Myocardial infarction is a severe and persistent ischemic myocardial necrosis caused by acute occlusion of the coronary artery. Traditional drugs have poor targeting for the treatment of myocardial infarction, resulting in insufficient dosages of drugs that can reach the injured area and shorter drug residence time. In recent years, research has shown that nanodrugs have achieved selective targeted delivery to diseased tissues. In the targeted treatment of myocardial infarction, nanodrugs utilize the unique physicochemical properties of nanocarriers to carry inflammatory factor receptors, inhibitors of inflammatory signaling pathways, antioxidants, therapeutic genes, specific antibodies and growth factors to the myocardial tissue to release drugs. Nanodrugs counteract inflammatory damage mediated by monocytes, macrophages, and neutrophils by neutralizing inflammatory factors, inhibiting inflammatory signaling pathways and inflammatory gene expression, and regulating macrophage phenotype. Antioxidant irritability injury can be prevented by eliminating peroxide with antioxidant. The regeneration and repair of myocardial tissue can be promoted via growth factors. Nanodrugs can repair the cardiac electric conduction function and promote the proliferation and differentiation of cardiac mesenchymal stem cells through hydrogel. Research has found that nanodrugs can protect ischemic myocardial tissue and improve cardiac function. This article provides a review of the research progress in the applications of nanodrugs in the treatment of myocardial infarction.

Age-related macular degeneration (AMD) is a degeneration of the macular area of the retina caused by a combination of factors, such as genetics, environments, eating habits and aging. This disease can be divided into the dry and wet type. The pathogenesis of dry AMD is unknown, but has been proven to be highly correlated with oxidative stress, inflammatory response, and lipid metabolism disorders. So far, there is no ideal dry AMD model. This review classifies the current research or existing dry AMD animal models according to the modeling mechanism, introduces the modeling methods, index changes, cycles of modeling, changes of specificity, modeling mechanism and applicability of animal models, comments on the strengths and weaknesses of various animal models, and helps to select the right animal models in order to facilitate future in-depth research on AMD.

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.

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.

OBJECTIVE  To evaluate the effect of hypidone hydrochloride (code: YL-0919) on depression- and anxiety-like behaviors of lipopolysaccharide (LPS)-treated mice, and on the transcription levels of inflammatory factors in LPS-treated BV2 cells. METHODS  ① Male C57BL/6J mice were divided into the normal control group, model group, model+YL-0919 group and model+YL-0919+BD1047 (a Sigma-1 receptor antagonist) group. On the first and sixth days of the experiment, mice were given LPS (0.5 mg·kg-1, ip). From the second to the sixth days of the experiment, YL-0919 (2.5 mg·kg-1, twice a day, ig) was administered alone or co-administered with BD1047 (4 mg·kg-1, once per day, ip). The spontaneous activity and anxiety-like behavior were evaluated via the open field test, and the depression-like behavior was tested via the forced swimming test in mice. Western blotting was used to detect the expressions of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and inflammatory factor interleukin-1β (IL-1β) in the prefrontal cortex of mice. ② BV2 cells were divided into the cell control group, LPS group (LPS 1 mg·L-1 incubation for 6 h), LPS+YL-0919 group (one hour of pre-administration of YL-0919 2 μmol·L-1, followed by six hours of LPS 1 mg·L-1 incubation) and LPS+YL-0919+NE-100 (a Sigma-1 receptor antagonist) group (one hour of pre-administration of YL-0919 2 μmol·L-1 and NE-100 10 μmol·L-1, followed by six hours of LPS 1 mg·L-1 incubation). Real-time quantitative PCR (RT-qPCR) was used to detect the effect of YL-0919 pretreatment on mRNA levels of inflammatory factors IL-6 and IL-1β of LPS-  treated (LPS 1 mg·L-1, 6 h) BV2 cells. RESULTS  ① Compared with the normal control group, there was no significant change in spontaneous activity in the LPS model group, but the time spent in the center zone and entries into the center were significantly reduced (P<0.05, P<0.01) in the open field test, while immobility time was significantly prolonged (P<0.01) in the forced swimming test. The expressions of NLRP3 and IL-1β in the prefrontal cortex were significantly up-regulated (P<0.05) following LPS treatment. Compared with the LPS model group, the above changes were significantly reversed in the model+YL-0919 group (P<0.05, P<0.01). The anti-depressant/anxiety behavioral effect of YL-0919 could be blocked by BD1047 (P<0.05, P<0.01). ②  Compared with the cell control group, the mRNA levels of IL-1β and IL-6 were up-regulated in the LPS model group (P<0.01). Compared with the model group, pretreatment with YL-0919 inhibited the increased mRNA levels of IL-1β and IL-6 by induced by LPS in BV2 cells (P<0.01), which was also blocked by the Sigma-1 receptor antagonist NE-100 (P<0.05). CONCLUSION  YL-0919 can counteract the anxiety-like/depression-like behavioral effects of LPS-treated mice and inhibit the increased transcription levels of inflammatory factors induced by LPS in BV2 cells, which might be mediated by the Sigma-1 receptor.

As a multifunctional temporary organ that connects the mother and  fetus, the placenta develops during pregnancy and keeps changing its structure and function during the process. As a place for material exchange, the placenta can also help ensure the healthy development of the fetus via its barrier effect against pathogens and exogenous compounds. Due to the physiological differences between species and human ethical constraints, there are still many limitations to current research. The construction of in vitro placental barrier models has brought more detailed information on the metabolism of complications and comorbidities during pregnancy, and the transport of drugs and toxicants across the placental barrier. This article evaluates the placental perfusion models, trophoblast cell lines and tissue culture models, organoid models, and placental microfluidic microarrays, and summarizes their applications in pharmacological and toxicological studies. The development of these models is of clinical significance for the assessment of drug efficacy during pregnancy, intrauterine treatment of the fetus, and perinatal development of new drugs.

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.

OBJECTIVE  To analyze the expression levels of solute carrier family 7 member 11 (SLC7A11) in neuroblastoma (NB) cell lines and in clinical samples with or without MYCN amplification and its correlation with the prognosis of children, and to investigate the role of SLC7A11 in the proliferation of MYCN-amplified high-risk NB. METHODS The correlation between the expression level of SLC7A11 mRNA and MYCN amplification status and the prognosis of NB children in NB clinical samples was analyzed using the GEO clinical database GSE49710 dataset, the TARGET database and  the R2 database GSE45547 dataset. The mRNA expression levels of SLC7A11 in different NB cell lines were detected by quantitative real-time PCR (RT-qPCR). In MYCN-amplified NB cell line SK-N-BE(2), the SLC7A11 mRNA level was targeted by two small interfering RNA (siRNAs) with different sequences. The knockdown efficiency of SLC7A11 mRNA and SLC7A11 protein and the MYCN mRNA and N-MYC protein levels were detected by RT-qPCR and Western blotting. NB cell proliferation was observed via crystal violet staining and real-time cell analysis (RTCA). The expression of the proliferation marker Ki-67 was detected by immunofluorescence. Stable knockdown of SLC7A11 was constructed in SK-N-BE(2) cells by short hairpin RNA (shRNA) lentiviral infection, and the stable knockdown efficiency of SLC7A11 and the mRNA and protein levels of MYCN were also detected by RT-qPCR and Western blotting. The ability of NB cells to form colonies was observed by colony-forming unit assay. RESULTS  By analyzing the datasets of NB clinical samples GSE49710 and GSE45547, it was found that the expression level of SLC7A11 mRNA in MYCN-amplified NB was significantly higher than in MYCN-non-amplified NB (P<0.05, P<0.01). The analysis of TARGET database and the R2 database found that SLC7A11 mRNA expression was negatively correlated with the survival rate of NB children (P<0.05, P<0.01). SLC7A11 mRNA level was significantly higher in MYCN-amplified NB cell lines SK-N-BE(2) and IMR32 than in MYCN-non-amplified cell lines CHLA-255 and SH-SY5Y (P<0.01). Transient knockdown of SLC7A11 resulted in slower proliferation of MYCN-amplified NB cells (P<0.01). The proportion of Ki-67 positive cells was significantly reduced in cells from the SLC7A11 knockdown group compared with the control group (P<0.05). Stable knockdown of SLC7A11 significantly inhibited the ability of colony formation of MYCN-amplified NB cells (P<0.01). Knockdown of SLC7A11 had little effect on the MYCN mRNA and N-MYC protein levels in SK-N-BE(2). CONCLUSION  SLC7A11 is highly expressed in MYCN-amplified NB cell lines and clinical samples, and its expression level is negatively correlated with the prognosis of children. Knockdown of SLC7A11 significantly inhibits the proliferation and colony formation of SK-N-BE(2) cells.

OBJECTIVE  To evaluate the neuroprotective effect of recombinant human nerve growth factor (rhNG) at the molecular level and explore the mechanism. METHODS  BV2 microglia were divided into the cell control group, lipopolysaccharide (LPS) group, and LPS+rhNGF 50 and 500 μg·L-1 groups. Except the cell control group, all the groups of BV2 cells were stimulated by LPS 1 mg·L-1 for 24 h to induce inflammatory reaction before LPS+rhNGF groups were cultured with rhNGF for another 48 h. After incubation, BV2 cells and microglial conditioned medium (MCM) were collected. The BV2 cells and MCM in each group were respectively co-cultured with mouse neuroblastoma cell Neuro-2a (N2a cells) for 12 or 24 h. The cell survival rate was detected by CCK-8 kit, the mRNA expression levels of pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and IL-1β, and anti-inflammatory cytokines IL-4 and IL-10 in BV2 cells were detected by real-time fluorescence quantitative PCR (RT-qPCR), and the concentrations of  IL-6, TNF-α, IL-4 and IL-10 in the BV2 cell culture supernatant were detected by CBA kit. The expressions of M1 phenotypic marker CD16 and M2 phenotypic marker CD206 in BV2 cells were detected using the immunofluorescence method. The apoptosis rate of N2a cells was detected with NeuN/TUNEL co-staining and Annexin V/7-AAD methods. RESULTS  Compared with the cell control group, the survival rate of BV2 cells in the LPS group decreased significantly (P<0.01), the cells became round and the cell body became larger, the mRNA levels of pro-inflammatory factors IL-1β, IL-6 and TNF-α, and anti-inflammatory factors IL-10 increased significantly (P<0.01), the mRNA level of anti-inflammatory factor IL-4 decreased (P<0.01), the protein levels of IL-6 and TNF-α increased significantly (P<0.01), but there was no significant difference in the protein level of IL-4. The percentages of CD16+  and CD206+ cells were significantly up-regulated (P<0.01), particularly the former. The survival rate of N2a cells co-cultured with BV2 cells or their MCM decreased significantly while the apoptosis rate increased significantly (P<0.05, P<0.01). Compared with the LPS group, the survival rate of BV2 cells in LPS+rhNGF 50 or 500 μg·L-1  groups increased significantly (P<0.01), the mRNA levels of IL-1β, IL-6 and TNF-α decreased significantly (P<0.05), the mRNA levels of IL-4 and IL-10 increased significantly (P<0.05, P<0.01), the protein levels of IL-6 and TNF-α decreased significantly (P<0.05, P<0.01), but the protein level of IL-4 hardly changed and the concentration of IL-10 was lower than the detection limit. The percentage of CD206+ cells increased significantly (P<0.01), and that of CD16+ cells decreased significantly (P<0.05). Compared with LPS-treated BV2 cell co-culture group or MCM-LPS-treated group, the survival rate of N2a cells co-cultured with BV2 cells treated with rhNGF 50 or 500 μg·L-1  and with MCM-rhNGF 50 or 500 μg·L-1 increased significantly while the apoptosis rate decreased significantly (P<0.05, P<0.01). CONCLUSION  rhNGF can induce the transformation of BV2 microglia into M2 phenotype and inhibit the apoptosis of N2a cells by inhibiting the inflammatory reaction of BV2 microglia.

OBJECTIVE  To study the analgesia effect and neurobiological mechanism of local administration of cannabidiol(CBD) in formalin-induced orofacial inflammatory pain. METHODS  The orofacial inflammatory pain model of mice was established by injection of 1% formalin into the right upper lip subcutaneously. ① Male C57BL/6J mice were randomly divided into the control group, model group and model+CBD (0.01, 0.03 and 0.06 mg) group. ② Male C57BL/6J mice were randomly divided into the normal control group, model group and model+CBD 0.06 mg group. Mice from ① were used to evaluate the analgesic effect of CBD (0.01, 0.03 and 0.06 mg) which was subcutaneously injected into the right upper lip. Following the conclusion of the behavioral experiment (equivalent to 90 minutes after formalin injection), 4 animals were randomly selected from the normal control group, model group and model+CBD 0.06 mg  group for immunofluorescence analysis. The immunofluorescence method was employed to assess the expression of c-Fos protein in both the spinal trigeminal caudal subnucleus (Sp5C) and anterior cingulate gyrus (ACC). The endocannabinoid probe was used in combination with optical fiber recording to detect the changes of endocannabinoid levels in central periaquaductal gray matter (PAG), basolateral amygdala (BLA) and ACC of mice from ② during the second phase of pain. RESULTS  In the formalin-induced orofacial inflammatory pain model, the total time of rubbing in model group was significantly longer than that of the control group, and subcutaneous administration of CBD (0.03, 0.06 mg) significantly decreased the total time of rubbing (P<0.01). CBD 0.06 mg significantly decreased the face-rubbing time of mice in the second pain period (P<0.01), but had no effect on the first pain period (P>0.05). The number of c-Fos positive neurons in PAG, BLA and ACC in the model group was significantly increased compared with the control group, and local administration of CBD could significantly reduce the expression level of c-Fos in the above three brain areas (P<0.01). In the second phase of pain, subcutaneous administration of CBD increased the level of endocannabinoid in BLA. CONCLUSION  In formalin-induced orofacial inflammatory pain, local administration of CBD can reduce the c-Fos expression of Sp5C and ACC, and up-regulate the function of the endocannabinoid system so as to achieve the anti-inflammatory and analgesic effects.

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.

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 investigate the effect of benzo[a] pyrene (BaP) on hepatic gluconeogenesis-related molecules and the molecular mechanism. METHODS  ① C57BL/6 mice were randomly divided into the normal control group, solvent control group, BaP 0.45, 0.9 and 1.80 mg·kg-1 groups.BaP was dissolved in corn oil for ig administration. The solvent control group was ig given corn oil 10 mL·kg-1 (vehicle) for 12 consecutive weeks. ② HepG2 cells in the logarithmic growth stage were randomly divided into the cell control group, BaP 0.1, 1.0 and 10.0 μmol·L-1 groups. The cells were inculated with the drug for 24 h. ③ HepG2 was divided into the cell control group, BaP 1 μmol·L-1, aryl hydrocarbon receptor (AhR) inhibitor CH223191 1 μmol·L-1, and BaP 1 μmol·L-1+CH223191 1 μmol·L-1 groups. The cells were inculated with the drug for 24 h. Protein levels of fibroblast growth factor 21 (FGF21) were detected in HepG2 cells of subgroups ② and ③ by cellular immunofluorescence. The mRNA expression levels of fructose-1,6-bisphosphatase 1 (FBP1), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxylase 1 (PCK1) and FGF21 were detected in mouse liver tissues and in HepG2 cells of subgroups ①, ② and ③ by real-time quantitative PCR; Western blotting was used to detect the protein expression levels of FBP1, G6Pase, PCK1 and FGF21 in mouse liver tissues and in HepG2 cells in subgroups ①, ② and ③. RESULTS  ① Compared with the vehicle control group, the protein and mRNA expressions of FBP1, G6Pase, PCK1 and FGF21 in liver tissues of C57BL/6 mice in BaP 0.90 and 1.80 mg·kg-1 groups were significantly increased (P<0.05, P<0.01). ② Compared with the cell control group, protein and mRNA expressions of FBP1, G6Pase, PCK1 and FGF21 in HepG2 cells of BaP 0.1, 1.0 and 10.0 μmol·L-1 groups were significantly increased (P<0.05,P<0.01). ③ Compared with the BaP 1 μmol·L-1 group, protein and mRNA expressions of FBP1, G6Pase, PCK1 and FGF21 in HepG2 cells in the BaP 1 μmol·L-1+AhR 1 μmol·L-1 group were significantly decreased (P<0.05, P<0.01). CONCLUSION  BaP can increase the mRNA and protein expressions of FBP1, G6Pase and PCK1, which in turn increases the level of hepatic gluconeogenesis by a mechanism that may be related to the activation of the AhR/FGF21 signalling pathway.

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.

Methamphetamine addiction is a serious medical and social issue, whose mechanisms have not been fully elucidated. Effective diagnostic and treatment methods are lacking. In recent years, there is increasing evidence that microRNA (miRNA) plays important regulatory roles in methamphetamine addiction. Non-clinical research suggests that miRNA may be involved in the occurrence and development of methamphetamine addiction via the regulation of neuroplasticity and damage to the nervous and immune systems. Clinical research suggests that miRNA have good prospects as potential biological markers for the diagnosis of methamphetamine addiction. Researchers are beginning to focus on the role of miRNA in extracellular vesicles in methamphetamine addiction, which can greatly contributes to the diagnosis of methamphetamine addiction and elucidation of the central-peripheral interaction mechanisms. This article provides a survey of the research progress in roles of miRNA in methamphetamine addiction, involving their regulatory roles, underlying mechanisms, therapeutic potential, and prospects as diagnostic biomarkers in order to advance our understanding of the neurobiological mechanisms of methamphetamine addiction and provide new perspective on releted diagnosis and treatment.

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.

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.

Epigallocatechin gallate (EGCG) is the main bioactive substance in tea polyphenols, which has good prospects of clinical application. However, the oral bioavailability of EGCG is poor in that most of it enters the intestines and is excreted through urine and feces. After intravenous administration, it is mainly distributed in the liver, lung, and small intestines. It can inhibit the activities of several liver drug metabolism enzymes and drug transporters. In clinical practice, the interactions between EGCG and related drugs deserves attention. Based on the pharmacokinetic characteristics of EGCG, this article reviews the research progress in the effects of EGCG on drug metabolism and drug interaction in the liver in hopes of providing reference for clinical applications of EGCG.

OBJECTIVE  To synthesize and evaluate N-terminal and C-terminal derivatives of CPI-1 in order to obtain light chain inhibitors with better activity and good pharmacokinetic performance. METHODS  Non-long-chain derivatives modified by different amino acids at the C-terminal of CPI-1 [CPI-1-L(1), CPI-I-GL(2), CPI-1-W(3), CPI-1-Y(4), CPI-1-R(5), CPI-1-K(6), CPI-1-E(7)] and long-chain derivatives modified by long-chain fatty acids at its C- or N-terminal [CPI-1-K(stearic)GG(8), CPI-1-LK(Stearic) GG(9), CPI-1-LGK(stearic)GG(10), C8-CPI-1(11), C12-CPI-1(12), C14-CPI-1(13), C16-CPI-1(14)] were synthesized using solid phase methods. After being deprotected, the linear peptide was folded into the cyclic target peptide in NH4HCO3 buffer. The long-chain modified CPI-1 derivatives were oxidized on resin to form disulfide bonds before being cleaved. All crude peptides were purified by reversed phase-high pressure liquid chromatography (RP-HPLC). The inhibitory activities of peptides against BoNT/A light chain LC424 (1-424) were determined by fluorescence resonance energy transfer assays, while the stability of enzymatic hydrolysis of peptides by trypsin was determined by RP-HPLC. Mice were randomly divided into the saline group, CPI-1 group and the CPI-1 derivative groups. 2×LD50 (2.5×LD50) BoNT/A was co-incubated with saline, (5 mg·kg-1) CPI-1 or CPI-1 derivatives before being intraperitoneally (ip) injected into mice. The survival rates of mice were recorded for 72 h. 2 and 6 h after ip injection of 2×LD50 BoNT/A, the mice were treated with saline, 5 mg·kg-1 CPI-1 or CPI-1 derivatives before the survival rates of mice were recorded for 72 h. RESULTS  Fourteen CPI-1 derivatives including 7 non-long-chain and 7 long-chain ones were synthesized. The relative molecular mass of all derivatives was consistent with the theoretical molecular mass by mass spectrometry. The results of inhibitory activities in vitro showed that the introduction of hydrophobic and basic amino acids [such as leucine(1), tryptophane(3), tyrosine(4) and arginine(5)] of at the C-terminal of CPI-1 resulted in a 3.5-0.8-fold increase in the inhibitory activity (IC50=0.06-0.15 μmol·L-1) compared to CPI-1 [IC50=(0.27±0.02) μmol·L-1] (P<0.01), but the introduction of acidic amino acid glutamic (7) led to a 78.6% decrease in the potency [IC50=(1.26±0.28) μmol·L-1](P<0.01). After the introduction of fatty acid at the N- or C-terminal of CPI-1, the potency (IC50>1.36 μmol·L-1) was decreased by at least 80% (P<0.01). The anti-enzymatic activity tests showed that after the introduction of fatty acid at the N-or C-terminal of CPI-1, the trypsin degradation rate of the CPI-1-K (stearic) GG and C12-CPI-1 was significantly decreased at 8 h (P<0.05, P<0.01). Detoxification experiments showed that after co-injection of 2×LD50 BoNT/A with 5 mg·kg-1 inhibitor  the survival rate of mice in CPI-1-L, CPI-1-W, CPI-1-Y, CPI-1-R, and CPI-1-K groups was significantly increased in 72 h compared to the blank control group (P<0.05), but not in the CPI-1 group and the survival rate of mice was more significantly increased  than in the CPI-1 group(P<0.05). After co-injection (ip) of 2.5×LD50 BoNT/A with 5 mg·kg-1 inhibitor, the survival rate of mice in CPI-1, CPI-1-LK (stearic) GG, C12-CPI-1, C14-CPI-1 and C16-CPI-1 groups was also more significantly increased than in the blank control group in 72 h (P<0.05) rather than in CPI-1-K (stearic)GG, CPI-1-LGK (stearic) GG and C8-CPI-1 groups. The survival rate of mice in other groups was not siginificantly different from that of the CPI-1 group. 2 and 6 h after the injection (ip) of BoNT/A (2×LD50) and treatment with the 5 mg·kg-1 inhibitor, the survival rate of mice in the C12-CPI-1 group was more significantly increased than in the blank control group in 72 h (P<0.05), but not in other groups. The survival rate of mice in other groups was not significantly different from that of the CPI-1 group. CONCLUSION  The introduction of hydrophobic amino acids and basic amino acids at the C-terminual of CPI-1 results in an increase in potency. The addition of fatty acid at C-or N-terminal enhances the anti-enzymatic activity of CPI-1. The CPI-1 derivative modified by dodecanoic acid displays apparent detoxification activity to BoNT/A.

OBJECTIVE  To study the cardiac and neurotoxic effects of fentanyl exposure on zebrafish larvae and the expression of related genes. METHODS  Well-developed embryos within 2 h of fertilization (2 hpf) were randomly divided into the 5, 10 and 20 mg·L-1 fentanyl-stained and normal control groups. The stereopicroscope was used to observe the deformational phenotype of embryoic development of the zebrafish. The deformational rate of zebrafish embryos or larvae was calculated at 24, 48, 72, 96 and 120 hpf, and the survival rate of juveniles was calculated at 120 hpf. The zebrafish microvisual behavior analysis system was used to analyze the heart rate (72, 96 and 120 hpf), pericardial area and SV-BA (venous sinus and arterial ball) distance (120 hpf) of the larvae. At 120 hpf, the HE  staining method was used to analyze structures of atria, ventricles and cardiomyocytes of zebrafish larvae, and Image J software was used to analyze the area and density of new neurons; the mRNA expressions of the heart and neurodevelopmental genes NK2 homeobox 5 (nkx2.5), neuronal PAS domain protein 4 (npas4a), natriuretic peptide B (nppb) and SRY-box transcription factor 9b (sox9b) were detected by fluorescence quantitative PCR. RESULTS  Fentanyl induced developmental malformations such as incomplete hatching, spinal curvature, tail deformity and pericardial edema in zebrafish. Compared with the normal control group, the deformability rates of larvae at 72 hpf in the 10, 20 mg·L-1 fentanyl group were significantly increased (both P<0.01), but there was no significant change in survival rates of larvae at 120 hpf in fentanyl all concentration groups. The results of heart rate analysis showed that the heart rate of zebrafish larvae at 72 hpf in the 20 mg·L-1 fentanyl group decreased significantly (P<0.01) compared with the control group, and the heart rate of larvae in each concentration group decreased significantly at 120 hpf (P<0.01). At 120 hpf, compared with the normal control group, the pericardial area and SV-BA distance of zebrafish larvae in the 20 mg·L-1 fentanyl group were significantly increased (P<0.01); the atrial ventricles of zebrafish larvae in the  20 mg·L-1 fentanyl group were significantly enlarged, and cardiomyocyte layers were small in number and thin; the area and density of neonatal neurons in the head of zebrafish juveniles in the 20 mg·L-1 fentanyl group were significantly decreased (P<0.05, P<0.01); the mRNA expressions of nkx2.5, npas4a and sox9b in the 20 mg·L-1 fentanyl group were down-regulated (P<0.01), while the mRNA expressions of nppb were up-regulated (P<0.01). CONCLUSION  Exposure to fentanyl during the development of zebrafish embryos can result in cardiac malformation and cardiac slowdown, as well as neonatal neuronal toxicity, which may be associated with changes in the expressions of genes related to cardiac and neural development.

OBJECTIVE  To investigate response dynamics of dopamine neurons in the ventral tegmental area (VTA) and dopamine neurotransmitters in the nucleus accumbens (NAc) to methamphetamine, cocaine and morphine. METHODS  The dopamine neurotransmitter fluorescent probe was expressed in the NAc of mice (n=28) and the calcium indicator protein carrying the calcium promoter of tyrosine hydroxylase was expressed in the VTA of mice (n=28). Then, the mice were equally divided the saline, methamphetamine (1 mg·kg-1, ip), cocaine (10 mg·kg-1, ip), and morphine (10 mg·kg-1, sc) into four groups, respectively. Using the fiber-optic recording system, the potency and kinetics of dopamine neurotransmitters and dopamine neurons were recorded and analyzed. The paraments included the area under curve (AUC), the peak and the latency time to reach the peak for dopamine neurotransmitter fluorescent probe in the NAc, besides, the event amplitude and duration were also analyzed for Ca2+ signals in the VTA. RESULTS  Both injection of methamphetamine andcocaine induced a rapid and sustained decrease of Ca2+ signals in dopamine neurons,whereas administration of morphine displayed a slow and sustained increaseof Ca2+ signals in dopamine neurons, and the extremes of the change curves in dopaminergic neuronexcitability after methamphetamine administration and the area under the curve were significantly lowerthan those of cocaine (P<0.05); the duration of action of methamphetamine on dopamine neuronsin the VTA was significantly longer than that of cocaine (P<0.05), whereas morphine showed the significantly longer duration of on dopamine neurons in the VTA compared with both methamphetamine and cocaine (P<0.01, P<0.05). But, all of above drugs significantly increased dopamine neurotransmitters in the NAc compared to saline treatment. However, they still showed different kinetic characteristics. The AUC induced by methamphetamine was significantly higher than cocaine and morphine (P<0.01, P<0.05); moreover, the time to the peak induced by methamphetamine or cocaine was significantly earlier than morphine treatment (P<0.01). CONCLUSION  These results illustrate different addictive drugs showed significantly distinct response dynamic for dopamine neurons in the VTA and dopamine in the NAc. This suggests that neural mechanisms underline the addiction induced by kinds of drugs may be marked difference.

OBJECTIVE To explore the role of chitinase-3-like protein 1(CHI3L1) in regulating the adipogenic and osteogenic differentiation abilities of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). METHODS hUC-MSCs were thawed and cultured. MSC surface markers CD14, CD34, CD45, CD73, CD90, and CD105 were detected by flow cytometry. The abilities of hUC-MSCs to differentiate into adipocytes and osteocytes were proved by oil red O staining and alkaline phosphatase (ALP) staining, respectively. The expressions of adipogenic and osteogenic key transcription factors were examined by real-time quantitative PCR (RT-qPCR). The lentiviral vector was used to construct hUC-MSCs with stable knockdown of CHI3L1 gene (shCHI3L1-MSCs) and the control group hUC-MSCs (shNC-MSCs). The effect of CHI3L1 on adipogenic differentiation of hUC-MSCs was investigated by in vitro induction experiments. The differences of lipid droplet numbers and ALP activity between shCHI3L1-MSC and shNC-MSC groups were detected by oil red O staining and ALP staining, respectively, and the key transcription factors of adipogenic and osteogenic differentiation were assessed by RT-qPCR. RESULTS The cultured hUC-MSCs showed high expressions of CD73, CD90 and CD105, and low or no expressions of CD14, CD34 or CD45. CHI3L1 knockdown did not affect the expressions of hUC-MSC surface markers. After osteogenic differentiation, the activity of ALP and the mRNA expression of osteogenic differentiation markers ALP and osteopontin (OPN) increased significantly in the induced group compared with the cell control group (P<0.01). After adipogenic differentiation, oil red O staining showed a large number of red lipid droplets in the induced group. RT-qPCR results showed that adipsin (ADI) and peroxisome proliferator activated receptor-γ (PPAR-γ) mRNA expressions were significantly increased in the induced group (P<0.01), suggesting that the hUC-MSCs whose CHI3L1 gene was knocked down remained capable of osteogenic and adipogenic differentiation. Meanwhile, compared with the shNC-MSC group, the number of lipid droplets and the mRNA expression levels of ADI and PPAR-γ were significantly increased (P<0.01), but ALP activity and the mRNA expression levels of OPN and ALP were significantly decreased (P<0.01) in the shCHI3L1-MSC group, indicating that hUC-MSCs with knockdown of CHI3L1 enhanced the adipogenic differentiation ability but weakened the osteogenic differentiation abibity. CONCLUSION CHI3L1 is involved in the regulation of adipogenic and osteogenic differentiation of hUC-MSCs in vitro.

OBJECTIVE  To explore the effect of neurotransmitter γ-aminobutyric acid (GABA) on differentiation and antibody production of B cells, and to recommend a new potential strategy to enhance humoral immune response after vaccination. METHODS Naïve B cells, germinal center (GC) B cells and splenic plasma cells (SPPCs) were sorted from the spleen of mice on the 7th day after immunization with sheep red blood cells (SRBCs), and RT-qPCR was used to detect the mRNA expressions of GABA A receptor, subunit α1 (Gabra1), Gabra2, GABA B receptor 1(Gabbr1), Gabbr2, solute carrier family 6 member 1 (Slc6a1), Slc6a11, Slc6a12 and Slc6a13. A plasma cell differentiation system in vitro was constructed. Purified naïve B cells were co-cultured with NB-21 feeder cells in the presence of mouse interleukin-4, and the production of CD138+ SPPCs was analyzed by flow cytometry with or without the treatment with GABA 0.2, 1.0 and 2.0 mmol·L-1. A classical antigen-immunized mouse model was prepared by ip injecting 4-hydroxy-3-nitrophenylacetyl conjugated to keyhole limpet hemocyanin (NP-KLH) 100 μg per C57BL/6 wild-type mouse mixed with 0.1 mL alum. The normal control group was injected with 0.1 mL double distillated water mixed with 0.1 mL alum. The NP-KLH immunized mice were ip injected with PBS or GABA 20 mg·kg-1, once a day, for 12 d after immunization before the percentages of GC B cells, SPPCs and IgG1+ SPPCs were tested by flow cytometry. The titer of antigen-specific IgG in serum was measured by ELISA on the 13rd day, and the abundance of antigen-specific bone-marrow plasma cells was tested by flow cytometry on the 27th day after immunization. RESULTS  Gabbr1 mRNA was highly expressed in naïve B cells, GC B cells and SPPCs in SRBC immunized mice. In the in vitro plasma cell differentiation system, naïve B cells could differentiate into CD138+ SPPCs constantly. Compared with the cell control group, the percentage of SPPCs was increased after GABA 1.0 and 2.0 mmol·L-1 treatment (P<0.01). Compared with the normal control group, the percentage of GC B cells in the NP-KLH immunized model+PBS group was increased (P<0.01), the percentages of SPPCs and IgG1+ SPPCs were significantly increased (P<0.05, P<0.01), the percentage of NP+IgM- bone-marrow plasma cells was increased (P<0.01), and the antigen-specific antibody response was enhanced (P<0.01). Compared with the NP-KLH immunized model+PBS group, the percentages of SPPCs (P<0.01) and IgG1+ SPPCs (P<0.05) in the NP-KLH immunized model+GABA group were increased, so was the percentage of NP+IgM- bone-marrow plasma cells (P<0.05), and the antigen-specific antibody response was enhanced (P<0.01). CONCLUSION  GABA can significantly promote the formation of plasma cells and the production of high affinity antibodies, which suggests that GABA may play an important role in regulating the differentiation of B cells.

Hepatocellular carcinoma (HCC) is a malignant tumor of the liver and a metabolic disease. As a macromolecular complex, lipoprotein participates in the process of lipid metabolism in vivo and regulates lipid homeostasis. When hepatocytes are damaged, lipoprotein metabolism is disturbed, lipid homeostasis becomes imbalanced, and excess lipid accumulates, accelerating hepatocellular carcinoma, while overexpression of lipoprotein receptors on the surface of already cancerous hepatocytes activates proliferation-related signaling pathways, accelerating the progression of HCC. The role of lipoproteins and their receptors in the development of HCC remains a hot spot, and the related drugs are mainly categorized into proprotein convertase subtilisin/kexin type 9 inhibitors and statins. This paper  reviews the role of lipoproteins in HCC progression in terms of HCC development and the current status of HCC therapeutic drugs, and provides data for targeting lipoproteins in the treatment of HCC.

Estradiol (E2) is the most important estrogen in humans, with various physiological and pharmacological activities. As a common hormone replacement medicine, E2 is applied to relieve menopausal symptoms in postmenopausal females. However, long-term and excessive use of E2 may increase breast cancer risk of females. Cytochrome P450 enzyme superfamily 1 (CYP1) is the main phase Ⅰ metabolizing enzyme involved in E2 metabolism, among which the increase in 4-hydroxylated metabolites catalyzed by the CYP1B1 subtype may promote the development of breast cancer, while the increase in 2-hydroxylated metabolites catalyzed by the CYP1A1 subtype may alleviate the development of breast cancer. This article reviews the relationships between the bioactivation of E2 mediated by CYP1 and the development of breast cancer in women so as to provide reference for the prevention of or protection against E2-triggered breast cancer.

OBJECTIVE  To investigate the correlation between neuroinflammation and exercise fatigue and to study the possible mechanism from the perspective of brain energy metabolism. METHODS  Male C57BL/6J mice were used in this study. ① To investigate the effect of drug administration on exercise fatigue of mice, the mice were divided into the vehicle group, lipopolysaccharides (LPS, 2.5 μg per mouse) group or minocycline (12 μg per mouse) or AZD3965 (50 nmol per mouse) or 4-CIN (40 nmol per mouse) groups. The rotate bar test was used to measure the time mice spent on the rotating bar 12 h after the mice were intracerebroventricularly injected (icv) with LPS, or 30 min after icv with minocycline, AZD3965 and 4-CIN, respectively. ② To investigate the effect of reducing neuroinflammation on exercise fatigue, the mice were divided into vehicle and 3 different doses of minocycline (3, 6 and 12 μg per mouse) groups, or vehicle and a single dose of minocycline (12 μg per mouse) groups. The running duration and distance covered by mice on the treadmill were determined with the treadmill test. The load swimming test was conducted 30 min after the mice were icv with minocycline. ③ To investigate the effect of LPS on exercise fatigue and the reversal effect of minocycline on LPS′s action, the mice were 
divided into the vehicle, LPS (2.5 μg per mouse) and LPS+minocycline (2.5 μg+12 μg per mouse) groups. The mice of the LPS+minocycline group were icv with minocycline 12 h after icv LPS. The treadmill test and load swimming test were used to assess the endurance exercise fatigue of the mice or the motor cortex was collected after the mice were sacrificed 12 h after they were icv with LPS or 30 min after icv with minocycline. The mRNA expression levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α), and monocarboxylate transporters-1 (MCT-1), MCT-2 and MCT-4 in the motor cortex were detected with reverse transcription and real-time quantitative PCR. ④ To investigate the effect of AZD3965 and 4-CIN on exercise fatigue, the mice were divided into the vehicle and 3 different doses of AZD3965 (12.5, 25 and 50 nmol per mouse) or 4-CIN (10, 20, 40 nmol per mouse) groups, or vehicle and a single dose of AZD3965 (50 nmol per mouse) or 4-CIN (40 nmol per mouse) groups. The treadmill test and forced swimming test were conducted 30 min after the mice were icv with AZD3965 or 4-CIN. Each group contained 12 mice. RESULTS  ① Compared with the vehicle group, LPS, minocycline, AZD3965 and 4-CIN had no obvious effect on the time mice spent on the rotating bar, suggesting that none of them affected the motor coordination of mice. ② Compared with the vehicle group, minocycline (12 μg per mouse) significantly increased the endurance time and distance on the treadmill and the endurance swimming time of mice (P<0.01). ③ Compared with the vehicle group, LPS (2.5 μg per mouse) significantly decreased the endurance time and distance on the treadmill and the endurance swimming time of mice (P<0.01), increased the mRNA expression levels of IL-1β, IL-6 and TNF-α (P<0.01), and decreased the mRNA expression levels in MCT-1, MCT-2 and MCT-4 (P<0.05) in the motor cortex of mice. Compared with the LPS group, minocycline (12 μg per mouse) significantly blocked the effect of LPS on exercise fatigue of mice (P<0.05), and reversed the changes of mRNA expression levels of IL-1β and TNF-α (P<0.05), and MCT-1, MCT-2 and MCT-4 (P<0.01) in the motor cortex of mice. ④ Compared with the vehicle group, AZD3965 (50 nmol per mouse) and 4-CIN (40 nmol per mouse) significantly decreased the endurance time and distance on the treadmill and the endurance swimming time (P<0.05). CONCLUSION  Neuroinflammation plays an important role in exercise fatigue, and the underlying mechanism might be associated with the astrocyte-neuron lactate shuttle that affects the neuron energy metabolic substrate supplement.

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.

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.

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.

In recent years, the incidence of chemical liver injury has remained high as one of the key causes of liver failure and death. However, the exact mechanisms are not fully understood and treatment options are quite limited. Recent studies have shown that NOD-like receptor protein 3 (NLRP3), as the most widely studied inflammasome, induces inflammatory response and plays a key role in the development of chemical liver injury. Therefore, targeting this inflammasome may be a promising pharmacological approach to chemical liver injury. Natural products are capable of clearing free radicals and inhibiting lipid peroxidation by targeting the NLRP3 inflammasome, thereby inhibiting the expressions of inflammatory factors and preventing pyroptosis. These products can help prevent and treat liver injury induced by a variety of chemical substances.

OBJECTIVE  To explore the microRNA (miRNA) regulation mechanism of up-regulated forkhead box protein A1(FOXA1) expression in benzo[a]pyrene (BaP) malignant transformed human bronchial epithelial cell T-16HBE-c1(THBEc1) and screen and identify the potential target genes of FOXA1. METHODS  TargetScan, ENCORI database and next-generation sequencing (NGS) results of miRNA between THBEc1 and non-transformed cell 16 HBE were used to comprehensively predict the miRNAs targeting FOXA1, and the predicted miRNAs were further screened via real-time quantitative PCR (RT-qPCR). The miRNA mimics were transfected into THBEc1 cells and the expression level of FOXA1 protein was determined by Western blotting to identify the miRNA targeting FOXA1. The potential target genes of FOXA1 were predicted by hTF, JASPAR and ENCODE databases combined with the NGS results of mRNA between FOXA1 knockout cell THBEc1-ΔFOXA1-c34 and control cell THBEc1-ctrl. RT-qPCR was used to further screen the predicted target genes [transmembrane protein 98 (TMEM98), IKAROS family zinc finger 2 (IKZF2), isocitrate dehydrogenase 1 (IDH1), TNF receptor associated factor 5 (TRAF5), bone morphogenetic protein receptor type 2 (BMPR2), heat shock protein B1 (HSPB1), RUNX family transcription factor 2 (RUNX2), golgin A7 family member B (GOLGA7B) and RAR related orphan receptor A (RORA)]. A cell model THBEc1-ΔFOXA1-c34-oe with stable expressions of FOXA1 was constructed via transfection of overexpression plasmid, that is, FOXA1 function was complemented, and the potential target genes of FOXA1 were identified by RT-qPCR and Western blotting. RESULTS  TargetScan and ENCORI databases predicted that 213 and 145 miRNAs might target FOXA1, respectively. A total of 351 miRNAs were found to be down-regulated in THBEc1 cells (fold change<0.5, and false discovery rate<0.05). Among them, hsa-miR-584-5p (miR-584-5p), hsa-miR-142-5p (miR-142-5p) and hsa-miR-211-5p (miR-211-5p) were predicted to target FOXA1 in the above two databases. Real-time quantitative PCR (RT-qPCR) results confirmed that the expression levels of miR-584-5p, miR-142-5p and miR-211-5p in THBEc1 were significantly lower than those in 16HBE (P<0.01). Transfection of miR-584-5p mimics or miR-211-5p mimics significantly down-regulated the expression of FOXA1 protein in THBEc1 cells (P<0.01), while transfection of miR-142-5p mimics had no significant effect on the expression of FOXA1 protein in THBEc1 cells. Twenty differentially expressed genes (fold change<0.5 or>2, and false discovery rate<0.05) between THBEc1-ΔFOXA1-c34 and THBEc1-ctrl cells were predicted as potential target genes of FOXA1 by hTF, JASPAR and ENCODE databases. RT-qPCR results showed that after knocking out FOXA1 in THBEc1, the mRNA expression levels of TMEM98, IKZF2, IDH1, TRAF5, BMPR2, HSPB1 and RUNX were down-regulated (P<0.01, P<0.05), while the mRNA 
expression levels of GOLGA7B and RORA were significantly up-regulated (P<0.01, P<0.05). After restoration of FOXA1 expression in THBEc1-ΔFOXA1-c34, the mRNA expression levels of IDH1 and HSPB1 were significantly up-regulated (P<0.01), but the mRNA expression levels of the remaining 7 genes remained unchanged. Western blotting results showed that the expression levels of IDH1 and HSPB1 in THBEc1-ΔFOXA1-c34 cells were significantly lower than those in THBEc1-ctrl cells (P<0.01). The expression levels of IDH1 and HSPB1 in THBEc1-ΔFOXA1-c34-oe cells were significantly up-regulated compared with THBEc1-ΔFOXA1-c34-ctrl cells (P<0.01). CONCLUSION  The miR-584-5p/miR-211-5p/FOXA1 axis is involved in the transcriptional regulation of IDH1 and HSPB1 in BaP transformed cells THBEc1.

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.

OBJECTIVE  To explore the effect and mechanism of liraglutide on CXC-chemokine ligand 16 (CXCL16) -induced lipid deposition in human podocytes. METHODS  ① Human podocytes were stimulated with high glucose (HG) 40 mmol·L-1 for 24 h and palmitic acid (PA) 250 μmol·L-1 for 6 h. Western blotting was used to detect the protein expression level of CXCL16 in podocytes. ② CXCL16 was used to stimulate podocytes at a concentration of 100 μg·L-1 for 0, 6, 12, and 24 h. Western blotting was used to detect the protein expression level of nephrin in podocytes. ③ Podocytes were divided into the cell control group, CXCL16 10 μg·L-1, CXCL16+liraglutide 10, 50, 100 nmol·L-1 group and CXCL16+simvastatin 100 nmol·L-1 group. After two hours of drug incubation, CXCL16 100 μg·L-1 was added and  cultured for 24 h. Oil Red O staining was used to detect the lipid droplet area in podocytes. ④ Podocytes were divided into the cell control group, CXCL16 10 μg·L-1 gruop, CXCL16+liraglutide 100 nmol·L-1 group and CXCL16+simvastatin 100 nmol·L-1 group. After two hours of incubation, CXCL16 was added and cultured for 24 h. Phalloidin staining was used to detect podocyte stress fibers. ELISA was used to measure the concentrations of tumor necrosis factor α (TNF-α), transforming growth factor-β (TGF-β) and interleukin-1β (IL- 1β) in cell culture supernatants. Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of TNF-α, TGF-β, and IL-1β. Western blotting was used to detect the protein expression levels of nephrin, sterol regulatory element-binding protein 1 (SREBP1), SREBP2, and adipose differentiation-related protein (ADRP) in podocytes. RESULTS  ① Compared with the cell control group, both high glucose and palmitic acid stimulation significantly increased CXCL16 expression levels in human podocytes (P<0.01).  ② Compared with the cell control group, recombinant CXCL16 stimulation significantly down-regulated the expression of nephrin in podocytes (P<0.01). ③ Compared with the cell control group, recombinant CXCL16 stimulation significantly increased the lipid droplet area in podocytes (P<0.01), and different concentrations of liraglutide or simvastatin could significantly alleviate this change (P<0.01).  ④ Compared with the cell control group, recombinant CXCL16 significantly decreased the percentage of podocyte stress fibers (P<0.01), increased the concentrations of TNF-α, TGF-β, and IL-1β in the culture medium (P<0.01), and significantly up-regulated the mRNA levels of TNF-α, TGF-β and IL-1β in podocytes (P<0.05, P<0.01), down-regulated the protein expression of nephrin (P<0.01), and significantly increased the protein expression levels of SREBP1, SREBP2, and ADRP (P<0.01). Compared with the CXCL16 group, liraglutide or simvastatin significantly restored the percentage of podocyte stress fibers (P<0.01), downregulated of the concentrations of TNF-α, TGF-β, and IL-1β in the culture medium (P<0.01), inhibit of the mRNA levels of TNF-α, TGF-β, and IL-1β in podocytes (P<0.05, P<0.01), upregulated the expression level of nephrin (P<0.01), and downregulated the expression levels of SREBP1, SREBP2, and ADRP (P<0.01). CONCLUSION  Liraglutide can reduce podocyte injury induced by CXCL16 by inhibiting lipid deposition and alleviating inflammation caused by lipid deposition. This may be related to the ability of liraglutide to inhibit the expressions of SREBPs and ADRP.

Cerebrovascular dysfunction is one of the main features of Alzheimer disease (AD). And the pathological change of the renin-angiotensin system (RAS) is an important indicator of vascular injury in AD patients. Brain RAS (BRAS) is independent of peripheral RAS and is capable of regulating neurons and glial cells, which is of great significance for the regulation of the neurovascular unit (NVU). The brain microvascular endothelial cells (BMECs) are important members of NVU. The blood-brain barriers composed of BMECs make it a pivotal link connecting the peripheral RAS with BRAS. This paper reviews the impact of BRAS on functions of neurons, BMECs and glial cells in AD as well as its contributions to the NVU. BRAS harmonizes AD pathology through energy metabolism, oxidative stress, and inflammatory response. Furthermore, this review summarizes the RAS antagonist agents used in clinic trials to emphasize the important role of BRAS in NVU regulation in AD. 

In recent years, the rise of various RNA therapeutics has promoted the development of microRNA(miRNA) drugs. With the in-depth study of miRNA mechanisms, a growing body of evidence shows that miRNA is a key regulator of cardiovascular function. miRNA mimics and miRNA inhibitors are the two main strategies for miRNA drug development. Nonclinical studies have identified multiple therapeutic miRNAs that play important roles in the treatment of common cardiovascular diseases such as heart failure, myocardial infarction, myocardial fibrosis, arrhythmia, and atherosclerosis. However, in addition to the complex pathological mechanisms of cardiovascular diseases and the limitations of 
preclinical studies, miRNA drug development is still facing such bottlenecks as off-target, side effects and targeted delivery. CDR132L, the first miRNA drug for the treatment of heart failure, has entered clinical trials, which indicates that miRNA drugs are expected to become the next generation of cardiovascular disease therapy.

OBJECTIVE  To integrate multiple behavioral parameters of mice undergoing observational fear learning (OFL) and to explore the gender differences in OFL among mice. METHODS  ① Female and male C57BL/6J mice were divided into the control group and model group respectively. In the OFL training stage, mice were allowed to witness the  partner suffering from plantar electric shock (model group) or not (control group) to establish an OFL model. After 24 hours, the observational fear retrieval test was conducted. The freezing behavior, avoidance behavior and social interaction behavior of these mice were recorded in the training stage and observational fear retrieval stage respectively, before gender differences in each behavior were studied. Three behavioral parameters were normalized and integrated by Z-value, and the gender differences in observational fear response in mice was revealed via the comprehensive emotional Z-value. ② Female mice underwent bilateral ovariectomy to prepare ovariectomized mice before three fear behaviors in OFL were detected. The differences between the ovariectomized model, sham-operated model group and their respective control groups, and between the sham-operated model group and ovariectomized model group were studied via the comprehensive emotional Z-value. RESULTS  ① During the training stage of OFL, the freezing time and corner zone time in female and male model groups increased significantly compared with their respective control groups (P<0.05), while the interaction zone entries of the female model group was significantly decreased compared with the control group (P<0.01), but there was no significant gender difference in freezing, open field avoidance or social interaction behaviors between female and male model groups. After 24 hours, the freezing time in the male model group remained significantly longer than that of the control group (P<0.01), but there was no significant difference between the female model group and the female control group. There was no significant difference in the open field avoidance and social interaction behaviors between the female model group and the male model group, but the freezing time in the male model group was significantly longer than that of the female model group (P<0.01). There was no significant difference in the open field avoidance between the male model group and the female model group, but the interaction zone entries of the male model group was significantly decreased compared with the female model group (P<0.01). According to the integrated comprehensive emotional Z-value, the fear response of the female and male model mice in the training stage was obviously stronger than that of their respective control mice (P<0.01), but there was no significant difference between male and female mice. After 24 hours, the fear response of the male model group remained significantly higher than that of the control group (P<0.05) and the female model group (P<0.01) in the 24 h fear retrieval stage. ② In the training stage, the fear response of the ovariectomized model group was significantly stronger than that of the ovariectomized control group (P<0.05), but there was no difference between the ovariectomized model group and the sham-operated model group. In the 24 h fear retrieval stage, the fear response of the ovariectomized model group remained stronger than that of the ovariectomized control group and the sham-operated model group (P<0.01). CONCLUSION  In OFL, both male and female mice can acquire fear by witnessing the suffering of their partners. The comprehensive emotional Z-value based on the integration of the three behavioral indicators proves that the observational fear learning of male mice is more lasting than that of female mice. After elimination of the influence of estrogen through bilateral ovariectomy, the observational fear learning response of ovariectomized female mice is similar to that of male mice.