OBJECTIVE: To explore the potential effects and mechanisms of Liang-Ge-San (LGS) for the treatment of acute respiratory distress syndrome (ARDS) through network pharmacology analysis and to verify LGS activity through biological experiments. METHODS: The key ingredients of LGS and related targets were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. ARDS-related targets were selected from GeneCards and DisGeNET databases. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed using the Metascape Database. Molecular docking analysis was used to confirm the binding affinity of the core compounds with key therapeutic targets. Finally, the effects of LGS on key signaling pathways and biological processes were determined by in vitro and in vivo experiments. RESULTS: A total of LGS-related targets and 496 ARDS-related targets were obtained from the databases. Network pharmacological analysis suggested that LGS could treat ARDS based on the following information: LGS ingredients luteolin, wogonin, and baicalein may be potential candidate agents. Mitogen-activated protein kinase 14 (MAPK14), recombinant V-Rel reticuloendotheliosis viral oncogene homolog A (RELA), and tumor necrosis factor alpha (TNF-α) may be potential therapeutic targets. Reactive oxygen species metabolic process and the apoptotic signaling pathway were the main biological processes. The p38MAPK/NF-κ B signaling pathway might be the key signaling pathway activated by LGS against ARDS. Moreover, molecular docking demonstrated that luteolin, wogonin, and baicalein had a good binding affinity with MAPK14, RELA, and TNF α. In vitro experiments, LGS inhibited the expression and entry of p38 and p65 into the nucleation in human bronchial epithelial cells (HBE) cells induced by LPS, inhibited the inflammatory response and oxidative stress response, and inhibited HBE cell apoptosis (P<0.05 or P<0.01). In vivo experiments, LGS improved lung injury caused by ligation and puncture, reduced inflammatory responses, and inhibited the activation of p38MAPK and p65 (P<0.05 or P<0.01). CONCLUSION LGS could reduce reactive oxygen species and inflammatory cytokine production by inhibiting p38MAPK/NF-κ B signaling pathway, thus reducing apoptosis and attenuating ARDS.
Drugs, Chinese Herbal/pharmacology* ; Respiratory Distress Syndrome/enzymology* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; NF-kappa B/metabolism* ; Animals ; Signal Transduction/drug effects* ; Molecular Docking Simulation ; Humans ; Male ; Network Pharmacology ; Apoptosis/drug effects* ; Mice

OBJECTIVE: To investigate the anti-inflammatory effect of rutaecarpine (RUT) on monosodium urate crystal (MSU)-induced murine peritonitis in mice and further explored the underlying mechanism of RUT in lipopolysaccharide (LPS)/MSU-induced gout model in vitro. METHODS: In MSU-induced mice, 36 male C57BL/6 mice were randomly divided into 6 groups of 8 mice each group, including the control group, model group, RUT low-, medium-, and high-doses groups, and prednisone acetate group. The mice in each group were orally administered the corresponding drugs or vehicle once a day for 7 consecutive days. The gout inflammation model was established by intraperitoneal injection of MSU to evaluate the anti-gout inflammatory effects of RUT. Then the proinflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and the proportions of infiltrating neutrophils cytokines were detected by flow cytometry. In LPS/MSU-treated or untreated THP-1 macrophages, cell viability was observed by cell counting kit 8 and proinflammatory cytokines were measured by ELISA. The percentage of pyroptotic cells were detected by flow cytometry. Respectively, the mRNA and protein levels were measured by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot, the nuclear translocation of nuclear factor κB (NF-κB) p65 was observed by laser confocal imaging. Additionally, surface plasmon resonance (SPR) and molecular docking were applied to validate the binding ability of RUT components to tumor necrosis factor α (TNF-α) targets. RESULTS: RUT reduced the levels of infiltrating neutrophils and monocytes and decreased the levels of the proinflammatory cytokines interleukin 1β (IL-1β) and interleukin 6 (IL-6, all P<0.01). In vitro, RUT reduced the production of IL-1β, IL-6 and TNF-α. In addition, RT-PCR revealed the inhibitory effects of RUT on the mRNA levels of IL-1β, IL-6, cyclooxygenase-2 and TNF-α (P<0.05 or P<0.01). Mechanistically, RUT markedly reduced protein expressions of tumor necrosis factor receptor (TNFR), phospho-mitogen-activated protein kinase (p-MAPK), phospho-extracellular signal-regulated kinase, phospho-c-Jun N-terminal kinase, phospho-NF-κB, phospho-kinase α/β, NOD-like receptor thermal protein domain associated protein 3 (NLRPS), cleaved-cysteinyl aspartate specific proteinase-1 and cleaved-gasdermin D in macrophages (P<0.05 or P<0.01). Molecularly, SPR revealed that RUT bound to TNF-α with a calculated equilibrium dissociation constant of 31.7 µmol/L. Molecular docking further confirmed that RUT could interact directly with the TNF-α protein via hydrogen bonding, van der Waals interactions, and carbon-hydrogen bonding. CONCLUSION RUT alleviated MSU-induced peritonitis and inhibited the TNFR1-MAPK/NF-κB and NLRP3 inflammasome signaling pathway to attenuate gouty inflammation induced by LPS/MSU in THP-1 macrophages, suggesting that RUT could be a potential therapeutic candidate for gout.
Animals ; NF-kappa B/metabolism* ; Male ; Indole Alkaloids/therapeutic use* ; Signal Transduction/drug effects* ; Mice, Inbred C57BL ; Inflammation/complications* ; Uric Acid ; Quinazolines/therapeutic use* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Humans ; Gout/chemically induced* ; Inflammasomes/metabolism* ; Cytokines/metabolism* ; THP-1 Cells ; Mitogen-Activated Protein Kinases/metabolism* ; Mice ; Molecular Docking Simulation ; Lipopolysaccharides ; Quinazolinones

OBJECTIVES: Pyroptosis plays a critical role in tubulointerstitial lesions of diabetic kidney disease (DKD). Annexin A2 (ANXA2) is involved in cell proliferation, apoptosis, and adhesion and may be closely related to DKD, but its specific mechanism remains unclear. This study aims to investigate the role and molecular mechanism of ANXA2 in high glucose-induced pyroptosis of renal tubular epithelial cells, providing new targets for DKD prevention and treatment. METHODS: Human renal tubular epithelial HK-2 cells were divided into a normal glucose group (5.5 mmol/L), a high glucose group (30.0 mmol/L), and a osmotic control group (24.5 mmol/L mannitol+5.5 mmol/L glucose). ANXA2 expression was modulated by overexpression of plasmids and small interfering RNA (siRNA). Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) assay, apoptosis by flow cytometry, and ANXA2, p50, and p65 subcellular localization by immunofluorescence. Western blotting was employed to detect α-smooth muscle actin (α-SMA), fibronectin (FN), and collagen type IV (Col-IV). Real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting were used to analyze nuclear factor-κB (NF-κB) subunits p50/p65 and the pyroptosis pathway factors NLR family Pyrin domain containing 3 (NLRP3), caspase-1, inferleukin (IL)-1β, and IL-18. Protein interactions between ANXA2 and p50/p65 were examined by co-immunoprecipitation, while chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were used to examine NF-κB binding to the ANXA2 promoter. RESULTS: High glucose upregulated ANXA2 expression and promoted its nuclear translocation (P<0.01). High glucose reduced cell proliferation, increased apoptosis, and elevated α-SMA, FN, and Col-IV expression (all P<0.05); ANXA2 overexpression aggravated these effects (all P<0.05), while ANXA2 knockdown reversed them (all P<0.05). High glucose activated NF-κB and increased NLRP3, caspase-1, L-1β, and IL-18 mRNA and protein expression (all P<0.05); ANXA2 overexpression further enhanced this, whereas knockdown suppressed NF-κB activation and downstream factors (all P<0.05). Co-immunoprecipitation confirmed ANXA2 directly binds the NF-κB subunit p65. ChIP assays revealed p65 binds specifically to ANXA2 promoter regions (ChIP-2, ChIP-4, and ChIP-6), and luciferase activity in corresponding mutant constructs (M2, M4, and M6) was significantly increased versus controls (all P<0.05), confirming positive transcriptional regulation of ANXA2 by p65. CONCLUSIONS ANXA2 and NF-κB form a positive feedback loop that sustains NLRP3 inflammasome activation, promotes pyroptosis pathway activation, and aggravates high glucose-induced renal tubular epithelial cell injury. Targeting ANXA2 or blocking its interaction with p65 may be a novel strategy to slow DKD progression.
Humans ; Pyroptosis/drug effects* ; Annexin A2/physiology* ; Epithelial Cells/cytology* ; Kidney Tubules/cytology* ; Glucose/pharmacology* ; Diabetic Nephropathies/metabolism* ; NF-kappa B/metabolism* ; Cell Line ; Cell Proliferation ; Transcription Factor RelA/metabolism* ; Feedback, Physiological

Stathmin 1 (STMN1) is a microtubule-binding cytoplasmic phosphoprotein that promotes microtubule depolymerization or inhibits microtubule assembly, thereby regulating cytoskeletal organization and cell cycle progression. STMN1 is upregulated in a variety of malignant tumors, where it drives proliferation, invasion, metastasis, and angiogenesis through classic pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and ferroptosis. STMN1 can also modulate the function of immune cells, thereby influencing antitumor immunity. Clinical data show that its high expression correlates positively with tumor drug resistance and poor prognosis, suggesting that STMN1 has potential as a tumor biomarker and therapeutic molecular target with important clinical significance.
Humans ; Stathmin/metabolism* ; Neoplasms/genetics* ; Biomarkers, Tumor/metabolism* ; NF-kappa B/metabolism* ; Cell Proliferation ; Drug Resistance, Neoplasm

Chronic obstructive pulmonary disease (COPD) currently lacks effective treatments to halt disease progression, making the search for preventive and therapeutic drugs a pressing issue. Natural products, with their accessibility, affordability, and low toxicity, offer promising avenues. Investigating the pharmacological effects and related signaling mechanisms of active components from natural products on COPD animal models induced by various triggers has become an important focus. In animal models induced by cigarette smoke, cigarette smoke combined with lipopolysaccharide (LPS), air pollution, elastase, bacterial or viral infections, the active compounds of natural products, such as flavonoids, terpenoids, and phenolics, can exert anti-inflammatory, antioxidant, mucus-regulating, and airway remodeling-inhibiting effects through key signaling pathways including nuclear factor-erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1), nuclear factor-kappa B (NF-κB), and mitogen-activated protein kinase (MAPK). These findings not only provide a theoretical basis for the clinical diagnosis and treatment of COPD but also point to new directions for future scientific research.
Pulmonary Disease, Chronic Obstructive/etiology* ; Animals ; Disease Models, Animal ; Biological Products/pharmacology* ; Humans ; NF-kappa B/metabolism* ; Flavonoids/pharmacology* ; Signal Transduction/drug effects* ; Anti-Inflammatory Agents/pharmacology* ; Heme Oxygenase-1/metabolism* ; Terpenes/pharmacology* ; Antioxidants/pharmacology* ; NF-E2-Related Factor 2/metabolism* ; Smoke/adverse effects* ; Phenols/therapeutic use*

OBJECTIVES: To investigate the expression of CDKN3 in gastric cancer and its impact on prognosis of gastric cancer patients. METHODS: We analyzed CDKN3 expression in clinical specimens from 114 gastric cancer patients and assessed its association with 5-year postoperative survival of the patients. GO and KEGG enrichment analyses were used to predict the biological function and possible mechanism of CDKN3. The effects of lentivirus-mediated CDKN3 knockdown on biological behaviors of gastric cancer cells were evaluated using Transwell assay, CCK-8 assay, TUNEL staining, flow cytometry, and Western blotting. RESULTS: CDKN3 expression was significantly higher in gastric cancer tissues than in the adjacent tissues with significant correlations with CEA level, CA19-9 level, and T and N staging (P<0.05). High CDKN3 expression was an independent risk factor affecting 5-year postoperative survival of the patients and predictive for long-term prognosis (P<0.01). Enrichment analyses suggested a probable association of CDKN3 with apoptosis. In MGC-803 cells, CDKN3 knockdown significantly lowered migration and invasion capacities of the cells, while CDKN3 overexpression produced the opposite effects. TUNEL staining revealed a significantly lower level of cell apoptosis in gastric cancer tissues than in adjacent tissues (P<0.01). CDKN3 knockdown obviously inhibited proliferation and increased apoptosis of MGC-803 cells. CDKN3 overexpression down-regulated the expressions of p53, p21 and Bax and up-regulated the expressions of p-p65 and Bcl-2. CONCLUSIONS CDKN3 is highly expressed in gastric cancer tissues and affects patient prognosis. CDKN3 overexpression promotes proliferation, invasion and migration and suppressed apoptosis of gastric cancer cells possibly through the p53/NF-κB signaling pathway.
Humans ; Stomach Neoplasms/pathology* ; Apoptosis ; Signal Transduction ; Tumor Suppressor Protein p53/metabolism* ; Cell Movement ; Cell Line, Tumor ; NF-kappa B/metabolism* ; Prognosis ; Cyclin-Dependent Kinase Inhibitor Proteins/metabolism* ; Cell Proliferation ; Neoplasm Invasiveness ; Male ; Female ; Dual-Specificity Phosphatases

OBJECTIVES: To investigate the therapeutic mechanism of Huoxue Shufeng Granules (HXSFG) for alleviating central sensitization in a mouse model of chronic migraine (CM). METHODS: We analyzed the main chemical components of HXSFG through literature review and explored their pharmacological mechanisms by bioinformatics analyses. In a male C57BL/6J mouse model of CM established by intraperitoneal injections of nitroglycerin (10 mg/kg) every other day (5 injections), the effects of gavage with low, and high doses of HXSFG or intraperitoneal injections of topiramate for ameliorating central sensitization were evaluated using Von Frey test and a hot plate apparatus; the changes in expressions of inflammatory factors, the proteins in the TLR4/NF‑κB signaling pathway, and activation of c-Fos and CGRP were detected using RT-qPCR, Western blotting and immunofluorescence staining. RESULTS: Network pharmacology analysis suggested that the main active components in HXSFG for alleviating CM included formononetin, paeoniflorin, quercetin, and tanshinone. Gene Ontology (GO) enrichment analysis identified 492 GO entries, comprising 366 biological processes, 46 cellular components, and 80 molecular functions. KEGG pathway enrichment analysis indicated that the Toll-like receptor and NF‑κB signaling pathways were crucial in mediating the therapeutic effects of HXSFG on CM. In the mouse models of CM, both topiramate and HXSFG treatments alleviated the symptoms of central sensitization, evidenced by improved mechanical and thermal pain thresholds in the mice. HXSFG significantly reduced the expression of c-Fos and CGRP, improved inflammatory markers, and downregulated the expressions of TLR4, p-NF‑κB, IL-1β, and TNF‑α proteins in the mouse models. CONCLUSIONS HXSFG effectively alleviates central sensitization in CM mice by modulating the inflammatory pathways and inhibiting the TLR4/ NF-κB signaling pathway, suggesting its potential as a therapeutic option for CM.
Animals ; Toll-Like Receptor 4/metabolism* ; NF-kappa B/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Mice ; Male ; Mice, Inbred C57BL ; Signal Transduction/drug effects* ; Migraine Disorders/metabolism* ; Disease Models, Animal ; Inflammation

OBJECTIVES: To investigate the role of activating transcription factor 3 (ATF3) in atherosclerotic plaques for regulating inflammatory responses during atherosclerosis (AS) progression. METHODS: Human coronary artery specimens from autopsy cases were examined for ATF3 protein expression and localization using immunofluorescence staining and Western blotting. Apolipoprotein E-deficient (ApoE^-/-) mouse models of AS induced by high-fat diet (HFD) feeding for 12 weeks were subjected to tail vein injection of adeno-associated virus serotype 9 (AAV9) to knock down ATF3 expression. After an additional 5 weeks of HFD feeding, the mice were euthanized for analyzing structural changes of the aortic plaques, and the expression levels of ATF3, inflammatory factors (CD45, CD68, IL-1β, and TNF-α), and NF-κB pathway proteins (P-IKKα/β and P-NF-κB p65) were detected. In the cell experiment, THP-1-derived foam cells were transfected with an ATF3-overexpressing plasmid or an ATF3-specific siRNA to validate the relationship between ATF3 and NF‑κB signaling. RESULTS: In human atherosclerotic plaques, ATF3 expression was significantly elevated and partially co-localized with CD68. ATF3 knockout in ApoE^-/- mice significantly increased aortic plaque volume, upregulated the inflammatory factors, enhanced phosphorylation of the NF‑κB pathway proteins, and increased the expressions of VCAM1, MMP9, and MMP2 in the plaques. In THP-1-derived foam cells, ATF3 silencing caused activation of the NF‑κB pathway, while ATF3 overexpression suppressed the activity of the NF-κB pathway. CONCLUSIONS AS promotes ATF3 expression, and ATF3 deficiency exacerbates AS progression by enhancing plaque inflammation via activating the NF-κB pathway, suggesting the potential of ATF3 as a therapeutic target for AS.
Animals ; Activating Transcription Factor 3/metabolism* ; Signal Transduction ; NF-kappa B/metabolism* ; Humans ; Mice ; Plaque, Atherosclerotic/metabolism* ; Inflammation/metabolism* ; Apolipoproteins E ; Atherosclerosis/metabolism* ; Diet, High-Fat

OBJECTIVES: To evaluate the protective effect of Bufei Yishen Formula (BYF) against cigarette smoke extract (CSE)-induced injuries in human bronchial epithelial BEAS-2B cells and explore the underlying mechanism. METHODS: BEAS-2B cells exposed to CSE were treated with normal rat serum, BYF-medicated rat serum at low or high doses, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor), PDTC combined with high-dose BYF-medicated serum, or S-carbomethyloysteine (S-CMC, as the positive control). CCK-8 assay was used to determine the optimal concentration and treatment time of CSE, BYF-medicated serum and S-CMC. The treated cells were examined for inflammatory factor levels in the supernatant and cellular expressions of MUC5AC and MUC5B using ELISA, cell ultrastructural changes with transmission electron microscopy, and cell apoptosis rate using flow cytometry. The expression levels of TLR4/NF‑κB pathway-associated mRNAs and proteins were determined by qRT-PCR and Western blotting. RESULTS: CSE exposure significantly increased secretions of IL-1β, IL-6 and TNF-α, mRNA and protein expressions of MUC5AC and MUC5B, and early and total apoptosis rates in BEAS-2B cells, where the presence of apoptotic bodies was detected. CSE also significantly enhanced the mRNA and protein expressions of TLR4, I-κB, and NF-κB and reduced mRNA and protein expressions of AQP5. Treatments of the CSE-exposed cells with BYF-medicated serum, PDTC and S-CMC all significantly lowered inflammatory factor levels, MUC5AC and MUC5B expressions, and early and total cell apoptosis rates, and partly reversed the changes in cellular ultrastructure and mRNA and protein expressions of the TLR4/NF-κB pathway, and the effects were the most conspicuous following the combined treatment with high-dose BYF-medicated serum and PDTC. CONCLUSIONS BYF can inhibit cell apoptosis, inflammation and mucus hypersecretion in CSE-induced BEAS-2B cells by inhibiting the TLR4/NF-κB signaling pathway.
Humans ; Epithelial Cells/cytology* ; Drugs, Chinese Herbal/pharmacology* ; NF-kappa B/metabolism* ; Bronchi/cytology* ; Smoke/adverse effects* ; Apoptosis/drug effects* ; Mucin 5AC/metabolism* ; Cell Line ; Toll-Like Receptor 4/metabolism* ; Mucin-5B/metabolism* ; Signal Transduction/drug effects* ; Nicotiana ; Rats ; Thiocarbamates/pharmacology* ; Animals

OBJECTIVES: To investigate pharmacologically active components of Yiqi Zishen Formula (YZF) and their mechanisms for alleviating airway inflammation in mice with chronic obstructive pulmonary disease (COPD). METHODS: Ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry was employed to characterize the chemical components in YZF and YZF-medicated rat serum. A compound-disease target network was constructed based on serum components of YZF to screen the key pathways and targets using enrichment analysis. A mouse model of cigarette smoke-induced COPD was used to evaluate the anti-inflammatory effect of YZF and validate the expression of key proteins in network pharmacology-enriched pathways. Fifty male C57BL/6J mice were randomized equally into control group, COPD model group, high- and low-dose YZF treatment groups, and N-acetylcysteine treatment group. Pulmonary function of the mice was assessed using whole-body plethysmography, and lung histopathology, alveolar structure, and airway remodeling were analyzed using HE staining. The levels of IL-1β, IL-6, and TNF‑α in bronchoalveolar lavage fluid (BALF) were determined with ELISA, and pulmonary expressions of PI3K, Akt, phosphorylated Akt (p-Akt), p65, and phosphorylated p65 (p-p65) were detected using immunohistochemistry. RESULTS: We identified a total of 156 chemical components (including 26 flavonoids or flavonoid glycosides, 27 alkaloids, and 11 saponins) in YZF and 43 prototype components in medicated rat serum. Network pharmacology revealed 704 YZF-related targets and 1199 COPD-associated targets. Integrated analysis suggested that the anti-COPD effects of YZF were associated with the PI3K-Akt signaling pathway. In mouse models of COPD, YZF treatment significantly increased mean alveolar number and peak expiratory flow (P<0.05), reduced mean linear intercept, bronchial wall thickness, lung coefficient, and BALF cytokine levels, and suppressed the expressions of PI3K, Akt, p-Akt, p65, and p-p65 in the lung tissues. CONCLUSIONS YZF alleviates COPD symptoms and airway inflammation in mice possibly by inhibiting the PI3K/Akt/NF‑κB pathway through its multiple components that interact with multiple targets.
Animals ; Pulmonary Disease, Chronic Obstructive/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Signal Transduction/drug effects* ; Male ; Mice, Inbred C57BL ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; NF-kappa B/metabolism* ; Inflammation/drug therapy* ; Rats