This study systematically investigated the effects of Xihuang Pill on exosomes (Exo) secretion and how Exo pretreated with Xihuang Pill affect intercellular communication, thereby inhibiting the activity of triple-negative breast cancer cells. First, the inhibitory affected of Xihuang Pill on the activity and migration of 4T1 and MDA-MB-231 cells were verified using MTT, scratch assays, and crystal violet assays. Then, Exo and Exo secreted by cells pretreated with Xihuang Pill (XHP-Exo) were obtained by ultracentrifugation. The Exo were characterized by transmission electron microscopy, potential, particle size, and marker proteins. The inhibitory effects of XHP-Exo on the activity and migration of 4T1 and MDA-MB-231 cells were detected using crystal violet and scratch assays. The expression of programmed death ligand 1 (PD-L1), glutathione peroxidase 4 (GPX4), reactive oxygen species (ROS), and reduced glutathione (GSH) in cells was detected by Western blot and immunofluorescence. The chemical components of Xihuang Pill and XHP-Exo were analyzed by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap mass spectrometry (UPLC-Q-Exactive-MS/MS). The results showed that Xihuang Pill could significantly inhibit the proliferation and migration of breast cancer cells, downregulate kidney and brain expressed adaptor protein (KIBRA) expression, and effectively reduce the secretion of exosomes from breast cancer cells. Furthermore, XHP-Exo induced ferroptosis in recipient tumor cells, characterized by increased intracellular ROS levels and decreased GSH content, accompanied by downregulation of PD-L1 expression. Component analysis revealed 19 common active chemical components in both Xihuang Pill and XHP-Exo. In summary, Xihuang Pill inhibits the secretion of exosomes from breast cancer cells, and component-containing exosomes derived from it induce ferroptosis in recipient cells and downregulate PD-L1 expression.

This study aimed to investigate the mechanism of the traditional Chinese medicinal formula Qijing Mingmu (QJMM) in the treatment of conjunctivochalasis (CCH) using network pharmacology and cellular experiments. Effective compounds and action targets of QJMM components were screened using the TCMSP and TCM-ID databases. Disease targets for CCH were obtained from the DisGeNET database, with gene names converted from the UniProt database. The Venny database yielded disease-drug intersection targets, which were input into the STRING database to generate protein-protein interaction (PPI) networks, followed by gene ontology (GO) and KEGG pathway analyses. Cytosccape software was employed to construct target networks for core components and therapeutic effects of QJMM, facilitating molecular docking. Conjunctival tissue was obtained from CCH and normal control groups (NC) (ethics approval number: PTEC-A-2024-30-1), subjected to immunohistochemical staining, and cultured as primary conjunctival fibroblasts. Fibroblasts were divided into NC, CCH, CCH+QJMM, and CCH+ nuclear factor κappa-B (NF-κB) inhibitor pyrrolidine dithiocarbamate (PDTC) groups. Immunofluorescence staining, cytokine assays, Western blot (WB), and quantitative reverse transcription PCR (qRT-PCR) were performed on each group. Results showed that 176 active compounds were screened from QJMM, yielding 293 target molecules in total. CCH identified 27 targets, with 12 overlapping targets identified via Venn diagram analysis. The PPI network diagram indicated tumor necrosis factor (TNF), matrix metalloproteinase (MMP)-2, MMP-9, and interleukin (IL)-6 as key targets. GO analysis yielded 91 enriched entries, while KEGG analysis identified 45 related entries and 110 signaling pathways, revealing the NF-κB signaling pathway. Molecular docking revealed that the core components of CCH exhibited strong binding affinity with the key target NF-κB, indicating potent biological activity. Subsequent immunohistochemical staining demonstrated significantly higher Toll-like receptor 4 (TLR4) and NF-κB expression in conjunctival tissues of the CCH group compared to the control group (P < 0.05). Conjunctival fibroblast experiments confirmed that NF-κB and its upstream factors TLR4 and myeloid differentiation primary response 88 (MYD88) exhibited higher protein and mRNA expression in the CCH group than in the NC group. Furthermore, expression of downstream inflammatory cytokines IL-6, IL-1β, and TNF-α was significantly elevated compared to the NC group (P < 0.05). Following administration of QJMM and PDTC, the expression of NF-κB and its upstream factors TLR4 and MYD88 protein, mRNA and immunofluorescence intensity decreased compared to the CCH group (P < 0.05). In conclusion, QJMM exerts multi-targeted, multi-pathway regulatory effects in treating CCH. Both network pharmacology and experimental studies confirm NF-κB plays a pivotal role in CCH pathogenesis and progression. QJMM effectively treats CCH by inhibiting the NF-κB signaling pathway.

This study aims to investigate the pharmacological effects of procyanidin B3 (Pro B3) on non-alcoholic fatty liver disease (NAFLD) and its molecular mechanism underlying the regulation of fatty acid oxidation (FAO) in hepatocytes during NAFLD, thereby providing experimental evidence for the clinical application of Pro B3 in NAFLD. Mice were fed a high-fat diet (HFD) for 12 weeks to induce NAFLD and received oral gavage administration for 12 weeks. The pharmacological effect of Pro B3 in improving NAFLD was assessed through serum biochemical indexes, liver pathological changes and lipid droplet formation in the liver using Hematoxylin-eosin (H & E) staining and oil red O staining. This study protocol was approved by the Experimental Animal Ethics Committee of Gannan Medical University (approval No.: 2025443). In the in vitro experiments, the HepG2 cells were induced with a mixture of palmitic acid and oleic acid (2∶1) to create a cellular model of NAFLD and evaluate the cytotoxicity and cell viability of ProB3 on HepG2 cells. The expressions of FAO-associated genes such as acyl-CoA synthetase long chain family member 5 (ACSL5), carnitine palmitoyltransferase 1 (CPT1), acetyl-CoA carboxylase (ACC), and GATA binding protein 3 (GATA3) were detected by immunohistochemical, immunofluorescence, qRT-PCR and Western blot. Mechanistically, the JASPAR database and dual-luciferase reporter gene assays were used to explore the transcription factors and binding sites of ACSL5. By silencing GATA3 using siRNA, the effects of Pro B3 on the expression of ACSL5 and lipid deposition were examined. The results demonstrated that Pro B3 significantly improves the NAFLD-related indicators in mice, reduces serum lipid levels and lipid droplet accumulation in HepG2 cells, and up-regulates the expression of FAO-related genes, such as ACSL5, CPT1, ACC. Furthermore, GATA3 might be a transcription factor of ACSL5, and the transcriptional regulatory site might be "ACATAG". Moreover, Pro B3 significantly upregulated the expression of GATA3 mRNA and protein levels, while silencing GATA3 significantly inhibited the expression of ACSL5 mRNA and protein levels, as well as weakened the inhibitory effect of Pro B3 on lipid accumulation in HepG2 cells. In conclusion, Pro B3 may be an effective lead compound for treating NAFLD by promoting FAO in hepatocytes through regulation of the GATA3/ACSL5 pathway, thereby ameliorating NAFLD.

Exploring cellular senescence inducers is one of the approaches to screen anti-tumor active lead compounds. Whether glaucocalyxin B induces cellular senescence remains to be elucidated. This study aims to investigate the effect and mechanism of glaucocalyxin B on the senescence of lung adenocarcinoma A549 cells. 2 and 5 μmol·L^-1 of glaucocalyxin B induced senescence in A549 cells. Glaucocalyxin B significantly increased the levels of cyclin-dependent kinase inhibitor 1A (p21) and the proportion of β-galactosidase staining-positive cells in A549 cells. Glaucocalyxin B had no significant effect on the levels of peroxiredoxin Ⅰ (Prdx Ⅰ) protein in A549 cells but significantly inhibited its enzymatic activity. Knockdown of Prdx Ⅰ induced senescence in A549 cells. Glaucocalyxin B inhibited the enzyme activity of Prdx Ⅰ, leading to the increase of reactive oxygen species (ROS), and the ROS scavenger N-acetylcysteine attenuated glaucocalyxin B-induced the senescence of A549 cells. Either glaucocalyxin B or Prdx Ⅰ knockdown significantly upregulated the levels of tumor protein p53 in A549 cells, while knockdown of p53 significantly inhibited glaucocalyxin B-induced the senescence of A549 cells. In conclusion, glaucocalyxin B can induce senescence in lung adenocarcinoma cells by inhibiting the Prdx Ⅰ/ROS/p53 pathway, which might be one of its anti-tumor mechanisms.

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that establishes latent infection in the trigeminal ganglia following primary infection. Its reactivation can be triggered by both internal and external factors, including heat stress, emotional stress, excessive consumption of warm-natured foods, and the administration of tonic and warming traditional Chinese medicines (TCMs). The fire-heat symptoms in the upper facial region induced by HSV-1 reactivation are consistent with the "Shanghuo" syndrome described in TCM. In this study, HSV-1 infection models established by corneal scarification and intranasal inoculation were systematically compared. Dynamic changes throughout the entire "primary infection-latency-reactivation" cycle were monitored using multiple parameters, including mortality, body weight, ocular manifestations, viral glycoprotein B (gB) mRNA expression, and restless behavior. This study aimed to provide a basis for model selection in investigating the biological mechanisms underlying TCM "Shanghuo" and for screening anti-HSV-1 therapeutics. The results showed that during the primary infection phase (0-14 d), mice in both groups exhibited body weight loss and ocular surface inflammation, with an earlier onset (3 d vs 5 d) and more severe ocular damage observed in the corneal scarification group. During the latency phase (28 days post-infection), latency-associated transcript (LAT) was detected in the trigeminal ganglia in both groups, with a higher latency rate in the corneal scarification group (100% vs 87.5%). Following heat stress-induced reactivation, the intranasal inoculation group exhibited a significantly higher reactivation rate (85.7% vs 66.6%), more pronounced restless behavior, and elevated levels of gB mRNA expression and viral titers in brain tissue compared with the corneal scarification group. In conclusion, both corneal scarification and intranasal inoculation successfully establish HSV-1 models encompassing the full cycle of primary infection, latency, and reactivation. Dynamic monitoring of infection stages can be achieved by detecting gB mRNA in eye wash fluid. The corneal scarification model, characterized by a high latency rate and prominent ocular manifestations, is suitable for studies of viral keratitis and related therapeutic evaluation. In contrast, the intranasal inoculation model, with a higher reactivation rate and marked restless behavior, exhibits similarities to the restless manifestations of the TCM "Shanghuo" heat pattern, suggesting its potential utility in evaluating the efficacy of heat-clearing TCMs. Animal experiments conducted in this study were approved by the Ethics Committee of Jinan University (approval No. IACUC-20260311-06).

It is well established that baicalin induces the polarization of macrophages toward the M1 phenotype via glycolytic pathways; however, the underlying regulatory mechanisms remain unclear. This study aimed to identify the key upstream targets and regulated transcription factors involved in baicalin's regulation of glycolytic metabolism. Using the RAW264.7 cell model, RNA-seq and ATAC-seq technologies were employed, combined with Western blot, qRT-PCR, siRNA, and dual-luciferase reporter assays for detection and validation. The results showed that baicalin, at concentration of 100 μmol·L^-1, promoted RAW264.7 cell M1 polarization accompanied by high expression of Trem1; co-treatment with the Trem1 inhibitor LP17 reversed baicalin-mediated RAW264.7 cell polarization and the activation of downstream glycolytic signaling pathways. Among the transcription factors associated with the Trem1 promoter region, Fos was shown to be involved in the regulation of RAW264.7 cell polarization by baicalin. This study reveals that baicalin promotes downstream glycolytic metabolism by regulating the transcription factor Fos to activate Trem1, thereby driving macrophage polarization toward the M1 phenotype, providing a new metabolic mechanism to support its therapeutic potential in tumor immunoregulation and inflammation-related diseases.

To rapidly screen natural antibacterial active components targeting lipopolysaccharide (LPS) through the detection of traditional Chinese medicine (TCM) monomers via plasmonic near-field sensing. In this study, a plasmonic near-field biosensor based on gold nanoislands (AuNIs) and LPS (LPS@AuNIs biosensor) was constructed, and the LPS antagonistic activity of natural products derived from TCM was evaluated by the change in phase response. The limit of detection (LOD) and limit of quantification (LOQ) of the LPS@AuNIs biosensor for polymyxin B (PB), which specifically binds to LPS, were 6.34 ± 0.002 374 and 61.7 ± 0.001 735 nmol·L^-1, respectively. Detection results of 14 TCM monomers showed that the LPS@AuNIs biosensor exhibited excellent concentration-dependent responses to both 1, 2, 3, 4, 6-O-pentagalloylglucose (β-PGG) and geniposide (GP). Comparative analysis revealed that β-PGG displayed potent binding activity, with a dissociation constant (k_a) superior to that of PB. Further verification via minimum inhibitory concentration (MIC) and displacement assays confirmed that β-PGG possessed strong binding affinity comparable to PB. The developed biosensor enables specific binding between LPS and analytes, featuring high sensitivity and detection efficiency for high-precision quantitative analysis. This study innovatively integrates nanophotonics with natural medicine research, establishing an integrated system of "drug screening-target validation" and providing a high-efficiency technical platform for the rapid and effective screening of natural antibacterial active components.

Polypyrimidine tract-binding protein 1 (PTBP1) is a critical regulatory target in cancers and neurological disorders. However, the development of its small-molecule inhibitors has been hampered by the lack of traditional druggable pockets on its RNA-binding domains. This study employed a computer-aided drug design (CADD) strategy focusing on a dynamic binding pocket mediated by the flexible linker3 within the PTBP1 RBD3/4 domain. A commercial library containing approximately 1.6 million compounds was subjected to structure-based virtual screening. Following initial filtering based on lead-likeness rules and subsequent molecular docking, 14 candidate compounds were synthesized. Their binding activities were experimentally validated using prokaryotically expressed and purified PTBP1-RBD3/4 recombinant protein via bio-layer interferometry (BLI). Six compounds with micromolar affinity were identified, among which C17414 exhibited the highest binding affinity (K_D = 1.6 µmol·L^-1). Molecular docking analysis revealed that C17414 efficiently occupies the target pocket through multiple interactions, including hydrogen bonds, salt bridges, π-π stacking and hydrophobic interactions. Systematic ADMET (absorption, distribution, metabolism, excretion and toxicity) property predictions indicated that C17414 and C7485 possess a favorable balance of lipophilicity, polar surface area and membrane permeability, suggesting promising drug-likeness and potential for central nervous system drug development. This work not only discovers a series of high-affinity PTBP1 inhibitor hits and validates the feasibility of targeting its dynamic pocket but also lays a solid foundation for developing novel therapies against PTBP1.

Nuclear receptor-binding SET domain protein 3 (NSD3) plays an important role in the initiation and progression of lung squamous cell carcinoma (LUSC), whereas small-molecule inhibitors targeting NSD3 remain limited. This study targeted the NSD3-PWWP1 domain and identified 37 potential NSD3-binding compounds through Glide-based hierarchical virtual screening followed by drug-likeness filtering. Surface plasmon resonance (SPR) analysis showed that compounds 2, 4, 20 and 27 could directly bind to NSD3, among which compound 2 exhibited the strongest equilibrium dissociation constant (K_D = 0.95 μmol·L^-1), approaching that of the positive control BI9321 (0.46 μmol·L^-1). Molecular docking analysis indicated that all four candidate compounds could occupy the NSD3-PWWP1 binding pocket and form stable interactions with key residues. At 30 μmol·L^-1, the four candidate compounds showed different degrees of inhibitory activity against three NSD3-high LUSC cell lines. Notably, compound 2 displayed a half maximal inhibitory concentration (IC₅₀) of 4.08 ± 1.01 μmol·L^-1 in HCC95 cells, which was superior to that of BI9321 (17.62 ± 3.11 μmol·L^-1). Meanwhile, these candidate compounds did not show obvious cytotoxicity toward HEK293T cells and exhibited favorable ADMET profiles and physicochemical property distributions. In conclusion, several potential NSD3 inhibitors were identified in this study. Among them, compound 2, a novel coumarin-based scaffold distinct from BI9321, represents a promising lead for further optimization as an anti-LUSC agent.

In this study ten new compounds Sd-01-Sd-10 were synthesized via acylation from sitagliptin and various fatty acyl chlorides. Animal experiments approved by the Institutional Animal Care and Use Committee of Jilin Provincial Institute of Pharmaceutical Research (approval No. 2025001). The target compounds were evaluated for their in vivo hypoglycemic activity using a type 2 diabetic mouse model, with subsequent structure-activity relationship analysis and further assessment of the most active compounds via an oral glucose tolerance test. Meanwhile, a maximum feasible dose test was conducted in healthy mice for preliminary safety evaluation. The results indicated that this series of compounds exhibited promising hypoglycemic activity and a high level of safety, which was supported by the observation of no abnormal behavior or mortality in healthy mice after a single oral dose of 2 000 mg·kg^-1. Among them, Sd-06 showed activity comparable to sitagliptin, whereas Sd-05 was more potent. Notably, Sd-05 at the lower dose of 30 mg·kg^-1 effectively improved glucose tolerance in normal mice, surpassing the efficacy of sitagliptin at 50 mg·kg^-1, highlighting its value for further development.