Objective To investigate the effects of LBL-007, a novel fully human lymphocyte activation gene 3 (LAG-3) monoclonal antibody, in combination with programmed cell death protein 1 (PD-1) antibody, on the invasion, migration and proliferation of tumor cells, and to elucidate the underlying mechanisms. Methods Human lymphocyte cells Jurkat were co-cultured with A549 and MGC803 tumor cell lines and treated with the isotype control antibody human IgG, LBL-007, anti-PD-1 antibody BE0188, or tumor necrosis factor-alpha (TNF-α, the NF-κB signaling pathway agonist). Tumor cell proliferation was assessed using a colony formation assay; invasion was measured by Transwell^TM assay; migration was evaluated using a wound healing assay. Western blotting was employed to determine the expression levels of NF-κB pathway-related proteins: IκB inhibitor kinase alpha (Ikkα), phosphorylated Ikkα (p-IKKα), NF-κB subunit p65, phosphorylated p65 (p-p65), NF-κB Inhibitor Alpha (IκBα), phosphorylated IκBα (p-IκBα), matrix metalloproteinase 9 (MMP9), and MMP2. Results Compared with the control and IgG isotype groups, LBL-007 and BE0188 significantly reduced tumor cell proliferation, invasion, and migration. They also decreased the phosphorylation of p-IKKα, p-p65 and p-IκBα, and the expression of MMP9 and MMP2 of tumor cells in the co-culture system. The combined treatment of LBL-007 and BE0188 enhanced inhibitory effects. Treatment with the NF-κB signaling pathway agonist TNF-α reversed the suppressive effects of LBL-007 and BE0188 on tumor cell proliferation, invasion, migration, and NF-κB signaling. Conclusion LBL-007 and anti-PD-1 antibody synergistically inhibit the invasion, migration, and proliferation of A549 and MGC803 tumor cells by blocking the NF-κB signaling pathway.
Humans ; Cell Proliferation/drug effects* ; Cell Movement/drug effects* ; Signal Transduction/drug effects* ; NF-kappa B/metabolism* ; Neoplasm Invasiveness ; Antibodies, Monoclonal/pharmacology* ; Programmed Cell Death 1 Receptor/antagonists & inhibitors* ; Cell Line, Tumor ; Antigens, CD/immunology* ; Lymphocyte Activation Gene 3 Protein ; A549 Cells ; I-kappa B Kinase/metabolism* ; Jurkat Cells ; Matrix Metalloproteinase 9/metabolism*

The new generation of artificial intelligence technology,represented by deep learning,has emerged as a crucial driving force in the advancement of new drug research and development.This article creatively proposes a workflow named"Molecular Factory"for the design and optimization of drug molecules based on artificial intelligence technology.This workflow integrates intelligent molecular generation models,high-performance molecular docking algorithms,and accurate protein-ligand binding affinity prediction methods.It has been integrated as a core module into DrugFlow,a one-stop drug design software platform,providing a comprehensive set of mature solutions for the discovery and optimization of lead compounds.Utilizing the"Molecular Factory"module,we conducted the research of second-generation inhibitors against Menin that can combat drug resistance.Through the integration of computational and experimental approaches,we rapidly identified multiple promising compounds.Among them,compound RG-10 exhibited the IC50 values of 9.681 nmol/L,233.2 nmol/L,and 40.09 nmol/L against the wild-type Menin,M327I mutant,and T349M mutant,respectively.Compared to the positive reference molecule SNDX-5613,which has entered Phase Ⅱ clinical trials,RG-10 demonstrated significantly enhanced inhibitory activity against the M327I and T349M mutants.These findings fully demonstrate the unique advantages of the"Molecular Factory"technology in practical drug design and development scenarios.It can rapidly and efficiently generate high-quality active molecules targeting specific protein structures,holding significant value and profound implications for advancing new drug discovery.

ObjectiveTo explore new targets and herbal medicines of total ginsenosides in ameliorating alcoholic hepatitis （AH） by data mining and experimental validation and to provide new directions for the clinical treatment of AH. MethodGSE28619 was selected as the test set from the GEO database and GSE83148 and GSE103580 were selected as the validation sets. The limma package and weighted gene co-expression network analysis （WGCNA） were employed to identify the AH-related differentially expressed genes and modular genes， and Venny was used to extract the common genes. The protein-protein interaction （PPI） network was constructed and the enrichment analysis was carried out. The hub genes were further screened and evaluated for their diagnostic value. After validation with the datasets， new potential targets of AH and traditional Chinese medicine were predicted. Molecular docking between the targets and active ingredients of traditional Chinese medicine was performed， and the results were validated by experiments. Eight out of 48 SD rats were randomly selected into a blank group and received an equal amount of normal saline. The rest rats were subjected to modeling with ethanol by gavage and then randomized into low- （10 mg·kg^-1）， medium- （20 mg·kg^-1）， and high-dose （40 mg·kg^-1） total ginsenosides， model， and positive control （metadoxine， 117 mg·kg^-1） groups. After 3 weeks of gavage， serum samples were collected for the measurement of aspartate aminotransferase （AST） and alanine aminotransferase （ALT） levels， and liver samples were collected for hematoxylin-eosin （HE） staining. Western blot and Real-time PCR were employed to determine the protein and mRNA levels， respectively， of potential targets in the liver tissue. ResultData mining predicted the potential genes： Proto-oncogene FOS and collagen type Ⅰ alpha 2 （COL1A2）. Experimental validation showed that the liver injury was alleviated after drug administration compared with that after modeling. The serum AST and ALT levels were reduced after drug administration. The protein and mRNA levels of FOS were significantly up-regulated， while those of COL1A2 were down-regulated after drug administration. ConclusionTotal ginsenosides ameliorate HA via FOS and COL1A2.

Objective To explore the mechanisms of circadian rhythm disorder(CRD)on behavior and testicular spermatogenic capacity in adolescent mice.Methods Thirty SPF grade C57 mice were selected and randomly di-vided into the control and CRD groups with 15 mice in each group.The control group kept 12 h dark/12 h bright circulating light,and the CRD group kept 24 h light.The trial lasted for 61 days.The growth curves of mice in each group were counted;the elevated plus maze test and open field test were performed to detect mice behavior;neuronal morphology was visualized by Nissl staining.The distribution of ionized calcium-binding adapter molecule 1(Iba1)and neuron-specific nuclear protein(NeuN)in the hypothalamus were detected by immunofluorescence and Western blot.Expression of testosterone synthesis-related genes steroidogenic acute regulatory protein(StAR)and hydroxy-delta-5-steroid dehydrogenase,3 beta-and steroid delta-isomerase 1(HSD3B1)and spermatogenesis-related genes gametogenetin binding protein 2(GGNBP2)and deleted in azoospermia-like(DAZL)were deter-mined by RT-qPCR.Results The weight of the CRD group was significantly higher than that of control group at 61 days;in the elevated plus maze test,the time,frequency,and percentage of time in the open arm of the CRD group were significantly less than those of the control group;in the open field test,there was no significant differ-ence in movement distance between the two groups;however,the residence time of the central area in the CRD group was significantly less than that in the control group;the frequency of entering the central area in the CRD group was significantly less than that in the control group.Nissl staining results showed that the positive cells in the CRD group were significantly lower than the control group.Immunofluorescence and Western blot results showed that Iba1 protein expression was up-regulated and NeuN protein expression was down-regulated in the hypothalamus of the CRD group.In the RT-qPCR experiment,the expression of HSD3B1 in the CRD group was significantly low-er than that of the control group;the expression of GGNBP2 and DAZL in the CRD group was significantly lower than that in the control group.Conclusion The CRD treatment can not only lead to depressive behavior in adoles-cent mice but also reduce the development of reproductive system in male adolescent mice.

Objective To explore the inhibitory effect of Sidaxue, a traditional Miao herbal medicine formula, on articular bone and cartilage destruction and synovial neovascularization in rats with collagen-induced arthritis (CIA). Methods In a SD rat model of CIA, we tested the effects of daily gavage of Sidaxue at low, moderate and high doses (10, 20, and 40 g/kg, respectively) for 21 days, with Tripterygium glycosides (GTW) as the positive control, on swelling in the hind limb plantar regions by arthritis index scoring. Pathologies in joint synovial membrane of the rats were observed with HE staining, and serum TNF-α and IL-1β levels were detected with ELISA. The expressions of NF-κB p65, matrix metalloproteinase 1 (MMP1), MMP2 and MMP9 at the mRNA and protein levels in the synovial tissues were detected using real-time PCR and Western blotting. Network pharmacology analysis was conducted to identify the important target proteins in the pathways correlated with the therapeutic effects of topical Sidaxue treatment for RA, and the core target proteins were screened by topological analysis. Results Treatment with GTW and Sidaxue at the 3 doses all significantly alleviated plantar swelling, lowered arthritis index scores, improved cartilage and bone damage and reduced neovascularization in CIA rats (P<0.05), and the effects of Sidaxue showed a dose dependence. Both GTW and Sidaxue treatments significantly lowered TNF-α, IL-1β, NF-κB p65, MMP1, MMP2, and MMP9 mRNA and protein expressions in the synovial tissues of CIA rats (P<0.05). Network pharmacological analysis identified MMPs as the core proteins associated with topical Sidaxue treatment of RA. Conclusion Sidaxue alleviates articular bone and cartilage damages and reduces synovial neovascularization in CIA rats possibly by downregulating MMPs via the TNF-α/IL-1β/NF-κB-MMP1, 2, 9 signaling pathway, and MMPs probably plays a key role in mediating the effect of Sidaxue though the therapeutic pathways other than oral administration.

Objective To investigate the role of silencing regulatory protein 1(Sirt1)in the regulation of Vibrio vulnificus sepsis-induced macrophage apoptosis and the molecular mechanisms.Methods Mouse RAW264.7 macrophages which stably overexpressed Sirt1 were constructed and screened by genistein G418.CCK-8 analysis was used to detect the proliferation of cells in the control group and Sirt1-Flag group.The changes of expression levels of apoptosis-associated protein poly ADP-ribose polymerase(PARP),cleaved-PARP,caspase3,cleaved-caspase3 and acetylated p53 in different treatment groups were detected via Western blotting.A Vibrio vulnificus sepsis model in mice was established,and the expression levels of apoptosis-associated protein cleaved-caspase3 in the lung,spleen and liver of mice of different treatment groups were detected by immunohistochemistry.Results Overexpression of Sirt1 reduced VVC-induced RAW264.7 cell damage.Overexpression of Sirt1 as well as RSV pretreatment lowered the expression of apoptosis-associated protein cleaved-PARP,cleaved-caspase3 and acetylated p53 in VVC-stimulated RAW264.7 cells and mouse peritoneal macrophages.In the mouse model of Vibrio vulnificus sepsis,therapeutic administration of RSV reduced the expression of apoptosis-associated protein marker cleaved-caspase3 in lung,spleen and liver tissues.Conclusion Sirt1 can inhibit p53 acetylation and reduces apoptosis in mouse macrophages,which helps protect against Vibrio vulnificus sepsis.

Objective To explore the inhibitory effect of Sidaxue, a traditional Miao herbal medicine formula, on articular bone and cartilage destruction and synovial neovascularization in rats with collagen-induced arthritis (CIA). Methods In a SD rat model of CIA, we tested the effects of daily gavage of Sidaxue at low, moderate and high doses (10, 20, and 40 g/kg, respectively) for 21 days, with Tripterygium glycosides (GTW) as the positive control, on swelling in the hind limb plantar regions by arthritis index scoring. Pathologies in joint synovial membrane of the rats were observed with HE staining, and serum TNF-α and IL-1β levels were detected with ELISA. The expressions of NF-κB p65, matrix metalloproteinase 1 (MMP1), MMP2 and MMP9 at the mRNA and protein levels in the synovial tissues were detected using real-time PCR and Western blotting. Network pharmacology analysis was conducted to identify the important target proteins in the pathways correlated with the therapeutic effects of topical Sidaxue treatment for RA, and the core target proteins were screened by topological analysis. Results Treatment with GTW and Sidaxue at the 3 doses all significantly alleviated plantar swelling, lowered arthritis index scores, improved cartilage and bone damage and reduced neovascularization in CIA rats (P<0.05), and the effects of Sidaxue showed a dose dependence. Both GTW and Sidaxue treatments significantly lowered TNF-α, IL-1β, NF-κB p65, MMP1, MMP2, and MMP9 mRNA and protein expressions in the synovial tissues of CIA rats (P<0.05). Network pharmacological analysis identified MMPs as the core proteins associated with topical Sidaxue treatment of RA. Conclusion Sidaxue alleviates articular bone and cartilage damages and reduces synovial neovascularization in CIA rats possibly by downregulating MMPs via the TNF-α/IL-1β/NF-κB-MMP1, 2, 9 signaling pathway, and MMPs probably plays a key role in mediating the effect of Sidaxue though the therapeutic pathways other than oral administration.

Objective:This study aimed to explore the clinical value of myostatin(MST) and follistatin(FST) as biological biomarkers in evaluating sarcopenia in elderly women.Methods:This was a retrospective cross-sectional study that enrolled 350 females aged 20-89 years who underwent physical examinations in Shanghai Huadong Hospital in 2021. Demographic characteristics, muscle mass, fat mass, bone mineral density, hand grip strength, gait speed, and serum indices of MST and FST were collected.Results:The serum levels of MST did not change significantly with age. However, the serum levels of FST increased with age. In women aged≥60 years, MST was positively correlated with total lean mass and appendicular skeletal muscle index(ASMI; r=0.236, P=0.041; r=0.289, P=0.014), while FST was negatively correlated with ASMI( r=-0.265, P=0.030). In multivariate stepwise regression analysis, after adjusting for age, body mass index, hip bone mineral density, and total fat mass, only FST was independently correlated with ASMI( β=-0.238, P=0.006), while MST was not correlated with ASMI. The receiver operating characteristic curve was plotted using muscle mass reduction as the state variable and serum FST level as the test variable. The area under the curve was 0.753. And when the FST cutoff value was 17.49 ng/mL, the maximum Jordan index was 0.46, with a sensitivity of 77.3% and a specificity of 68.7%. Women aged ≥60 years were divided into three groups based on serum FST levels. Compared to the upper third of the serum FST level group, the low third of the FST level group had a significantly reduced risk of suffering from sarcopenia( OR=0.098, P =0.036). Conclusions:Serum FST lever has a better correlation with muscle mass among elderly women, making it a promising biomarker for evaluating muscle mass.

Neurodegenerative diseases(NDs)are closely related to the central nervous system and characterized by morphological abnormalities and progressive loss of function in specific neuron groups.The main NDs include Alzheimer's disease,Parkinson's disease,amyotrophic lateral sclerosis,multiple sclerosis and Huntington's disease.However,no direct therapies for NDs exist.In recent years,single cell RNA-sequencing(scRNA-seq)has been widely used in various NDs.The pathogenesis of NDs is closely related to morphology of immune cells,and the pathogenesis mainly involves mitochondrial function,glucose metabolism,inflammation,and synaptic transmission.Induced pluripotent stem cells are a potential therapy for NDs.Ultimately,we review the application of scRNA-seq to various NDs and provide a reference for prevention and treatment of NDs.

【Objective】 To establish a co-expression lncRNA-mRNA ceRNA network and explore the potential molecular mechanism of lncRNA in dengue fever. 【Methods】 DENV-2-infected and normal pHUVEC were sequenced and screened for differentially expressed lncRNA and mRNA by gene microarray technology. Differentially expressed mRNA was analyzed by protein-protein interaction (PPI), and significantly related co-expressed lncRNA-mRNA was screened by Pearson’s correlation coefficient. The microRNA (miRNA) that bound to co-expressed lncRNA-mRNA was predicted by the database. The ceRNA network of co-expressed lncRNA-mRNA was constructed by Cytoscape software. Finally differentially expressed mRNAs and co-expressed lncRNA-mRNA were analyzed by GO and KEGG enrichment, and co-expressed lncRNA-mRNA was verified by RT-qPCR. 【Results】 At 48 h and 72 h after infection, 105 and 51 differentially expressed mRNAs were obtained, respectively, while 59 and 29 differentially expressed lncRNAs were obtained, respectively. Furthermore, at the two time intervals, there were 10 differential mRNAs and 5 differential lncRNAs, respectively. PPI analysis of differential mRNAs showed that isocratic values of interleukin 6 (IL6), interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), and 2’-5’-oligoadenylate synthetase 2 (OAS2) were relatively high. The pairing results of lncRNA-mRNA co-expression analysis with the highest correlation coefficients at 48 h and 72 h after infection were XLOC_001966-SMTNL1 and XLOC_001966-ESR2, respectively. According to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the functions of differentially expressed mRNA and co-expressed lncRNA-mRNA were mainly involved in virus epidemic prevention response, immune response, and signal transduction, as well as the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, type I interferon, and cytokine receptor interaction. RT-qPCR revealed that lncRNA XLOC-I2-8991 was upregulated in the co-expressed lncRNA-mRNA, whereas all the other lncRNA and mRNA were downregulated. 【Conclusion】 This study initially revealed the potential lncRNA-mRNA co-expression network during dengue virus infection, and found that co-expressed lncRNA-mRNA was mainly enriched in the immune regulation and signal transduction pathways during virus infection. The findings will help further exploration into the infection mechanism of DENV-2.