model group, the coverage rate of α-SMA/CD31 in the model+sunitinib group and model+DMXAA group increased significantly (P<0.05). Compared with the model group, there was no significant change in the tumor hypoxia area in the model+sunitinib group, but this in the model+DMXAA group decreased significantly (P<0.01). CONCLUSION  DMXAA significantly inhibits the growth and metastasis of LLC in mice, and its mechanism may be related to its improvement of tumor vascular normalization and hyposic microenvironments.

Cationic liposomes, as non-viral vectors, are widely used in gene therapy and gene silencing. Although numerous cationic liposomes have various structures, they can all improve the performance of gene delivery. As gene therapy is increasingly studied, it may be foreseen that new cationic lipoplexes will be explored. In this review, we aim to discuss four constituent domains of cationic lipids (headgroup, hydrophobic domain, linker and helper lipids) in gene delivery. This article attempts to demonstrate that various lipid structures show different transfection efficiency and cytotoxicity by summarizing the similarities and differences between the four parts of cationic lipids. Furthermore, their major influencing factors are covered. Finally, three clinical cases of ionizable lipids are described to reveal their characteristics and differences from cationic lipids. This paper is intended to provide a conceptual framework for the design of cationic liposomes and for the selection of cationic lipids.

Fibroblasts are stromal cells widely distributed in tissues and organs throughout the body. Fibroblasts are involved in the synthesis and remodelling of the extracellular matrix. Fibroblasts participate in physiopathological processes such as tissue damage repair, inflammatory responses and immune regulation. Large numbers of activated fibroblasts have been found in fibrosis, autoimmune diseases and tumor lesions. Activated fibroblasts participate in tissue fibrosis and influence the tumor microenvironment mainly by secreting collagen and fibronectin. They also secrete a variety of inflammatory and growth factors that play an immunomodulatory role in autoimmunity and tumors. In recent years, it has been found that modulating fibroblast activation can effectively delay the development of these diseases, and that targeting fibroblast activation biomarkers can assess the development and treatment of these diseases. Therefore, the use of fibroblast-targeted drugs and therapeutic tools is expected to achieve new breakthroughs in the clinical management of fibrosis,  autoimmune diseases and tumors.

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.

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.

RNA methylation is a common epigenetic post-transcriptional modification with various patterns, such as N1-methyladenosine (m1A), 5-methylcytidine (m5C) and N6-methyladenosine (m6A). RNA methylation can perform specific biological functions in corresponding targets, dynamical and reversible, thus bridging exogenous environmental factors and different disease outcomes. Exogenous chemicals can induce oxidative stress, inflammation, autophagy and cell cycle disorders, which are regulated by specific RNA methylation modifications and bring about epigenetic toxic effects. These alterations act as new key molecular events during cellular senescence and the development of aging and age-related disorders. The correlation between RNA methylation and cell senescence will provide a new line of thought for prevention of and interventions in aging.

OBJECTIVE  To evaluate the mechanisms underlying the antidepressant effect of ZBH2012001, a novel serotonin and norepinephrine reuptake inhibitor (SNRI), in general and its ability to enhance monoaminergic transmission and suppress neuroinflammation in particular. METHODS  ① Male ICR mice were divided into vehicle (distilled water), duloxetine (DLX, 10 or 20 mg·kg^-1) and ZBH2012001 (5, 10 and 20 mg·kg^-1) groups. One hour following ig administration, the antidepressant effect of ZBH2012001 was evaluated using the tail suspension test (TST) and forced swimming test (FST). ② Radioligand binding assay was conducted to evaluate the affinity of ZBH2012001 for human serotonin transporters (hSERTs) and human norepinephrine transporters (hNETs). ③ Mice were divided into vehicle (distilled water), DLX (10 or 20 mg·kg^-1) and ZBH2012001 (5, 10 and 20 mg·kg^-1) groups. One hour following drug administration, the 5-hydroxytryptophan (5-HTP)-induced head-twitch test or yohimbine-induced lethality test were performed to evaluate the effect of ZBH2012001 on the function of the 5-hydroxytryptamine (5-HT) and norepinephrine (NE) systems. ④ Mice were divided into vehicle (distilled water+0.1% acetic acid), reserpine model (distilled water+reserpine 5 mg·kg^-1), DLX (DLX 20 mg·kg-1+reserpine 5 mg·kg^-1) and ZBH2012001 (ZBH2012001 5, 10 and 20 mg·kg^-1+reserpine 5 mg·kg^-1) groups. One hour following drug administration, reserpine was injected intraperitoneally to establish a monoamine-depletion model. The ptosis, akinesia, and hypothermia assays were performed to evaluate the effect of ZBH2012001 on the down-regulation of the reserpine-induced monoamine system. The TST in mice was used to evaluate the effect of ZBH2012001 on reserpine-induced depressive-like behavior while high-performance liquid chromatography with electrochemical detection (HPLC-ECD) was used to measure the levels of monoamines and their metabolites in the hippocampal tissue of reserpine-induced monoamine-depletion mice. ELISA was employed to detect the contents of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in the hippocampal tissue of reserpine-induced monoamine-depletion mice. Western blotting was used to assess the expressions of ionized calcium-binding adapter molecule-1 (Iba-1) and nuclear factor-kappa B (NF-κB) in the hippocampal tissue of 
reserpine-induced monoamine-depletion mice. RESULTS  ① Compared with the vehicle group, ZBH2012001 (5, 10 and 20 mg·kg^-1) significantly reduced the immobility time both in the TST in mice (P<0.01, respectively), and ZBH2012001 (20 mg·kg^-1) and in the FST in mice (P<0.05). ② ZBH2012001 competitively inhibited the binding of [³H]-imipramine to hSERTs and [³H]-nisoxetine to hNETs, with the half maximal inhibitory concentration (IC₅₀) values of 84.95 and 712.90 nmol·L^-1, respectively. ③ Compared with the vehicle group, ZBH2012001 (10 and 20 mg·kg^-1) significantly increased the head twitches induced by 5-HTP in mice (P<0.01, respectively) and increased the mortality rate in mice induced by yohimbine (P<0.05, P<0.01). ④ In the reserpine-induced monoamine-depletion model in mice, compared with the vehicle group, mice in the reserpine model group exhibited ptosis, akinesia and hypothermia feature (P<0.01, respectively), significantly prolonged immobility time in the TST (P<0.01), significantly decreased the levels of NE, 5-HT and dopamine (DA) (P<0.05, P<0.01), significantly increased the metabolic conversion rate of 5-HT and DA (P<0.01, respectively), significantly elevated levels of TNF-α and IL-6 (P<0.05, respectively), and significantly increased expressions of Iba-1 and NF-κB (P<0.05, 
respectively) in the hippocampus. Compared with the model group, ZBH2012001 (5, 10 and 20 mg·kg^-1) significantly antagonized ptosis and hypothermia behaviors induced by reserpine (P<0.01, respectively), ZBH2012001 (10 and 20 mg·kg^-1) significantly shortened the immobility time in reserpine-treated mice (P<0.05, P<0.01), ZBH2012001 (20 mg·kg^-1) significantly increased the levels of NE and 5-HT in the hippocampus of reserpine-treated mice (P<0.05, respectively), decreased the metabolic conversion rate of 5-HT (P<0.05), significantly reduced the contents of TNF-α and IL-6 in the hippocampus of reserpine-treated mice (P<0.05, respectively), ZBH2012001 (5, 10 and 20 mg·kg^-1) significantly 
reduced the expression of Iba-1 protein in the hippocampus of reserpine-treated mice (P<0.01, respectively), and ZBH2012001 (20 mg·kg^-1) significantly reduced the expression of NF-κB protein in the hippocampus of reserpine-treated mice (P<0.05). CONCLUSION  ZBH2012001 exerts its antidepressant effect through a dual mechanism involving monoamine enhancement and inflammation suppression.

With the gradual entry of gene therapy products into the market, there is an urgent need to establish viral vector-mediated genotoxicity evaluation methods. The two most widely used viral vectors are lentiviral vectors and adeno-associated viral vectors. Lentiviral vectors can stably integrate into the host genome while the viral vector genome mainly exists in the form of episomes in the nucleus of the host cell, and can still be randomly integrated into the host cell genome at a low frequency, the integration modes for which include random integration and targeted integration. This paper reviews the existing methods for evaluating the genotoxicity mediated by viral vectors, including whole-genome analysis of off-target modifications, integration site analysis, karyotype analysis, and tests to assess the potential for cell transformation. This article also offers a vision of the establishment of an accurate and rapid evaluation system for viral vector-mediated genotoxicity in the hopes of providing reference for improvement and development of new methods for evaluating the genotoxicity of viral vectors.

OBJECTIVE  To establish a cisplatin induced acute kidney injury (AKI) model based on human kidney organoids. METHODS  ① Kidney organoids containing various cell types were designed and constructed based on human induced pluripotent stem cells (hiPSC) induction techniques, and the tissue structure and cell types were identified by HE staining and immunofluorescence. ② Based on the constructed human kidney organoid model, the broad-spectrum anticancer drug cisplatin was used as the experimental drug, and three concentration gradients of 20, 50 and 75 μmol·L^-1 were applied to the human kidney organoid model. The samples were collected 48 h later, and the cell viability was detected by observing the morphological changes of kidney organoids. The expression levels of renal injury factor-1 (Kim-1) and inflammatory cytokine interleukin-8 (IL-8) were detected with real-time quantitative PCR to determine the degree and location of renal injury. RESULTS  ① Histological and immunofluorescence results showed that mature human renal organoids could be produced after iPSC-induced differentiation. The organoids had primitive tubular structure and included various cell types 
including the proximal tubular tubular, distal tubular tubular, podocytes, interstitial endothelial cells and tubular epithelial cells. ② Single administration of three concentrations of cisplatin resulted in the destruction of cell morphology and structure, and a large number of tubular structures disappeared. Live/Dead staining showed that renal organoids exhibited dose-dependent apoptosis to cisplatin. With cisplatin at the concentration of 20 μmol·L-1, cell viability fell below 50%, and approached 0 at a maximum concentration of 75 μmol·L^-1. ③ Kim-1 and IL-8 were significantly increased at different concentrations of cisplatin induced AKI models. CONCLUSION  This study has constructed a human kidney organoid model and verified the feasibility of human kidney organoids being used to simulate AKI induced by chemotherapy drugs in vitro. Kim-1 combined with IL-8 is expected to provide data for clinical prediction of the possibility of AKI induced by such drugs and countermeasures against adverse reactions.

Neuropsychiatric disorders such as Parkinson disease, Alzheimer disease, and schizophrenia may induce hallucinations, delusions, and other psychiatric symptoms during the course of disease development. These symptoms are highly prevalent and difficult to cure, and have a impact on the lives of patients. Classical antipsychotic drugs such as chlorpromazine, sulpiride and perphenazine can control related symptoms, but they can also cause uncontrollable extrapyramidal system reactions and side effects such as hyperprolactinemia. In recent years, it has been found that non-classical antipsychotics such as olanzapine, clozapine, risperidone and pimovanserin can treat hallucinatory symptoms in neuropsychiatric disorders by antagonising the 5-hydroxytryptamine 2A (5-HT_2A) receptor, or by antagonising both the 5-HT_2A receptor (strong) and the dopamine 2 (D₂) receptor (weak). In preclinical studies, non-classical antipsychotic drugs have shown great therapeutic effects against hallucination  induced by multiple factors. Clinical studies have confirmed that these drugs improve psychotic symptoms (mainly hallucinations and delusions) significantly. In patients who are insensitive or tolerant to clozapine and risperidone, pimavanserin still shows therapeutic effects. At the same time, the incidence and severity of adverse reactions to non-classical antipsychotic drugs are reduced, and they are well tolerated. This article reviews the research progress in the role of 5-HT_2A receptor antagonists in attenuating hallucinogenic symptoms so as to provide reference for the design and development of new therapeutic drugs.

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.

OBJECTIVE  To investigate the effect of the 5-hydroxytryptamine 2A receptor (5-HT_2AR) agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) on field potentials in area V2 of the secondary visual cortex in freely moving mice. METHODS  C57BL/6J mice were embedded with electroencephalocortical (ECOG) electrodes and recovered for 7 d after surgery. After 1% DMSO (vehicle) was ip given and recording of local field potentials (LFP) in area V2 of the secondary visual cortex for 30 min, DOI (2.0 mg·kg^-1, ip) was administered and LFP in area V2 was recorded for 30 min before the 5-HT_2AR antagonist volinanserin (0.3 mg·kg^-1, ip) was administered and LFP in area V2 was recorded for 30 min. The recorded data were imported into Neuro Explorer software for spectral analysis. GraphPad Prism 8.0.2 software was used to statistically analyze the changes in power spectral density of γ and θ oscillations before and after administration while Matlab software was used to analyze the phase amplitude coupling of θ and γ oscillations. RESULTS  Compared with the vehicle group, DOI (2.0 mg·kg^-1, ip) significantly increased the power of γ oscillations (30-80 Hz) and θ oscillations (4-7 Hz) in area V2 of the secondary visual cortex (P<0.05), and the modulation index of phase amplitude coupling between θ and γ oscillations was significantly reduced (P<0.01). The administration of volinanserin reversed the DOI-induced enhancement of the power of γ and θ oscillations in area V2 of the secondary visual cortex (P<0.05). CONCLUSION  DOI may exert its hallucinogenic effects by increasing the power of γ oscillations and θ oscillations in area V2 of the secondary visual cortex and by decreasing the phase amplitude coupling of θ oscillations to γ oscillations in mice. 

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.

In recent years, drug repurposing has emerged as an effective strategy for identifying potential treatments for the outbreak of the Corona Virus Disease 2019 (COVID-19). Artificial intelligence (AI) has been widely employed in the field of drug repurposing, enabling rapid computation and screening of extensive drug databases. Based on different algorithm design principles, AI methodologies for drug repurposing in the context of COVID-19 can be categorized into three types:① network-based models, which emphasize the identification of associations between drugs and diseases to reveal potential therapeutic mechanisms; ② structure-based methods, which employ the analysis of structural interactions between drugs and targets for precise screening; and ③ machine learning/deep learning approaches, which utilize multidimensional processing of complex nonlinear data for candidate drug prediction. Despite the significant role of AI in drug repurposing, the quality and quantity of data have a notable impact on the computational results of AI. Experimental studies alone cannot fully simulate the complex physiological environment of the human body, which may limit the precise validation of candidate drugs in the preclinical stage. Optimization of drugs originally indicated for other conditions may also affect the effectiveness of candidate drugs for COVID-19. Moreover, treatment timing and individual differences may influence clinical outcomes. This review provides an overview of the application and challenges of AI in the field of drug repurposing for COVID-19 in order to provide reference for wider use of AI technology in COVID-19 treatment.

Calcium-activated chloride channels (CaCCs) are a class of channel proteins that transport chloride ions activated by intracellular calcium, which play a crucial role in regulating membrane potential, intracellular calcium balance, and cell excitability, particularly in neurons and muscle cells. In the Anoctamin (Ano) family, Ano1 is the most classic CaCC. Targeted modulators of Ano1 have potential therapeutic effects against such diseases as cancer, cystic fibrosis, hypertension, diarrhea, and asthma. Since the discovery of Ano1 in 2008, several methods for screening CaCC-specific modulators have emerged including high-throughput primary screening of fluorescent proteins, electrophysiological patch clamp technique and virtual screening, and identification of small molecule modulators with 
diverse pharmacological effects. This paper summarizes the principles, advantages and disadvantages of the mainstream screening methods, and reviews the chemical structures and potential applications of Ano1-specific modulators discovered to date.

OBJECTIVE  To investigate the effect of Jiawei Tianwang Buxin Dan (JWBXD) on insomnia in rats exposed to simulated high-altitude conditions. METHODS  ① Thirty SD rats were randomly divided into the normal control, model, model+Jiawei Tianwang Buxin Dan (JWBXD, 9.6 mg·kg^-1), model+Tianwang Buxin Dan (TWBXD, 9.6 mg·kg^-1), and model+diazepam (DZP, 3 mg·kg^-1) groups. Rats, except for the normal control group, were subjected to a low-pressure, low-oxygen animal experimental chamber simulating a 5000 m altitude. Respective drugs were ig administrated once daily at 9:00 for seven days, and signal acquisition and sleep analysis were conducted by a wireless physiological signal telemetry system. ② Forty rats were randomly divided into five groups as described in ①. Throughout the experiment, the general condition and body mass of the rats were observed daily. Drug administration lasted for seven days, and grip strength was tested one hour after the final administration. ELISA was used to measure the levels of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), corticosterone (CORT), and melatonin (MLT) in serum. Western blotting was performed to measure the expression levels of core clock proteins period circadian regulator 2 (Per2), circadian locomotor output cycles (Clock), cryptochrome 2 (Cry2), brain-muscle arnt-like protein 1 (Bmal1), nuclear receptor subfamily 1, group D member 1 (NR1D1), glycogen synthase kinase-3β (GSK-3β), as well as acetylserotonin O-methyltransferase (ASMT) in the hypothalamus and pineal gland, respectively. RESULTS  ① Compared with the normal control group, the model group exhibited a decrease in total sleep time (P<0.01), an increase in wakefulness (P<0.01), a significant reduction in slow wave sleep (SWS) (P<0.05) and the mean bouts duration (P<0.05). Compared with the model group, both DZP and JWBXD (P<0.01) prolonged sleep time and suppressed wakefulness (P<0.01) in the hypoxic environment. DZP and JWBXD prolonged SWS (P<0.05, P<0.01), while TWBXD had no significant effect. JWBXD improved the mean bouts duration of SWS in the model rats (P<0.01), whereas no such improvement was observed in model+DZP and model+TWBXD groups. ② Compared with the normal control group, the model group showed a significant decrease in forelimb grip strength (P<0.01), increased levels of serum ACTH (P<0.05), CRH, and CORT (P<0.01), and decreased MLT levels (P<0.05). The expression levels of Per2, Cry2, GSK-3β, and NR1D1 in the hypothalamus were downregulated (P<0.05, P<0.01), while Bmal1 and Clock were upregulated (P<0.05, P<0.01). ASMT expression in the pineal gland was decreased (P<0.05). Compared with the model group, JWBXD and TWBXD enhanced forelimb grip strength (P<0.01), reduced serum CORT and ACTH levels (P<0.05), decreased CRH levels (P<0.01), and restored MLT levels (P<0.01). JWBXD upregulated the expression levels of Per2, Cry2, GSK-3β and NR1D1 in the hypothalamus (P<0.05, P<0.01), but downregulated Bmal1 and Clock expression (P<0.05, P<0.01). TWBXD downregulated Bmal1 expression in the hypothalamus (P<0.01) and increased NR1D1 expression (P<0.05). DZP significantly enhanced the expression levels of Per2, Cry2 and NR1D1 in the hypothalamus (P<0.01). JWBXD, TWBXD and DZP improved ASMT expression in the pineal gland (P<0.05). CONCLUSION  JWBXD can improve sleep structure and prolong the duration of SWS in rats exposed to simulated high-altitude conditions. The mechanisms may involve the regulation of core clock protein expressions in the hypothalamus, promotion of melatonin secretion, and inhibition of HPA axis hyperactivity.

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.

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.

"Toxicity Testing in the 21st Century — A Vision and Strategy" proposed by the National Research Council of US has brought innovative directives and objectives for toxicity evaluation and risk assessment, pushing forward the next generation of toxicity testing and risk assessment. In this initiative, the concept of adverse outcome pathways (AOPs) has emerged as a prominent methodology, capturing the attention of toxicologists and researchers due to its promising applications in recent years. The quantitative AOP (qAOP) is an extension of the adverse outcome pathway, which is built upon the foundational qualitative adverse outcome pathway model and leverages mathematical frameworks to depict dose-response and/or response-response relationships.This article reviews the principles and advancement surrounding qAOP, introduceds two prevalent methodologies for constructing qAOP, Bayesian network models and regression models,  and demonstrates diverse applications of qAOP. Actual cases are used to underscore the transformative role of qAOP in contemporary toxicology and risk assessment practices.

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.

OBJECTIVE  To investigate the protective effect of artesunate (Art) against apoptosis and mitophagy induced by NaF in osteocytes MLO-Y4, and to explore the molecular mechanism. METHODS  MLO-Y4 cells were treated with NaF (2 mmol·L-1) for 48 h to establish an in vitro model of osteocytes injuries, and the cells were divided into the cell control group, NaF (2 mmol·L-1) group and NaF+Art 0.25, 0.50 and 1.00 μmol·L-1 groups. The cells were pretreated for 2 h and NaF was added for 48 h.The cell survival of MLO-Y4 cells was detected by MTT assay. The cell viability of MLO-Y4 cells was measured by Calcein-AM staining. The lactate dehydrogenase (LDH) content in the supernatant was examined by the LDH detection kit. The level of intracellular reactive oxygen species (ROS) was examined by DCFH-DA staining. The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were detected by chemical colorimetry. Apoptosis was measured by Hoechst33342 staining and Annexin-V/PI staining. The level of mitochondrial membrane potential (MMP) was measured by JC-1 staining. The formation of autophagic vacuoles and morphological mitochondrial changes were observed via Lyso-tracker staining and Mito-Tracker staining. The ATP content was detected with the luciferase method. The expression of microtubule-associated protein light chain 3 (LC-3) in mitochondria was examined by immunofluorescence staining. Protein expressions of LC-3, P62, E3 ubiquitin-ligase (Parkin) and PTEN-induced putative kinase 1 (PINK1) were detected by Western blotting. RESULTS  Compared with the cell control group, the cell survival rate and cell viability were significantly reduced in the NaF group (P<0.01), LDH content in the supernatant, the level of intracellular ROS, the MDA content, apoptosis rate and autophagic vesicle formation were remarkably increased (P<0.01), protein levels of Parkin and PINK1, and the conversion of LC-3Ⅱ from LC-3Ⅰ were markedly upregulated along with the elevation of LC-3 in damaged mitochondria (P<0.01), while P62 levels, SOD activity, MMP and ATP contents were reduced in NaF cells (P<0.05, P<0.01). Compared with NaF group,  the cell viability and survival rate of MLO-Y4 cells in NaF+Art 0.25, 0.50 and 1.00 μmol·L-1 groups were significantly increased (P<0.01); the content of LDH in supernatants was decreased obviously (P<0.01); the levels of intracellular ROS and MDA content were markedly reduced (P<0.05, P<0.01); the apoptosis rate and autophagic vesicle formation were remarkably decreased (P<0.05, P<0.01); protein levels of Parkin and PINK1, and the conversion of LC-3Ⅱ from LC-3Ⅰ were markedly down-regulated along with the accumulation of LC-3 in damaged mitochondria (P<0.01); MMP and ATP content were also reduced (P<0.05, P<0.01); while SOD activityand P62 levelwere significantly increased (P<0.05, P<0.01). CONCLUSION  Art has a protective effect against oxidative damage induced by NaF in MLO-Y4 cells, which might be related to the inhibition of apoptosis and mitophagy. 

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.

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.

Clostridium perfringens (CP) is a common zoonotic pathogen that widely exists in nature and takes a heavy toll on human health and animal husbandry through its secreted exotoxins. The main pathogenic toxins of CP are α, β, ε and τ toxins, enterotoxins and necrotizing enteritis B-like toxins. The diseases caused by these toxins vary. Antibiotic therapy is currently the main method for the treatment of CP infection, but due to the growing drug resistance, it is increasingly difficult to control CP infection. In recent years, based on the rapid development of genetic engineering technologies, vaccines or antibodies against different exotoxins may become a new generation of immunotherapy for CP infection. Studies have shown that the vaccines against CP α-toxin can prevent type A CP infection in mice. Anti-α-toxin single chain antibodies, bivalent single chain antibodies and nano-antibody can specifically bind to α-toxin, effectively neutralize the phospholipase C activity of α-toxin, and protect mice against lethal doses of α-toxin challenge. This article reviews the main pathogenic factors and mechanisms of CP and the research progress in genetic engineering antibody therapies so as to provide reference for the prevention and treatment of CP infection.

OBJECTIVE  To investigate the effect of erianin on the angiogenesis of glomerular endothelial cells in diabetic nephropathy (DN) rats and the role of slit homolog 2 protein (Slit2)/roundabout homolog 1 (Robo1) consecutive signaling pathway. METHODS  Rats were fed with high sugar and high fat feed for 8 weeks, before being intraperitoneally injected with streptozotocin solution (35 mg·kg-1) to prepare a DN rat model. DN rats were divided into the model group and model+erianin 10, 20 and 40 mg·kg-1 groups, with 10 rats in each, while another 10 rats served as normal control group. The urine protein quantification kit was used to measure the 24 h urine protein level of rats in each group while the automatic biochemical analyzer was used to detect the fasting plasma glucose (FPG) and serum creatinine (Scr) levels of rats in each group. PAS staining was applied to observe the pathological changes in the renal tissue of rats in each group. Immunofluorescence was used to detect the expressions of platelet endothelial cell adhesion molecule-31 (CD31) and podocalyxin (PCX) in kidney tissue of rats in each group. Western blot was adopted to detect the expressions of Slit2 and Robo1 proteins in the renal tissues of rats in each group. RESULTS  Compared with normal control group, the CD31 protein expressions, FPG, Scr, 24 h urine protein levels, and renal tissue Slit2 and Robo1 protein expressions were significantly increased in the model group (P<0.05). Pathological and immunofluorescence results suggested that rats in the model group developed many neoplastic glomerular capillaries, glomerular hypertrophy, and dilated mesangial areas, with non-tubular CD31 staining lacking adjacent PCX staining, and partial staining of tubular areas of CD31 lacking adjacent PCX staining. Compared with the model group, the CD31 glomerular endothelial area, FPG, Scr, 24 h urine protein levels, and protein expressions of Slit2 and Robo1 in renal tissues were significantly reduced in the model+erianin 10, 20 and 40 mg·kg-1 groups 
(P<0.05). Pathological and immunofluorescence results showed new glomerular capillaries, glomerular hypertrophy and dilatation of the thylakoid area were attenuated in rats, and CD31 tubular region staining was essentially adjacent to the PCX foot cell region staining in the model+erianin 10, 20 and 40 mg·kg-1 groups. CONCLUSION  Erianin may inhibit angiogenesis in glomerular endothelial cells of DN model rats by inhibiting the Slit2/Robo1 signaling pathway.

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.

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.

Lipid nanoparticles (LNPs), composed of ionizable lipids, are currently the most promising non-viral nucleic acid drug delivery vectors in clinical practice, and have great potential in gene therapy drug delivery and vaccine delivery. Ionizable lipids, the main components of LNPs, play a decisive role in the endosome escape rate, transfection efficiency, organ targeting and safety of LNPs. Among them, the hydrophilic head group of ionizable lipids contains tertiary amine groups, which can improve the buffering capacity of LNPs and thus change the pKa value, and imidazole can enhance the stability and transfection activity of mRNA-LNP. The ligand contains ester groups, which can induce gene silencing efficiently and improve the degradation rate and safety. When the number of hydrophobic tails is 3-4, with 1-2 unsaturation and 8-18 carbon chain length, LNPs can effectively induce gene silencing. Meanwhile, the presence of branching or asymmetric hydrophobic tails can improve the transfection efficiency of LNPs. Based on the chemical structure of ionizable lipids, this review summarizes the influence of the structure of ionizable lipids on the transfection efficiency and safety of nucleic acid carrying drugs LNPs, and the structure-activity relationship of ionizable lipids so as to provide reference for studies on novel ionizable lipids.

OBJEACTIVE  To investigate the effect of interleukin-32 (IL-32) on adipogenic differentiation of human umbilical cord-derived mesenchymal stem cells (HuMSCs). METHODS  ① HuMSCs were activated by treatment with tumor necrosis factor-α (TNF-α) (20 μg·L^-1) and interferon-γ (IFN-γ) (20 μg·L^-1) for 24 h to obtain stimulated-HuMSCs (S-HuMSCs). The surface markers CD14, CD34, CD45, CD73, CD90 and CD105 of HuMSCs and S-HuMSCs were detected by flow cytometry to identify their phenotypes. After ten days of adipogenic induction, oil red O staining was performed to detect lipid droplets in HuMSCs and S-HuMSCs. Real-time quantitative PCR (RT-qPCR) was used to detect the mRNA expressions of adipogenic transcription factors peroxisome proliferator activated receptor γ (PPARγ), CCAAT enhancer binding protein α (C/EBPα) and adiponectin (ADI ) to determine their adipogenic differentiation ability. After 14 d of osteogenic induction, alkaline phosphatase (ALP) staining was performed to detect the ALP staining area. RT-qPCR was used to detect the mRNA expressions of osteogenic related transcription factors RUNX family transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and distal-less homeobox 5 (DLX5) to determine the osteogenic differentiation ability. ② The lentiviral vector containing the over-expressed IL-32 gene sequence with green fluorescent protein (GFP) and puromycin resistance gene and the negative control (NC) empty vector were constructed and harvested. HuMSCs were infected with these viruses to obtain IL-32^highHuMSCs and NC-HuMSCs, respectively. HuMSCs, NC-HuMSCs and IL-32^highHuMSCs were induced to differentiate into adipocytes. Oil red O staining was used to detect the area of lipid droplet formation in cells at 0, 3, 5, 7, 10 and 14 days after induction, and RT-qPCR was used to detect the expressions of transcription factors PPARγ, C/EBPα and ADI mRNA related to adipogenic differentiation. RESULTS  ① The results of flow cytometry showed that HuMSCs and S-HuMSCs had high expressions of CD73, CD90 and CD105, low or no expressions of CD14, CD34 and CD45, and both phenotypes were consistent with the biological characteristics of MSCs. After adipogenic and osteogenic differentiation of HuMSCs and S-HuMSCs, compared with the self-differentiated group, the area of ALP staining and oil red O staining in the induced group increased (P<0.01). The mRNA expressions of osteogenic differentiation related transcription factors RUNX2, ALP and DLX5 and adipogenic differentiation related transcription factors PPARγ, C/EBPα and ADI were also significantly increased (P<0.01), indicating that both of them had the characteristics of MSCs. Compared with the HuMSCs induction group, the area of lipid droplet formation in the S-HuMSCs induction group was significantly reduced (P<0.01), the mRNA expressions of adipogenic differentiation-related transcription factors PPARγ, C/EBPα and ADI were also significantly decreased (P<0.05), but S-HuMSCs highly expressed IL-32 (P<0.01). ② After adipogenic differentiation of HuMSCs, NC-HuMSCs and IL-32^highHuMSCs in vitro, oil red O staining was performed at 0, 3, 5, 7, 10 and 14 days. From the 3rd day, the area of lipid droplet formation in the IL-32^highHuMSCs induction group was significantly smaller than that in the HuMSCs and NC-HuMSCs induction groups (P<0.01), and the mRNA expressions of adipogenic transcription factors PPARγ, C/EBPα and ADI were also significantly decreased (P<0.05). CONCLUSION  Overexpression of IL-32 can significantly reduce the adipogenic differentiation ability of HuMSCs.

OBJECTIVE  To establish a mouse model of diabetes mellitus (DM) combined with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection to investigate the important pathophysiological changes in the development of DM combined with SARS-CoV-2 infection. METHODS  Wild-type (WT) mice and transgenic mice expressing the human angiotensin-converting enzyme 2 receptor driven by the cytokeratin-18 gene promoter (K18-hACE2) were randomly divided into the control group, DM group, SARS-CoV-2 spike protein (S) infection group and DM combined with S protein 
infection group, with 10 to 12 mice in each group. All the mice were induced by 10 weeks of high-fat diet combined with 40 mg·kg^-1 streptozotocin (STZ) for 3 days by ip, except those in the control group or S protein infection group. The control group was given the same volume of 0.1 mol·L^-1 sodium citrate buffer. Mice in the S protein infection group and DM+S protein infection group were additionally given 50 μL mixture of 15 μg SARS-CoV-2 spike protein and 1 g·L^-1 polyinosinic-polycytidylic acid (poly[I:C]) via intranasal drops, while the control group was given an equal volume of sterile water. The glucose tolerance level and pancreatic islet β cell function of mice were evaluated via oral glucose tolerance test at the 6th week of high-fat feeding and 1 week after the administration of STZ by ip. From the 6th week of high-fat feeding to 2 weeks after the administration of STZ, the random blood glucose and fasting blood glucose of mice were measured by a blood glucose meter. Blood samples were taken from submandibular veins of 3 mice in each group at 24, 48 and 120 h after S protein infection, and lung tissues were taken after euthanization. The pathological changes of lungs of DM mice before and after S protein infection were observed by HE staining. Except for the DM group, blood samples were collected before S protein infection and at 6, 24, 48, 72 and 120 h after infection. The levels of plasma interleukin 1β (IL-1β), IL-2, IL-6, IL-10, IL-17, interferon gamma-induced protein 10 (IP-10), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), monocyte chemotactic protein-1 (MCP-1) and granulocyte-colony stimulating factor (G-CSF) were detected by Luminex. The plasma levels of heparan sulfate (HS) were measured by enzyme-linked immunosorbent assay. The levels of cytokines and HS were correlated with the degree of pathological damage by Spearman correlation analysis. RESULTS  STZ and high-fat diet could induce DM-like expression in mice, and the random blood glucose (P<0.01) and fasting blood glucose (P<0.05) after 1 week in the hACE2-DM group were significantly higher than  in the WT-DM group, and the degree of islet function damage in hACE2-DM mice was significantly higher than that of WT-DM mice (P<0.05). Compared with the DM group, the DM+S group showed more severe pulmonary pathological changes after S protein infection, accompanied by a large number of inflammatory infiltrations and thickening of lung interstitial. Compared with the control group, the levels of pro-inflammatory cytokines G-CSF, IL-6 and IP-10 in the plasma of the WT-S group were significantly increased at 6 h after S protein infection (P<0.01), and those of pro-inflammatory cytokine IL-17 and anti-inflammatory cytokine IL-10 were significantly increased at 24 h after S protein infection (P<0.05). Compared with the control group, the plasma levels of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, MCP-1, G-CSF and IP-10 in the hACE2-S group were significantly increased at 6 h after S protein infection (P<0.05, P<0.01). IL-17 was significantly increased at 24 h and 6 h after S protein infection in the WT-DM+S group and hACE2-DM+S group, respectively (P<0.01, P<0.05). In the hACE2-DM+S group, IFN-γ and IL-1β were significantly increased in delay to 48 h (P<0.05, P<0.01), and MCP-1 was significantly increased in delay to 72 h (P<0.05). Compared with the control group, the level of HS in the plasma of the WT-S group increased significantly (P<0.05, P<0.01) at 6 h and 24 h after S protein infection, but began to decrease at 48 h. At the same time, compared with the WT-S group, the HS level in the WT-DM+S group was slightly increased at 6 h after infection and decreased at 24 h. Compared with the control group, the HS level in the hACE2-S group was significantly increased at 24 h (P<0.01), as was the case with the WT-S group 24 h, 48 h and 120 h after S protein infection. At 6 h, 24 h and 48 h after S protein infection, the plasma HS level of the hACE2-DM+S group was significantly increased (P<0.01, P<0.05), and the duration of the increase was longer than in the hACE2-S group. Moreover, the levels of IL-1β, IL-10, MCP-1, IP-10, G-CSF and HS in plasma were positively correlated with the degree of lung damage in the DM+S group. CONCLUSION  In this study,  the mouse model of diabetes combined with SARS-CoV-2 spike protein infection has mimicked part of the pathophysiological features of clinical patients, mainly manifested as blunted immune response and elevated HS levels with longer duration to infection alone. IL-1β, IL-10, MCP-1, IP-10, G-CSF and HS may keep track of the course of disease in patients with diabetes combined with SARS-CoV-2 infection.

OBJECTIVE  To investigate the damage effect and potential toxic mechanism of SARS-CoV-2 spike protein (S protein) on human neuroblastomacells (SH-SY5Y). METHODS  SH-SY5Y were treated with S protein at concentrations of 25, 50, 75, and 100 mg·L^-1 for 24 h. Cell viability of SH-SY5Y was detected using the CCK-8 assay. The cytotoxic lactate dehydrogenase (LDH) detection kit was used to measure the release rate of LDH, and the 5-ethynyl-2′-deoxyuridine (EdU)-488 cell proliferation kit was used to assess cell proliferation. The ATP detection kit was used to measure intracellular ATP content. The JC-1 fluorescent probe method was employed to detect the mitochondrial membrane potential (MMP) of cells. Seahorse XF was used to measure mitochondrial respiratory and glycolytic capacity. RESULTS  Compared with the cell control group, cell viability was significantly reduced in S protein 25, 50, 75 and 100 mg·L^-1 groups (P<0.01), and the half-inhibition concentration (IC₅₀) was 65.05 mg·L^-1. The LDH release rate wassignificantly increased (P<0.01) and the proportion of EdU positive cellswas significantly reduced (P<0.01) in S protein 25, 50, 75 and 100 mg·L^-1 groups. S protein significantly reduced intracellular ATP content (P<0.01) at the concentrations of 75 and 100 mg·L^-1, while significantly reduced intracellular MMP (P<0.05, P<0.01) at the concentrations of 50 and 75 mg·L^-1.  S protein 50 mg·L^-1 increased the maximum value of basal glycolysis levels and glycolytic capacity (P<0.05, P<0.01), and S protein 25 and 50 mg·L^-1 increased the maximum value of respiration capacity (P<0.05, P<0.01). SH-SY5Y cell viability was positively correlated with the intracellular ATP content and the MMP level (r²=0.9209, P=0.001; r²=0.6170, P=0.0025), and negatively correlated with the maximum level of basal glycolysis and glycolytic capacity (r²=0.5194, P=0.0285; r²=0.6664, P=0.0073), and negatively correlated with ATP production capacity (r²=0.8204, P=0.0008). CONCLUSIONS  protein decreases the viability of SH-SY5Y cells and inhibited cell proliferation. The mechanism may be closely related to the disorder of energy metabolism.

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.

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.

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.

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.

OBJECTIVE  To investigate the effect of eukaryotic translation elongation factor 1A1 (eEF1A1) on the replication of vesicular stomatitis virus (VSV) and Herpes simplex virus 1 (HSV-1) to identify a potential target for broad-spectrum regulation of viruses. METHODS  Small interfering RNA (si-eEF1A) was transfected into human skin fibroblasts (BJ-5ta) to inhibit the expression of eEF1A1, and the negative control group was set up. The transfection efficiency was detected by real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting, the cell model of eEF1A1 gene silencing was constructed. The cell model was infected with VSV, the gene copy number and protein expression of VSV in the cells were detected. The cell model was infected with HSV-1, the mRNA and protein expression of HSV-1 in the cells were detected. The cell models were transfected with polyinosinic acid〔Poly(I:C)〕or sodium deoxyribonucleic acid (HT-DNA), respectively. The mRNA expression of interferon-β (IFN-β), C-X-C Motif Chemokine 10 (CXCL10 ) and interferon-stimulated gene 56 (ISG56 ) were detected by RT-qPCR. The phosphorylation expression of interferon regulatory factor 3 (IRF3) and TANK binding kinase 1 (TBK1) were detected by Western blotting. RESULT  Compared with the negative control group, the mRNA and protein expression of eEF1A1 in the eEF1A1 gene silencing group were significantly decreased (P<0.01), the cell model of eEF1A1 gene silencing was successfully constructed. Compared with the negative control group, the VSV gene copy number of the eEF1A1 gene silencing group decreased by 70%-80%. The VSV protein expression decreased significantly (P<0.01 ). The mRNA expression of HSV-1 was decreased by 50 % -60 %, and the protein expression of HSV-1 was significantly decreased (P<0.01 ). After stimulation with Poly (I : C) or HT-DNA, compared with the negative control group. there was no significant difference in the mRNA expressions of IFN-β, ISG56 and CXCL10 and the protein phosphorylation expression of IRF3 and TBK1 in the eEF1A1 gene silencing group. CONCLUSION  eEF1A1 silencing can inhibit VSV and HSV-1 virus replication, suggesting that eEF1A1 has a potential broad-spectrum regulatory effect on RNA viruses and DNA viruses, and may not recognize activated immune pathways through intracellular nucleic acid recognition.

OBJECTIVE  Based on fibroblast activation protein (FAP) gene promoter as the response element, to develop a new dual luciferase reporting system for  the screening of drugs related to myocardial fibrosis. METHODS  The promoter fragment of mouse FAP gene was synthesized in vitro and cloned into plasmid psiCHECK2 to replace HSV-TK promoter, and then a new recombinant plasmid psiCHECK2-FAP was obtained. After the recombinant plasmid psiCHECK2-FAP was digested by restriction endonucliase Hind Ⅲ, the product digested was identified by agar-gel electrophoresis and sequencing. After psiCHECK2-FAP was transient transfected into mouse cardiac fibroblasts (MCFs), and continued cultured for 24 h, and MCFs were treated with Ransforming growth factor β1 (TGF-β1, 5 μg·L-1) or angiotensinⅡ(Ang Ⅱ, 1 μmol·L-1) or palmitic acid (PA, 100 μmol·L-1) for 0, 12, 24, 48 h, 
respectively, the double luciferase reporter gene assay was used to detect luciferase activity; After psiCHECK2-FAP was transient transfected into MCFs, the cells were pretreated with Dapa (1 μmol·L-1) for 1 h, and supplemented with TGF-β1 (5 μg·L-1) or Ang Ⅱ(1 μmol·L-1) or PA (100 μmol·L-1), continued treatment for 24 h, the double luciferase reporter gene assay was used to detect luciferase activity, and the expression of collagenⅠand collagen Ⅲ protein was detected with Western blotting. RESULTS  The recombinant plasmid psiCHECK2-FAP was digested into two fragments by Hind Ⅲ with the expected strip size, and the sequencing results were completely consistent with the theoretical sequence; Compared with control group, the collagenⅠand collagen Ⅲ protein expression were significantly increased by TGF-β1 or Ang Ⅱ or PA in MCFs (P<0.05, P<0.01). However, compared with TGF-β1 or Ang Ⅱ or PA group, the intervention of Dapa significantly alleviated the promoter activity of FAP gene and the expression of  collagenⅠand collagen Ⅲ protein (P<0.05, P<0.01); Compared with control group, luciferase activity was significantly increased by TGF-β1 or Ang Ⅱ or PA (P<0.05, P<0.01), reaching the peak at 24 h. Compared with TGF-β1 or Ang Ⅱ or PA group, the intervention of Dapa significantly decreased luciferase activity (P<0.05, P<0.01). CONCLUSSION  Based on the promoter of FAP gene as the response element, a noval dual luciferase reporter gene system was established and showed a good sensitivity to the promyocardial fibrosis factor in MCFs, which can provide strategies for the development of antimyocardial fibrosis drugs.

OBJECTIVE  To investigate the impact of mercuric sulfide nanoparticles (HgS-NP) on various neurotransmitter-type neurons in Caenorhabditis elegans (C. elegans). METHODS  Wild-type N2 C. elegans, as well as the transgenic C. elegans expressing green fluorescent protein specifically in gamma-aminobutyric acid (GABA)-ergic, glutamatergic, dopaminergic, cholinergic, and serotoninergic neurons (strains EG1285, DA1240, BZ555, LX929, and GR1366), were synchronized at the L4 stage and treated with different concentrations of HgS-NP via solid exposure. ① Five transgenic strains of C. elegans were 
exposed to HgS-NP 0 (control group), 250, 500, and 1000 mg·L^-1 for 72 h. Fluorescence microscopy was used to observe the expression of green fluorescence in different neurotransmitter-type neurons of the corresponding transgenic C. elegans. ② Wild-type N2 C. elegans were exposed to HgS-NP 0 (control group) and 1000 mg·L^-1  for 72 h. Real-time quantitative PCR (RT-qPCR) was performed to detect the mRNA expression levels of 33 genes related to the GABAergic and glutamatergic neurotransmitter systems. RESULTS  ① Compared with the control group of the same genotype, the EG1285 C. elegans exhibited soma shrinkage, dendrite fragmentation, and neuronal loss, with a significant decrease in the relative fluorescence intensity of GABAergic neurons (P<0.05) after exposure to different concentrations of HgS-NP. After exposure to HgS-NP 1000 mg·L^-1, the DA1240 C. elegans showed a loss of glutamatergic neurons in the head region and a significant decrease in the relative fluorescence intensity (P<0.01). However, there were no significant changes in the morphology or relative fluorescence intensity of dopaminergic, cholinergic, and serotoninergic neurons in BZ555, LX929, and GR1366 C. elegans after exposure to HgS-NP, respectively.② Compared with the control group, wild-type N2 C. elegans exposed to HgS-NP 1000 mg·L^-1  showed increased mRNA expression levels of glr-7 and glr-8, which encoded non-N-methyl-D-aspartate glutamate receptors (P<0.05), while the expression levels of the remaining 31 genes related to the GABAergic and glutamatergic neurotransmitter systems showed no significant changes. CONCLUSION  Exposure to a high dose of HgS-NP for 72 h may potentially damage the GABAergic and glutamatergic neurons in C. elegans, but have no significant effect on dopaminergic, cholinergic or serotoninergic neurons.