There have been breakthroughs in the development and clinical application of immunotherapeutic agents in recent years. As the clinical targets of second-generation immune checkpoint inhibitors (ICIs), programmed death-1 (PD-1) and its ligand (programmed death-ligand 1, PD-L1) have become one of the hot spots in drug discovery. The Food and Drug Administration of the USA and National Medical Products Administration in China have approved a variety of PD-1/PD-L1 monoclonal antibody drugs (such as nivolumab, pembrolizumab and atezolizumab), which are marketed for the treatment of small-cell lung cancer, rectal cancer, colon cancer, and melanoma, among other diseases. However, in the clinical application of PD-1/PD-L1 monoclonal antibody drugs, it is found that they can cause different degrees of immune-related adverse reactions in the lung, liver, kidney, gastrointestinal system, endocrine system and skin, and even the emergence of hyperprogressive disease. Effective monitoring and management of clinical applications of PD-1/PD-L1 monoclonal antibody drugs and rational use of some drugs can improve the immunotherapy process and reduce the effects of adverse reactions and hyperprogressive diseases in patients under immunotherapy.

In recent years, increasing attention has been paid to the study on the pathogenesis of cerebral ischemia in terms of the overall structure and function of neurovascular unit (NVU), which has become one of the hot spots in the field of brain sciences and major brain diseases. This paper is intended to outline the roles of the four main NVU cells (neurons, astrocytes, microglia and cerebral microvascular endothelial cells) in brain function and pathogenesis of cerebral ischemia, which are closely related in structure, work together to maintain cerebral homeostasis in function, and play an important role in brain function and cerebral ischemic injuries. NVU injury leads to microvascular and blood brain barrier integrity impairment, neuronal cell death, glial reaction and immune cell infiltration, and even tissue injury and brain edema. This paper also aims to elucidate the roles of NVU structure and function in the pathogenesis of cerebral ischemia, and offer new ideas and strategies for the research related to drugs for prevention and treatment of cerebral ischemia based on NVU structure and function.

In case of ischemic stroke（IS）, thrombus in the vascular system can cause ischemia and hypoxia in brain tissue, produce inflammatory cytokines and cause brain tissue damage, while reactive oxygen species during ischemia-reperfusion cause stress injury. Conventional drug administration is limited by the selective permeability of the blood-brain barrier and the low bioavailability of the drug itself, and its therapeutic effect against IS is unsatisfactory. Nanomedicine is expected to bring hope in that it has a unique mechanism of action and can cross the blood-brain barrier to reach the periphery of the infarct, release drugs or therapeutic genes, and exert a therapeutic effect. Nanomedicine inhibits platelet aggregation, enhances the efficacy of thrombolytic drugs, dissolves thrombus, increases blood supply to ischemic areas, eliminates reactive oxygen species and weakens injury response by fighting inflammatory cytokines. Loaded therapeutic genes regulate the differentiation process of neural stem cells, increase the number of neurons, induce the occurrence of blood vessels, and enhance the repair function of brain tissue. Nanomedicines can not only improve pharmacokinetics and pharmacodynamics to achieve more effective drug treatment, but also use nanoimaging technology to achieve real-time monitoring and condition assessment of therapy.

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.

OBJECTIVE  To assess the profiles of elements in benzo[a]pyrene (BaP) induced carcinogenesis, and explore the joint effects of copper with cisplatin or vinorelbine on cell proliferation. METHODS Forty-four elements were measured using an inductively coupled plasma mass spectrometer in 16HBE cells and BaP malignantly transformed 16HBE (T-16HBE-C1) cells. Partial least square was used to validate the robustness of cell classification of elements. Cell viability was measured by MTT assay for copper (0, 237, 340, 487, 1000 and 1432 μmol·L-1), cisplatin (0, 4.4, 6.1, 8.6, 12.0 and 16.8 μmol·L-1), and vinorelbine (0, 3.8, 9.8, 25.0, 40.0 and 64.0 μmol·L-1) to acquire their half maximal inhibitory concentration (IC50). Mixtures of copper and chemotherapeutics were prepared according to the ratio of each IC50. Their joint effects on cell viability were assessed by MTT assay and isobolographic analysis. Inhibition effect of copper (0, 50, 100, 200, 400 and 800 μmol·L-1) with IC50 of cisplatin or vinorelbine on proliferation of T-16HBE-C1 cells was also assessed. RESULTS  A total of 29 elements were quantified in 16HBE and T-16HBE-C1 cells, among which concentrations of copper, zinc, silver, selenium and rubidium decreased (P<0.05, P<0.01), while those of molybdenum, arsenic, lithium, germanium, strontium, nickel, lanthanum, mercury, iron, and cesium increased (P<0.05, P<0.01) in T-16HBE-C1 cells. Element concentration could be used to distinguish T-16HBE-C1 cells from 16HBE cells. Copper concentration-dependently inhibited proliferation of both cells, with a statistically significant lower IC50 of (613±16) μmol·L-1 in 16HBE cells than (776±15) μmol·L-1 in T-16HBE-C1 cells (P<0.01). Mixtures of copper and cisplatin (1∶69.5) or vinorelbine (1∶33.4) could inhibit cell proliferation, and copper had additive effects with cisplatin or vinorelbine. When copper concentration was higher than 400 μmol·L-1, copper combined with IC50 of cisplatin or vinorelbine inhibited cell proliferation of T-16HBE-C1 cells compared with IC50 of cisplatin (11.2 μmol·L-1) or vinorelbine (23.2 μmol·L-1) alone. CONCLUSION  Element profiles and correlations can change significantly after 16HBE cells are malignantly transformed by BaP. Copper could inhibit the proliferation of T-16HBE-C1 cells and have additive effects with cisplatin or vinorelbine in higher concentration.

Letrozole, a third generation non-steroidal aromatase inhibitor, has been approved for the treatment of breast cancer in women. In recent years, it has been used in the field of growth and development in children, such as childhood dwarfism, somatic delayed pubertal growth and precocious puberty, but the long-term effects on liver and kidney function, lipid metabolism, reproductive function and bone metabolism are unclear. Studies have shown that letrozole can cause abnormal testicular morphology, changes in seminiferous tubules and interstitial tissues, reduce bone density, affect the balance of bone metabolism, and cause cognitive impairment and apoptosis of nerve cells. The mechanism of reproductive toxicity of letrozole may be related to its influence on the development and maturation of testicular cells, the expression of sex hormones and gonadotropins in vivo, and the distribution and expression of estrogen receptors in testicular tissues. The mechanism of bone metabolic toxicity is related to its increase in the proliferation and differentiation of osteoclasts induced by receptor activator of NF-κB ligand, as well as the increase of apoptosis, oxidative stress and NF-κB activity of osteoblasts. The mechanism of cognitive toxicity is related to its regulation of classical and nonclassical effects of the hippocampus, reduction of glutamate uptake by astrocytes, and reduction of L-type calcium channel blockade of caspase 3 activation. This article is to provide reference for safe and effective use of letrozole in clinical pediatrics.

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.

OBJECTIVE  To study the protective effect of saikosaponin b2 (SSb2) on corticosterone (CORT) induced PC12 cell injury and its mechanism. METHODS  ① PC12 cells were divided into the cell control group (24 h of culture with RPMI-1640 medium), CORT group (24 h of culture with CORT 100-800 μmol·L-1) and SSb2 group (24 h of culture with SSb2 1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 μmol·L-1). MTT assay was used to detect the cell survival rate. ② PC12 cells were divided into the cell control group (24 h of culture with RPMI 1640 medium), model group (24 h of culture with CORT 400 μmol·L-1), and model+SSb2 group (3 h pretreatment with SSb2 1.5625, 3.125, 6.25, 12.5 and 25 μmol·L-1, removal of the supernatant before cells were co-incubated with CORT 400 μmol·L-1 and corresponding concentrations of SSb2 for 24 h). MTT assay was used to detect the cell survival rate while micro-plate assay was used to detect the lactate dehydrogenase (LDH) leakage rate of PC12 cells. ③ PC12 cells were divided into the cell control group, model group and model+SSb2 12.5 μmol·L-1 group. AnnexinV-FITC/PI flow cytometry assay was used to detect PC12 cell apoptosis, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) cell metabonomics was used to detect metabolic profile changes and colorimetric assay was employed to detect the glutamic acid content and glutaminase activity in PC12 cells. RESULTS  Compared with the cell control group, the cell viability decreased to (55±6)% (P<0.01) when the concentration of CORT was 400 μmol·L-1. When the concentration of SSb2 was higher than 50 μmol·L-1, there was significant toxicity to PC12 cells (P<0.01). ② Compared with the cell control group, the cell survival rate was significantly decreased (P<0.01), while the release rate of LDH was significantly increased (P<0.01) in the model group. Compared with the model group,  the cell survival rate significantly increased (P<0.05, P<0.01), while the LDH release rate significantly decreased (P<0.01) in the model+SSb2 group. ③ Compared with the cell control group, cell apoptosis was significantly increased in the model group (P<0.05). Compared with the model group, cell apoptosis was significantly decreased (P<0.05) in the model+SSb2 group. Metabolomics results show that SSb2 significantly back-regulated nine differential metabolites of glutamate, creatine, N-acetylaspartate, L-tyrosine, citric acid, L-isoleucine, lactic acid, glutamine and choline. Further network analysis of the key metabolites regulated by SSb2 yielded five major metabolic pathways: D-glutamine and D-glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, alanine, aspartate and glutamate metabolism, tyrosine metabolism and arginine biosynthesis. Compared with the cell control group, the content of glutamate and activity of glutaminase were significantly decreased  in the model group (P<0.01). Compared with the model group, the content of glutamate (P<0.01) and activity of glutaminase (P<0.05) were significantly increased in the model+SSb2 group. CONCLUSION  SSb2 has a neuroprotective effect on CORT-injured PC12 cells, and the mechanism of which is related to inhibition of apoptosis and regulation of metabolic disorders.

OBJECTIVE  To study the sedative and hypnotic effects of zolpidem and the content of amino acid neurotransmitters in the thalamus and hypothalamus after treatment with zolpidem. METHODS  Experiments on the loss of righting reflex (LORR) induced by the upper-threshold dose pentobarbital sodium (50 mg·kg^-1, ip) were conducted to establish a hypoxic insomnia model in mice by simulating an altitude of 5500 m. Based on this model, the synergistic effect of zolpidem (0.33, 1, 3, 9 and 27 mg·kg^-1, ip) and the subthreshold (20 mg·kg^-1, ip) and upper-threshold pentobarbital sodium, as well as the sedative hypnotic effect of zolpidem (10, 13, 17, 20, 23, 30 and 40 mg·kg^-1, ip) were evaluated via the LORR in normoxic and hypoxic environments. One hour after ip given zolpidem, the levels of glutamic acid (Glu) and γ-aminobutyric acid (GABA) in the thalamus and hypothalamus of mice in either environment were determined by the high-performance liquid chromatography (HPLC) with fluorescence detection. RESULTS  One-day treatment with hypoxia significantly shortened the duration of LORR induced by the upper-threshold dose pentobarbital sodium. Compared with normoxia vehicle and hypoxia induced insomnia vehicle groups, zolpidem 9 and 27 mg·kg^-1 significantly shortened the latency to LORR (P<0.01, P<0.05) and prolonged duration of LORR induced by subthreshold and upper-threshold pentobarbital sodium (P<0.01, P<0.05). The median effective dose (ED₅₀) of LORR induced by zolpidem was 16.21 and 20.55 mg·kg^-1 in normoxic and hypoxic environments, respectively. The results of neurotransmitter level detection showed that Glu contents in the thalamus and hypothalamus and  the ratio of Glu/GABA in the hypothalamus were decreased after treatment with zolpidem 40 mg·kg^-1 in a normoxic environment (P<0.01, P<0.05). Compared with the normoxia control group, Glu content and the ratio of Glu/GABA in the hypothalamus were significantly increased after treatment with hypoxia (P<0.01, P<0.05), and zolpidem 40 mg·kg-1 could reverse their elevation. CONCLUSION  The sedative-hypnotic effect of zolpidem is weakened in a hypoxic environment, and the effect of zolpidem on the levels of Glu and GABA in the hypothalamus may play an important role in the sedative-hypnotic effect of zolpidem.

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.

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.

OBJECTIVE  To observe whether mitochondria can be transferred from mesenchymal stem cells (MSCs) to irradiated cells by establishing a mitochondrial fluorescent reporting system. METHODS  The lentiviral vector pSIN-EF1α-COX8A-DsRed2 (named COX8A-DsRed2) that might guide the expression of red fluorescence protein in the membrane of mitochondria was constructed. A lentivirus (named Lv-COX8A-DsRed2) was prepared in 293T cell line. Dental pulp stem cells (DPSCs) (named DPSC-COX8A-DsRed2) was infected with Lv-COX8A-DsRed2. The intracellular expression of the red fluorescence protein in DPSC was observed under fluorescence microcopy. The mitochondrial localization of the expressed red fluorescent probe in DPSC-COX8A-DsRed2 was confirmed according to TOMM20 immunostaining and MitoTracker Green staining results, which could specifically label mitochondria. The IEC-6 cells that received 10 Gy X-ray radiation were used as an injured cell model. The co-culture system was established by supplementing DPSC-COX8A-DsRed2 into the culture plate with the irradiated IEC-6 labelled by CFSE for 24 h. RESULTS  The imaging results of fluorescent microcopy observation showed that DPSC-COX8A-DsRed2 expressed the mitochondrial fluorescent reporting system, which was co-located with TOMM20 protein and Mito Tracker Green. The imaging results of confocal fluorescence microcopy showed that the mitochondria with red fluorescent protein were transferred from DPSC-COX8A-DsRed2 to the irradiated IEC-6 cells, suggesting that the established mitochondrial fluorescent reporting system could indicate mitochondrial transfer from donor cells to injured ones. CONCLUSION  DPSC-COX8A-DsRed2 stably expressing the mitochondrial fluorescent reporting system is established, which can be used to track mitochondrial transfer.

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.

Drugs and physical stimulation, including light, electricity, and magnetic fields, can be used to influence how neurons operate,among which chemogenetic and optogenetic technologies are most widely used. In recent years, magnetogenetic technology has also been developed that can activate neurons in magnetic fields through magnetic sensitive actuators, leading to non-invasive and instantaneous activation of specific brain regions. This article reviews the evolution of and problems with chemogenetic and optogenetic techniques commonly used in brain science research. It also outlines the latest progress in magnetogenetic technologies, which are not full-fledged yet, as well as the role of transcranial electrical stimulation, transcranial magnetic stimulation, deep brain stimulation and transcranial ultrasound stimulation technology in the functional regulation of brain diseases. Constant adjustment and improvement can make it possible for these technologies to be used more widely for the study of brain sciences and the treatment of brain diseases.

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.

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.

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 investigate the potential developmental toxicity and delayed toxicity of Fuganline oral liquid (FGLOL) after long-term administration in juvenile SD rats via a three-stage juvenile animal study (JAS). METHODS  Stage 1: according to the proposed clinical dose for infants within one year of age, FGLOL 3.88, 11.64, 38.75 g·kg-1 was orally administered to rats of postnatal day 4 (PND4) rats for 18 days, and the drug was stopped for 3 weeks. Stage 2: according to the proposed clinical dose for children ages 1 to 6, FGLOL 3.88, 11.64, 38.75 g·kg-1 was orally administered to PND15 rats for 31 d, and the drug was discontinued for 3 weeks. Stage 3: according to the proposed clinical dose for children aged 7 to 12, FGLOL 29.06, 58.13, 116.25 g·kg-1 was orally administered to PND40 rats for 66 d, and the drug was stopped for 4 weeks. The effects of FGLOL on health status, food intake, body mass, growth and development, nerve reflex development, learning and memory ability, physical development (body length), bone development (bone mineral density), hematology and coagulation (white blood cells, red blood cells and platelet count), blood biochemistry (glutamate dehydrogenase, urea nitrogen and triglycerides) and histopathology were investigated in young rats. RESULTS  In the three-stage JAS test, long-term administration of FGLOL did not cause rat death, and no toxicological effects were observed on body mass, growth and development, nerve reflex development, physical development, bone development, hematology and coagulation, blood biochemistry and histopathology of juvenile rats compared with the vehicle control group. CONCLUSION  The no observed adverse effect level (NOAEL) of FGLOL is 38.75 g·kg-1 for the JAS test corresponding to humans between 1 and 6 years old, while the NOAEL of FGLOL is 116.25 g·kg-1 for the JAS test and repeated drug toxicity test corresponding to humans aged 7 to 12.

OBJECTIVE  To study the toxicokinetics and tissue distribution characteristics of alpha-amanitin in rats. METHODS  The tail venous blood was collected from SD rats before and 5, 10, 20, 30 and 45 min, 1, 1.5, 2.5, 4 and 8 h after intraperitoneal injection of alpha-amanitin (1.5 mg·kg-1), and the concentration of alpha-amanitin in blood was determined by liquid chromatography-mass spectrometry (LC-MS/MS). DAS 2.0 software was used to analyze and plot the drug-time curve with toxicokinetic parameters. Based on the toxicokinetics results, 18 SD rats were randomly divided into three groups. The rats were sacrificed, and left ventricular arterial (LVA) blood and 9 types of tissue samples involving the heart, liver, spleen, lung, kidney, whole brain, small intestine, stomach wall and testis were collected 15 min, 40 min and 2.5 h after dosing, and the concentrations of alpha-amanitin were measured by LC-MS/MS to obtain the tissue distribution results of alpha-amanitin in SD rats. RESULTS  Toxicokinetics studies revealed that the peak blood concentration (Cmax) was (633±121) μg·L-1, the elimination half-life (T1/2) was (0.72±0.37) h, and the peak time (Tmax) was (0.52±0.16) h. The total clearance rate (CLz) was (1.62±0.26) L·h·kg-1, the area under the curve (AUC0-t) was (946±183) μg·h·L-1, and the mean retention time (MRT0-t) was (1.18±0.17) h. The apparent volume of distribution (Vz) was (1.65±0.86) L·kg-1. The results of tissue distribution study showed that alpha-amanitin was widely distributed in SD rats with the highest concentration in the kidney, followed by the lung, small intestines, stomach wall, LVA blood and liver, but was low in the heart, spleen, testicles and other tissues, and very low in the brain. Alpha-amanitin was absorbed and eliminated quickly, peaked at 40 min in each tissue, and the concentration was minimized after 2.5 h. CONCLUSION  The absorption and elimination of alpha-amanitin by intraperitoneal injection are rapid in SD rats, and the blood concentration reaches the peak about 31 min after administration, but can not be detected 4 h later. Alpha-amanitin is mainly distributed in the kidney, followed by the tissues and metabolic organs with rich blood flow, such as the lung, small intestines, stomach wall, LVA blood and liver. The content of alpha-amanitin is low in the heart, spleen, testicles and other tissues, and very low in the brain. It is speculated that it may have toxic targeting effect on the kidney and low blood-brain barrier permeability.

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 study the effect of fluoropezil on embryo-fetal developmental toxicity and toxicokinetics in rabbits, and provide reference for clinical medication. METHODS  According to the sequence of pregnancy, pregnant rabbits were divided into five groups: vehicle control group (1% hydroxypropyl methylcellulose+1.5% polyethylene glycol 400 aqueous solution), positive control group (cyclophosphamide 18 mg·kg-1), and fluoropezil (3.6, 9.0 and 22.5 mg·kg^-1) groups. The vehicle control group and the fluoropezil groups were ig administrated on the 6^th to 18^th day of gestation (GD_6-18) while the positive control group was ig given cyclophosphamide on GD_6-20. The pregnant rabbits were sacrificed on GD₂₈, and the embryo-fetal development was detected. Sex hormone levels of pregnant rabbits on GD₅, GD₁₈ and GD₂₈ were detected by ELISA method. Blood samples with toxokinetics were collected for concomitant toxic generation at the first and last administration, and drug concentrations in fetal, placenta and amniotic fluid were detected with liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS  Fluoropezil 3.6, 9.0 and 22.5 mg·kg^-1 had no significant effect on body mass, mass gain, food consumption, pregnancy outcomes, fetal appearance, viscera, skeletal and physical growth and development of pregnant rabbits. Only on GD₁₈ or GD₂₈, the levels of follicle stimulating hormone, estradiol and progesterone in each dose group fluctuated to some extent. The combined toxokinetics results indicated that fluoropezil could cross the placental barrier of the rabbits, but did not accumulate in pregnant rabbits or fetuses. Fetal mass, crown-rump length and uterus mass in the cyclophosphamide group were lower than those in the vehicle control group. The appearance and bone of the cyclophosphamide group were positive. CONCLUSION  The no observed adverse effect level (NOAEL) of fluoropezil toxicity on rabbit embryo-fetal development is 22.5 mg·kg^-1, which is 125 times of the effective dose. At the dosage level of 22.5 mg·kg^-1, C_max is 1093 μg·L^-1, and AUC_{(0-24 h)} 6650 μg·h·L^-1 on GD₁₈.

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.

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.

OBJECTIVE  To construct an insulin-resistant (IR) small intestinal organoid model of mice and study the protective effect of flavanomarein (FM) on the intestinal mucosal barrier in the model. METHODS  ① Small intestinal organoid models of C57BL/6J and db/db of mice were constructed. The expressions of Ki-67, E-cadherin (E-cad), lysozyme (Lyz) and mucin-2 (Muc-2) in small intestinal organoids were detected by 3D immunofluorescence. RT-qPCR was used to detect the expressions of fibronectin (Fn), glucagon-like peptide-1 (GLP-1) and peotide YY (PYY) mRNA while Western blotting was used to detect the expressions of Fn, GLP-1 and PYY protein. The Lyz secretion level was detected by ELISA. ②  Small intestinal organoids were divided into five groups: C57BL/6J mice ′small intestinal organoids as the normal control group, db/db mice′ intestinal organoids as the IR model group, db/db mice small intestinal organoids with flavanomarein 25, 50 and 100 μmol·L^-1 intervention for 48 h as IR model+FM groups. RT-qPCR was used to detect the expression of Lyz mRNA while Western blotting was used to detect the expression of Lyz protein. RESULTS  ① On the 6^th day of small intestinal organoid culture, a ring structure with a clear luminal structure was formed and an IR mouse small intestinal organoid model was established. 3D Immunofluorescence detection showed that the established small intestinal organoids all expressed Ki-67, E-cad, Lyz and MUC-2. Compared with the normal control group, the expression of Fn mRNA in the IR model group was significantly increased (P<0.05) while the expressions of GLP-1 and PYY mRNA were significantly decreased (P<0.05). Compared with the normal control group, the expression of Fn protein in the IR model group was significantly decreased (P<0.05) while the expressions of GLP-1 and PYY protein were significantly increased (P<0.05). ELISA results showed that compared with the normal control group, the secretion levels of Lyz in the IR model group were significantly decreased (P<0.01). ② RT-qPCR results showed that compared with the normal control group, the expression of Lyz mRNA in the IR model group was significantly decreased (P<0.01). Compared with the IR model group, the expression of Lyz mRNA in the IR model+FM 50 and 100 μmol·L^-1 groups was significantly increased (P<0.05, P<0.01). Western blotting results showed that compared with the normal control group, the expression of Lyz protein in the IR model group was significantly decreased (P<0.01). Compared with the IR model group, the expression  of Lyz protein in the IR model+FM 50 and 100 μmol·L^-1 groups was significantly increased (P<0.05, P<0.01). CONCLUSION  The constructed IR mouse small intestinal organoid model provides a more complete in vitro research model for exploring the pathophysiological mechanism by which drug interventions help repair the intestinal mucosal barrier. FM may maintain the intestinal mucosal barrier by reversing the decrease in Lyz expression levels in IR mice, thereby improving IR.

Activity-based protein profiling (ABPP) and affinity-based protein profiling (AfBPP) are reliable chemical proteomics techniques that exhibit significant advantages in identifying the direct acting targets of small molecule drugs and toxicants, which can help researchers understand the pharmacological and toxicological mechanisms of active small molecules. This paper introduces ABPP and AfBPP technologies and summarizes the applications of this technique in drug off-target effect and target identification of toxicants in recent years, such as drug off-target effect of crenolanib, BIA 10-2474, orlistat and target identification of VX, fenitrothion, acrolein. It is hoped that this review will give readers a better idea of ABPP/AfBPP and offer a line of thinking for researchers in the fields of pharmacology, toxicology and chemical biology.

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.

Circular RNA (circRNA) is an emerging class of endogenous non-coding RNA, which is widely expressed in the brain and plays an important role in a variety of biological processes. Research has shown that circRNA plays a key role in physiological and pathological processes of the brain, such as neurodevelopment, synaptic plasticity and neurodegenerative diseases through a variety of mechanisms such as adsorption of microRNA, binding to proteins and translation of peptides. In the field of drug addiction, the expression of circRNA is significantly changed in animal models and brains of addicts, and the regulation involves neural adaptation in brain regions that form the reward circuit such as the nucleus accumbens and prefrontal cortex. Additionally, addiction-related circRNAs are closely associated with neurotransmitter systems, signaling pathways, and neuroinflammatory responses, and they influence the formation and maintenance of drug addiction by modulating gene expression networks related to drug addiction. Here, the biogenesis and regulatory mechanism of circRNA as well as its important role in brain function and drug addiction are reviewed in order to provide a new perspective for explorations of the pathological mechanism of drug addiction.

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. 

Exposure to ionizing radiation intervenes in genomic stability and gene expression, resulting in the disruption of normal metabolic processes in cells and organs by causing complex biological responses. Altered genomic variations, gene expression and metabolite concentrations in blood or tissue samples reflect systemic radiation damage. With the application of new techniques and extensive study on the mechanisms for ionizing radiation damage, related indicators such as chromosomal variation, gene expression, lipid and metabolism are being recognized and promise to be the markers for early diagnosis and prognosis of radiation exposure. Therefore, this article reviews recent progress in and potential applications of biomarkers related to ionizing radiation injury.

Ebola viruses belong to the Filoviridae family, which is highly contagious and likely to cause a variety of symptoms, including severe haemorrhagic fever in humans and primates, with a case fatality rate of up to 90%. Niemann-Pick C1 (NPC1) protein is an important receptor expressed in the endosomal membrane of host cells during Ebola virus infection, and its interaction with the glycoprotein (GP) cleaved by cathepsin of Ebola virus is a key link in the viral infection host, mediating the fusion of the viral envelope and endosomal membrane before releasing the viral genome to the host cell. Recent years have seen some small molecule inhibitors and monoclonal antibody gene therapy drugs developed by using NPC1 protein as a target of broad-spectrum anti-filovirus drugs. This article introduces the structure of NPC1 and its role in Ebola virus infection, and summarizes small molecule inhibitors, monoclonal antibody drugs and gene therapy drugs targeting NPC1.

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 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.

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.

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.

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 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. 

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 investigate the effect and mechanism of acute exposure to sodium cyanide (NaCN) on brain nerve damage induced by closed hypoxia in mice. METHODS  ① Mice were randomly divided into hypoxia+NaCN 0 (hypoxia control group), 2.56, 3.8, and 5.1 mg·kg^-1 groups. After ip administration of different concentrations of NaCN, the mice were immediately placed into a closed hypoxic tank and the hypoxia survival time was observed. ② Mice were divided into normal control, NaCN 3.8 mg·kg^-1, hypoxia (30 and 60 min) and NaCN 3.8 mg·kg^-1+hypoxia (30 and 60 min) groups. After grouping, the pH, oxygen saturation (sO₂), oxygen tension (pO₂) and carbon dioxide partial pressure (pCO₂) of arterial blood of mice were detected using an arterial blood gas analyzer. The cortical cerebral blood flow of mice was detected using a laser speckle imager. The dry and wet brain tissue were weighed separately, and the brain moisture content was calculated. The kit was used to detect the activity of total superoxide dismutase (T-SOD) and the content of malondialdehyde (MDA) in the hippocampus. TUNEL staining was used to detect the apoptosis rate of cells in the hippocampus. HE staining was used to detect pathological changes in the hippocampus. RESULTS  ① Compared with the hypoxic control group, the survival time of mice in the hypoxic+NaCN groups was significantly prolonged (P<0.01). ② Compared with the normal control group, the hypoxia 30 min group showed upregulation of arterial blood pCO₂ (P<0.05), downregulation of pO₂ (P<0.05). The hypoxia 60 min group showed upregulation of arterial blood pCO₂ (P<0.05) and downregulation of cortical cerebral blood flow (P<0.05). In the NaCN 3.8 mg·kg_-1 group, arterial blood pO₂ and sO₂ were significantly downregulated (P<0.05), so was cortical cerebral blood flow (P<0.01), but MDA content and T-SOD activity were significantly upregulated (P<0.01), and the brain moisture content was increased (P<0.01). Compared with the hypoxia 30 min group, sO₂ and pO₂ of arterial blood in the NaCN+hypoxia 30 min group were significantly upregulated (P<0.05), while pCO₂ was significantly downregulated (P<0.05). Compared with the hypoxia group at corresponding time points, the NaCN+hypoxia 30 or 60 min groups showed significant downregulation of cerebral blood flow (P<0.01), significant upregulation of MDA content and T-SOD activity (P<0.01), and significant upregulation of brain moisture content (P<0.01). HE staining results showed that the NaCN 3.8 mg·kg^-1 group and the NaCN+hypoxia group (30 or 60 min) showed significant cell swelling and vacuolization in cells in the hippocampal tissue, a decrease in the number of neurons, nuclear pyknosis and deep staining. TUNEL fluorescence results showed that the NaCN 3.8 mg·kg^-1 group significantly increased the apoptosis rate of the mouse hippocampus compared with the normal control group (P<0.05). The NaCN+
hypoxia 30 and 60 min groups significantly increased the apoptosis rate of the mouse hippocampus compared with the hypoxia group at corresponding time points (P<0.05). CONCLUSION  NaCN can exacerbate hypoxia induced decrease in cerebral blood flow, oxidative stress in brain tissue, and neuronal apoptosis in mice, thereby reducing oxygen consumption in closed hypoxic tanks and prolonging their survival time. The mechanism is related to reduced utility of cell oxygen, delaying CO₂ accumulation  and increasing free oxygen in vivo.

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.