BACKGROUND:During bone remodeling,bone formation and bone resorption are spatially and temporally coordinated,involving intricate interactions between osteoclasts and osteoblasts.Isoginkgetin,a flavonoid found in Ginkgo biloba,has a wide range of anticancer activity and anti-reactive oxygen species activity;however,the effect of isoginkgetin on osteoclast differentiation is unknown.OBJECTIVE:To study the effect and mechanism of action of isoginkgetin on osteoclastogenesis.METHODS:In vitro studies were performed on mouse bone marrow-derived macrophages,and cell counting kit-8 cytotoxicity assay was used to detect the effect of isoginkgetin on cell viability of bone marrow-derived macrophages.Macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand were used to induce the differentiation of bone marrow-derived macrophages to osteoclasts.Network pharmacology and molecular docking and molecular dynamics simulations were used to predict the processes and targets of the effects of isoginkgetin on the differentiation of osteoclasts.Tartrate-resistant acid phosphatase staining and F-actin staining were used to detect the effects of isoginkgetin on the differentiation and function of osteoclasts.Western blot and RT-PCR were used to detect the effects of isoginkgetin on the expression of genes and proteins related to osteoclast differentiation,reactive oxygen species,and PI3K/AKT pathways.Fluorescent probes were used to detect cellular and mitochondrial reactive oxygen species levels.Flow cytometry technology was used to detect reactive oxygen species levels in cells.RESULTS AND CONCLUSION:(1)Network pharmacology results showed that isoginkgetin affected osteoporosis mainly through the PI3K-AKT pathway and cellular response to drugs and hypoxia,and GSK3β,ESR1,MCL1 and CCNA2 were the key targets.(2)Cell counting kit-8 and tartrate-resistant acid phosphatase staining results showed that isoginkgetin at 8 μmol/L had the most significant inhibitory effect on osteoclastogenesis in vitro,and F-actin results showed that isoginkgetin inhibited osteoclast cytoskeletal actin ring formation in a concentration-dependent manner.(3)Molecular dynamics simulations showed that isoginkgetin bound well to osteoclastogenesis marker proteins(NFATc1,c-Fos,CTSK,and MMP9).Western blot and RT-PCR results indicated that isoginkgetin inhibited the expression of osteoclastogenesis marker proteins and genes(NFATc1,c-Fos,CTSK,and MMP9).(4)Western blot results showed that isoginkgetin inhibited the phosphorylation level of PI3K/AKT/GSK3β and suppressed osteoclastogenesis by activating the PI3K-AKT-GSK3β pathway.(5)The results of reactive oxygen species assay showed that isoginkgetin significantly reduced receptor activator of nuclear factor kappa-B ligand-induced cellular and mitochondrial reactive oxygen species production,and inhibited the differentiation of bone marrow-derived macrophages to osteoclasts.

BACKGROUND:During bone remodeling,bone formation and bone resorption are spatially and temporally coordinated,involving intricate interactions between osteoclasts and osteoblasts.Isoginkgetin,a flavonoid found in Ginkgo biloba,has a wide range of anticancer activity and anti-reactive oxygen species activity;however,the effect of isoginkgetin on osteoclast differentiation is unknown.OBJECTIVE:To study the effect and mechanism of action of isoginkgetin on osteoclastogenesis.METHODS:In vitro studies were performed on mouse bone marrow-derived macrophages,and cell counting kit-8 cytotoxicity assay was used to detect the effect of isoginkgetin on cell viability of bone marrow-derived macrophages.Macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand were used to induce the differentiation of bone marrow-derived macrophages to osteoclasts.Network pharmacology and molecular docking and molecular dynamics simulations were used to predict the processes and targets of the effects of isoginkgetin on the differentiation of osteoclasts.Tartrate-resistant acid phosphatase staining and F-actin staining were used to detect the effects of isoginkgetin on the differentiation and function of osteoclasts.Western blot and RT-PCR were used to detect the effects of isoginkgetin on the expression of genes and proteins related to osteoclast differentiation,reactive oxygen species,and PI3K/AKT pathways.Fluorescent probes were used to detect cellular and mitochondrial reactive oxygen species levels.Flow cytometry technology was used to detect reactive oxygen species levels in cells.RESULTS AND CONCLUSION:(1)Network pharmacology results showed that isoginkgetin affected osteoporosis mainly through the PI3K-AKT pathway and cellular response to drugs and hypoxia,and GSK3β,ESR1,MCL1 and CCNA2 were the key targets.(2)Cell counting kit-8 and tartrate-resistant acid phosphatase staining results showed that isoginkgetin at 8 μmol/L had the most significant inhibitory effect on osteoclastogenesis in vitro,and F-actin results showed that isoginkgetin inhibited osteoclast cytoskeletal actin ring formation in a concentration-dependent manner.(3)Molecular dynamics simulations showed that isoginkgetin bound well to osteoclastogenesis marker proteins(NFATc1,c-Fos,CTSK,and MMP9).Western blot and RT-PCR results indicated that isoginkgetin inhibited the expression of osteoclastogenesis marker proteins and genes(NFATc1,c-Fos,CTSK,and MMP9).(4)Western blot results showed that isoginkgetin inhibited the phosphorylation level of PI3K/AKT/GSK3β and suppressed osteoclastogenesis by activating the PI3K-AKT-GSK3β pathway.(5)The results of reactive oxygen species assay showed that isoginkgetin significantly reduced receptor activator of nuclear factor kappa-B ligand-induced cellular and mitochondrial reactive oxygen species production,and inhibited the differentiation of bone marrow-derived macrophages to osteoclasts.

By consulting herbal texts， medical records， formula collections， and other relevant literature from various historical periods， as well as modern and contemporary research materials， different aspects of the historical evolution of Zijingpi， including its name， origin， scientific name verification， medicinal part， genuine producing areas， harvesting， processing， and preparation， properties and flavors， and primary indications， were systematically reviewed and verified， providing a basis for the development of famous classical formula preparations containing this medicinal material. According to the textual research， Cercis chinensis was first recorded under the name "Zijingmu" in the Rihuazi Bencao from the Five Dynasties period. From the Song Dynasty to the Qing Dynasty， it was known by various names such as "Zijing"， "Zijingpi"， and "Zijingmupi". In modern and contemporary times， it has been officially named "Zijingpi"， with aliases such as "Mantiaohong"， "Zihuashu"， and "Qingminghua". Historically， the mainstream source of Zijingpi was the dried bark of Cercis chinensis Bunge， a species of the legume family. However， there were also instances of confusion with the Lythraceae plant Lagerstroemia indica L. The producing areas of Zijingpi have no special geographical limitation， and the plant is currently distributed throughout most parts of China. There were no special requirements for harvesting time in ancient times， while modern records indicate harvesting time in spring， summer， and autumn. Ancient processing methods were rarely recorded， with only mentions of stir-frying Zijingpi. Modern practice mostly uses the raw material medicinally. Modern standards prefer it to be "dry， long strips， and thick". The functions of Zijingpi， mainly to promote blood circulation， relieve strangury， and detoxify， have remained consistent from ancient to modern times. Based on the textual research findings， it is recommended that when developing and exploiting the famous classical formulas containing Zijingpi， the bark of C. chinensis should be selected as the source. The processing method should be chosen according to the formula requirements， and if no specific requirements are indicated， it is suggested to use the raw material medicinally.

OBJECTIVE To investigate the stability and compatibility of butorphanol， nicardipine， urapidil， and tirofiban. METHODS A micro-infusion pump was used to simulate the infusion process. Samples were collected in real time from the needle tip at 0， 2， 4， 6， and 8 h after mixing of the four drugs. The appearance， pH， and number of insoluble particles were examined. The contents of butorphanol， nicardipine， urapidil， and tirofiban were determined using the ultra-high-performance liquid chromatography method. RESULTS Within 8 hours， all mixed solutions remained clear and transparent， with no visible turbidity， precipitation， color change， or gas generation. The pH fluctuation ranged from 0.02 to 0.09， both within 0.1 pH units. The number of insoluble particles ≥10 μm in size in each compatibility solution was ≤9， and the number of insoluble particles ≥25 μm was ≤2， meeting the requirements of the 2025 edition of the Chinese Pharmacopoeia （Volume Ⅳ）. The relative percentage contents of butorphanol， nicardipine， urapidil， and tirofiban at 8 hours after compatibility mixing ranged from 92.38% to 113.40%. CONCLUSIONS Within 8 hours under simulated clinical micro-infusion pump infusion conditions， the stability and compatibility of the mixed solution containing butorphanol， nicardipine， urapidil， and tirofiban were satisfactory.

ObjectiveTo explore the host immunomodulatory effect and its molecular mechanism of gallic acid （GA） in Bacillus Calmette-Guérin （BCG） infected macrophages， and to provide experimental evidence for host-directed anti-tuberculosis research against Mycobacterium tuberculosis （Mtb）. MethodsThe minimum inhibitory concentration （MIC） of GA against BCG was determined by resazurin colorimetric assay. Bacterial growth-curve test and MTT colorimetric assay were used to evaluate the effects of GA on BCG proliferation and invasion. A BCG-infected RAW264.7 macrophage model was established， including infection control group （Ctrl）， isoniazid （INH） group， and low-， medium- and high-dose GA groups. Flow cytometry combined with colony-plate counting was performed to detect intracellular BCG survival. Cell apoptosis was measured by Annexin V-APC/7-AAD double staining， and qRT-PCR was used to detect the mRNA expression of apoptosis-related genes Caspase-3 and Bcl-2. Macrophage polarization phenotypes were analyzed by flow cytometry. Intracellular reactive oxygen species （ROS） were detected using DHE fluorescent probe. The Griess assay was applied to determine supernatant nitric oxide （NO） concentration. ELISA was used to detect the levels of TNF-α， IFN-γ， IL-6 and IL-10 in cell culture supernatant. ResultsThe MIC of GA against BCG was 1 250 μmol/L. GA inhibited BCG proliferation and invasion in a concentration-dependent manner （P＜0.05）. Safe concentrations of 20， 40 and 80 μmol/L were selected for subsequent cellular experiments. Thirty-six-hour GA pretreatment markedly reduced the intracellular BCG bacterial load （P＜0.05）. GA exerted bidirectional regulation on macrophage apoptosis： it inhibited apoptosis at the early-stage infection and promoted apoptosis at the late-stage infection. GA induced M1-type polarization in early infection and facilitated M2-type polarization in late infection （P＜0.05）. Meanwhile， GA decreased intracellular ROS and NO levels， down-regulated the secretion of pro-inflammatory cytokines and up-regulated the secretion of anti-inflammatory cytokine IL-10 （P＜0.05）. ConclusionGA inhibits BCG proliferation and invasion in vitro. It remodels host immune microenvironment via bidirectional regulation of macrophage apoptosis and polarization as well as anti-oxidative and anti-inflammatory effects， showing potential value for host-directed anti-mycobacterial therapy.

ObjectiveTo explore the host immunomodulatory effect and its molecular mechanism of gallic acid （GA） in Bacillus Calmette-Guérin （BCG） infected macrophages， and to provide experimental evidence for host-directed anti-tuberculosis research against Mycobacterium tuberculosis （Mtb）. MethodsThe minimum inhibitory concentration （MIC） of GA against BCG was determined by resazurin colorimetric assay. Bacterial growth-curve test and MTT colorimetric assay were used to evaluate the effects of GA on BCG proliferation and invasion. A BCG-infected RAW264.7 macrophage model was established， including infection control group （Ctrl）， isoniazid （INH） group， and low-， medium- and high-dose GA groups. Flow cytometry combined with colony-plate counting was performed to detect intracellular BCG survival. Cell apoptosis was measured by Annexin V-APC/7-AAD double staining， and qRT-PCR was used to detect the mRNA expression of apoptosis-related genes Caspase-3 and Bcl-2. Macrophage polarization phenotypes were analyzed by flow cytometry. Intracellular reactive oxygen species （ROS） were detected using DHE fluorescent probe. The Griess assay was applied to determine supernatant nitric oxide （NO） concentration. ELISA was used to detect the levels of TNF-α， IFN-γ， IL-6 and IL-10 in cell culture supernatant. ResultsThe MIC of GA against BCG was 1 250 μmol/L. GA inhibited BCG proliferation and invasion in a concentration-dependent manner （P＜0.05）. Safe concentrations of 20， 40 and 80 μmol/L were selected for subsequent cellular experiments. Thirty-six-hour GA pretreatment markedly reduced the intracellular BCG bacterial load （P＜0.05）. GA exerted bidirectional regulation on macrophage apoptosis： it inhibited apoptosis at the early-stage infection and promoted apoptosis at the late-stage infection. GA induced M1-type polarization in early infection and facilitated M2-type polarization in late infection （P＜0.05）. Meanwhile， GA decreased intracellular ROS and NO levels， down-regulated the secretion of pro-inflammatory cytokines and up-regulated the secretion of anti-inflammatory cytokine IL-10 （P＜0.05）. ConclusionGA inhibits BCG proliferation and invasion in vitro. It remodels host immune microenvironment via bidirectional regulation of macrophage apoptosis and polarization as well as anti-oxidative and anti-inflammatory effects， showing potential value for host-directed anti-mycobacterial therapy.

[This corrects the article DOI: 10.1016/j.apsb.2021.07.004.].

The continuous emergence of SARS-CoV-2 variants as well as other potential future coronavirus has challenged the effectiveness of current COVID-19 vaccines. Therefore, there remains a need for alternative antivirals that target processes less susceptible to mutations, such as the formation of six-helix bundle (6-HB) during the viral fusion step of host cell entry. In this study, a novel high-throughput screening (HTS) assay employing a yeast-two-hybrid (Y2H) system was established to identify inhibitors of HR1/HR2 interaction. The compound IMB-9C, which achieved single-digit micromolar inhibition of SARS-CoV-2 and its Omicron variants with low cytotoxicity, was selected. IMB-9C effectively blocks the HR1/HR2 interaction in vitro and inhibits SARS-CoV-2-S-mediated cell-cell fusion. It binds to both HR1 and HR2 through non-covalent interaction and influences the secondary structure of HR1/HR2 complex. In addition, virtual docking and site-mutagenesis results suggest that amino acid residues A930, I931, K933, T941, and L945 are critical for IMB-9C binding to HR1. Collectively, in this study, we have developed a novel screening method for HR1/HR2 interaction inhibitors and identified IMB-9C as a potential antiviral small molecule against COVID-19 and its variants.

The oxytocin receptor (OXTR) has garnered increasing attention for its role in regulating both mature behaviors and brain development. It has been established that OXTR mediates a range of effects that are region-specific or period-specific. However, the current studies of OXTR expression patterns in mice only provide limited help due to limitations in resolution. Therefore, our objective was to generate a comprehensive, high-resolution spatiotemporal expression map of Oxtr mRNA across the entire developing mouse brain. We applied RNAscope in situ hybridization to investigate the spatiotemporal expression pattern of Oxtr in the brains of male mice at six distinct postnatal developmental stages (P7, P14, P21, P28, P42, P56). We provide detailed descriptions of Oxtr expression patterns in key brain regions, including the cortex, basal forebrain, hippocampus, and amygdaloid complex, with a focus on the precise localization of Oxtr⁺ cells and the variance of expression between different neurons. Furthermore, we identified some neuronal populations with high Oxtr expression levels that have been little studied, including glutamatergic neurons in the ventral dentate gyrus, Vgat⁺Oxtr⁺ cells in the basal forebrain, and GABAergic neurons in layers 4/5 of the cortex. Our study provides a novel perspective for understanding the distribution of Oxtr and encourages further investigations into its functions.
Animals ; Receptors, Oxytocin/metabolism* ; Male ; Brain/growth & development* ; Mice ; Mice, Inbred C57BL ; Neurons/metabolism* ; Single-Cell Analysis ; Gene Expression Regulation, Developmental ; RNA, Messenger/metabolism* ; Animals, Newborn

ObjectiveTo investigate the change in the expression of magnesium transporter 1 （MagT1） in peripheral blood CD8⁺ T cells in patients with chronic hepatitis B virus （HBV） infection， as well as the correlation of serum Mg²⁺ and MagT1 with HBV DNA load. MethodsA total of 102 patients with HBV infection who were admitted to Tangshan Workers’ Hospital from January 2022 to December 2023 were enrolled， and a case-control study was conducted. According to the stage of disease progression， the subjects were divided into chronic hepatitis B group with 40 patients， compensated liver cirrhosis group with 32 patients， and hepatocellular carcinoma group with 30 patients， and 32 healthy volunteers matched by age and sex were enrolled as normal control group. The serum concentration of Mg²⁺ was measured； RT-qPCR was used to measure the mRNA expression level of MagT1 in CD8⁺ T cells and serum HBV DNA load； flow cytometry was used to measure the expression levels of programmed death-1 （PD-1）， T-cell immunoglobulin and mucin-domain containing-3 （Tim-3）， and natural killer cell group 2D （NKG2D） on the surface of CD8⁺ T cells. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups， and the SNK-q test was used for comparison between two groups； the chi-square test was used for comparison of categorical data between groups； a Pearson linear correlation analysis was used to investigate the correlation between the mRNA expression level of MagT1 and other related indicators. ResultsThere were significant differences in the serum level of Mg²⁺ and the mRNA expression level of MagT1 between the normal control group， the chronic hepatitis B group， the compensated liver cirrhosis group， and the hepatocellular carcinoma group （F=29.014 and 145.578， both P<0.001）. The Pearson correlation analysis showed that the serum level of Mg²⁺ and the mRNA expression level of MagT1 were positively correlated with HBV DNA load （r=0.335 and 0.394， both P<0.05）. The hepatocellular carcinoma group had the highest levels of PD-1 and Tim-3， followed by the compensated liver cirrhosis group， the chronic hepatitis B group， and the normal control group； the normal control group had the highest level of NKG2D， followed by the chronic hepatitis B group， the compensated liver cirrhosis group， and the hepatocellular carcinoma group （all P<0.001）. The Pearson correlation analysis showed that the mRNA expression level of MagT1 was negatively correlated with PD-1 and Tim-3 （r=-0.643 and -0.640， both P<0.05） and was positively correlated with NKG2D （r=0.655， P<0.05）. ConclusionThere is a reduction in the mRNA expression level of MagT1 in peripheral blood CD8⁺ T cells in patients with HBV infection， which is positively correlated with serum HBV DNA load. The reduction in the expression of MagT1 may contribute to CD8⁺ T cell exhaustion by impairing Mg²⁺ influx， manifesting as the upregulation of PD-1 and Tim-3 and the downregulation of NKG2D.