OBJECTIVES: To investigate the effect of avitinib for suppressing NLRP3 inflammasome activation and alleviating septic shock and explore the underlying mechanism. METHODS: Mouse bone marrow-derived macrophages (BMDM), human monocytic leukemia cell line THP-1, and peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers were pre-treated with avitinib, followed by activation of the canonical NLRP3 inflammasome using agonists including nigericin, monosodium urate (MSU) crystals, or adenosine triphosphate (ATP). Non-canonical NLRP3 inflammasome activation was induced via intracellular transfection of lipopolysaccharide (LPS). Western blotting was used to detect the secretory protein markers of NLRP3 inflammasome activation and assess pyroptosis, and the levels of inflammatory cytokines in cell culture supernatant were determined with ELISA. In a mouse model of LPS-induced septic shock, the effect of avitinib treatment on the levels of inflammatory cytokines in serum and peritoneal lavage fluid were examined with ELISA, and survival curves of the mice were plotted using the Kaplan-Meier method. RESULTS: Avitinib significantly inhibited NLRP3 inflammasome activation in multiple cell types, and dose-dependently reduced IL-1β secretion and caspase-1 cleavage while suppressing GSDMD-mediated pyroptosis without obviously affecting IL-6 or TNF-α levels. In the mouse models of LPS-induced septic shock, avitinib significantly lowered IL-1β levels in serum and peritoneal fluid and extended survival time of the mice. CONCLUSIONS Avitinib suppresses NLRP3 inflammasome activation and alleviates septic shock in mice.
Animals ; Shock, Septic/metabolism* ; Mice ; NLR Family, Pyrin Domain-Containing 3 Protein ; Inflammasomes/drug effects* ; Humans ; Macrophages/metabolism* ; Interleukin-1beta/metabolism* ; Lipopolysaccharides

OBJECTIVES: To explore the toxic mechanism of Clostridium perfringens Beta1 toxin mediated by P2X7 receptor-induced calcium dyshomeostasis. METHODS: Ten-day-old BALB/c mice were randomly divided into control group, recombinant Beta1 toxin (rCPB1) group, PD151746 group, and PD151746+rCPB1 group, and all the treatment agents were administered by gavage. The changes in expressions of inflammatory factors in the jejunum of the mice were detected using antibody chip technology to explore the regulatory role of calcium dyshomeostasis in Beta1 toxin-induced inflammatory injury level. In the cell experiment, THP-1 cells were transfected with a si-RNA targeting P2X7 receptor and treated with rCPB1, and the changes in cell survival rate, levels of Ca²⁺, ROS and ATP, and expressions of pyroptosis and ferroptosis markers were determined. RESULTS: Oral administration of rCPB1 significantly increased the levels of inflammatory cytokines in the jejunal tissue of the neonatal mice, but their levels were significantly decreased after treatment with PD151746. In THP-1 cells, rCPB1 treatment significantly decreased cell survival and increased the levels of Ca²⁺, ROS, ATP and the expressions of pyroptosis and ferroptosis markers, and these changes were obviously attenuated by P2X7 receptor knockdown. CONCLUSIONS P2X7 receptor-mediated functional pore formation by Beta1 toxin can further lead to calcium dyshomeostasis, thereby triggering excessive accumulation of ROS to subsequently induce the co-occurrence of pyroptosis and ferroptosis.
Animals ; Pyroptosis/drug effects* ; Receptors, Purinergic P2X7/metabolism* ; Mice ; Mice, Inbred BALB C ; Ferroptosis/drug effects* ; Humans ; Calcium/metabolism* ; Macrophages/drug effects* ; Bacterial Toxins/toxicity*

OBJECTIVES: To investigate the mechanism through which pinostrobin (PSB) alleviates dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: C57BL/6 mice were randomized into control group, DSS model group, and PSB intervention (30, 60, and 120 mg/kg) groups. Colitis severity of the mice was assessed by examining body weight changes, disease activity index (DAI), colon length, and histopathology. The expressions of tight junction proteins ZO-1 and claudin-1 in the colon tissues were examined using immunofluorescence staining, and macrophage infiltration and polarization were analyzed with flow cytometry. ELISA and RT-qPCR were used for detecting the expressions of inflammatory factors (TNF‑α and IL-6) and chemokines (CCL₂, CXCL₁₀, and CX₃CL₁) in the colon tissues, and PI3K/AKT phosphorylation levels were analyzed with Western blotting. In cultured Caco-2 and RAW264.7 cells, the effect of PSB on CCL₂-mediated macrophage migration was assessed using Transwell assay. Network pharmacology analysis was performed to predict the key pathways that mediate the therapeutic effect of PSB. RESULTS: In DSS-induced mouse models, PSB at 60 mg/kg optimally alleviated colitis, shown by reduced weight loss and DAI scores and increased colon length. PSB treatment significantly upregulated ZO-1 and claudin-1 expressions in the colon tissues, inhibited colonic macrophage infiltration, and promoted the shift of macrophage polarization from M1 to M2 type. In cultured intestinal epithelial cells, PSB significantly inhibited PI3K/AKT phosphorylation and suppressed chemokine CCL₂ expression. PSB treatment obviously blocked CCL₂-mediated macrophage migration of RAW264.7 cells, which could be reversed by exogenous CCL₂. Network pharmacology analysis and rescue experiments confirmed PI3K/AKT and CCL₂ signaling as the core targets of PSB. CONCLUSIONS PSB alleviates DSS-induced colitis in mice by targeting intestinal epithelial PI3K/AKT signaling, reducing CCL₂ secretion, and blocking macrophage chemotaxis and migration, highlighting the potential of PSB as a novel natural compound for treatment of inflammatory bowel disease.
Animals ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases/metabolism* ; Colitis/drug therapy* ; Dextran Sulfate ; Proto-Oncogene Proteins c-akt/metabolism* ; Macrophages ; Chemokine CCL2/metabolism* ; Humans ; Signal Transduction/drug effects* ; Caco-2 Cells ; RAW 264.7 Cells ; Epithelial Cells/drug effects* ; Intestinal Mucosa/metabolism*

Periodontitis is a common oral disease characterized by progressive alveolar bone resorption and inflammation of the periodontal tissues. Dimethyl fumarate (DMF) has been used in the treatment of various immune-inflammatory diseases due to its excellent anti-inflammatory and antioxidant functions. Here, we investigated for the first time the therapeutic effect of DMF on periodontitis. In vivo studies showed that DMF significantly inhibited periodontal destruction, enhanced mitophagy, and decreased the M1/M2 macrophage ratio. In vitro studies showed that DMF inhibited macrophage polarization toward M1 macrophages and promoted polarization toward M2 macrophages, with improved mitochondrial function, inhibited oxidative stress, and increased mitophagy in RAW 264.7 cells. Furthermore, DMF increased intracellular mitochondrial Tu translation elongation factor (TUFM) levels to maintain mitochondrial homeostasis, promoted mitophagy, and modulated macrophage polarization, whereas TUFM knockdown decreased the protective effect of DMF. Finally, mechanistic studies showed that DMF increased intracellular TUFM levels by protecting TUFM from degradation via the ubiquitin-proteasomal degradation pathway. Our results demonstrate for the first time that DMF protects mitochondrial function and inhibits oxidative stress through TUFM-mediated mitophagy in macrophages, resulting in a shift in the balance of macrophage polarization, thereby attenuating periodontitis. Importantly, this study provides new insights into the prevention of periodontitis.
Dimethyl Fumarate/pharmacology* ; Mitophagy/drug effects* ; Animals ; Mice ; Macrophages/metabolism* ; Periodontitis/prevention & control* ; RAW 264.7 Cells ; Oxidative Stress/drug effects* ; Peptide Elongation Factor Tu/metabolism* ; Mice, Inbred C57BL ; Male ; Mitochondria/drug effects*

This study delves into the mechanism of total flavone of Abelmoschus manihot(TFA) in treating ulcerative colitis(UC) and depression via inhibiting M1 polarization of macrophages and reshaping intestinal flora and glycerolphospholipid metabolism. The study established a mouse model of UC and depression induced by chronic restraint stress(CRS) and dextran sulfate sodium(DSS). The fecal microbiota transplantation(FMT) experiment after TFA intervention was conducted. Mice in the FMT donor group were modeled and treated, and fecal samples were taken to prepare the bacterial solution. Mice in the FMT receptor group were treated with antibiotic intervention, and then administered bacterial solution by gavage from mice in the donor group, followed by UC depression modeling. After the experiment, behavioral tests were conducted to evaluate depressive-like behaviors by measuring the levels of 5-hydroxytryptamine(5-HT) and brain-derived neurotrophic factor(BDNF) in the hippocampus of mice. The levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β)in the brain and colon tissue of mice were also measured, and the polarization status of macrophages was evaluated by measuring the mRNA levels of CD86 and CD206. 16S ribosomal RNA(16S rRNA) sequencing technology was used to analyze changes in the intestinal flora of mice. Wide target lipidomics was used to detect serum lipid metabolite levels in mice after FMT,and correlation analysis was conducted between lipids and differential intestinal flora significantly regulated by TFA. In vitro experiments, representative glycerophospholipid metabolites and glycerophospholipid inhibitors were used to intervene in Raw264.7 macrophages, and the mRNA levels of TNF-α,IL-6,IL-1β,CD86,and CD206 were detected. The results showed that TFA and FMT after intervention could significantly improve depressive-like behavior and intestinal inflammation in mice with UC and depression, significantly downregulate pro-inflammatory cytokines and CD86 mRNA expression in brain and colon tissue, inhibiting M1 polarization of macrophages, and significantly upregulate CD206 mRNA expression, promoting M2 polarization of macrophages. In addition, the high-dose group had a more significant effect. After TFA intervention, FMT significantly corrected the metabolic disorder of glycerophospholipids in mice with UC and depression, and there was a significant correlation between differential intestinal flora and glycerophospholipids. In vitro experiments showed that glycerophospholipid metabolites, especially lysophosphatidylcholine(LPC),significantly upregulated pro-inflammatory cytokines and CD86 mRNA expression, promote M1 polarization of macrophages, while glycerophospholipid inhibitors had the opposite effect. The results indicate that TFA effectively treats depression and UC by correcting intestinal flora dysbiosis and reshaping glycerophospholipid metabolism, thereby inhibiting M1 polarization of macrophages.
Animals ; Mice ; Gastrointestinal Microbiome/drug effects* ; Abelmoschus/chemistry* ; Macrophages/metabolism* ; Colitis, Ulcerative/immunology* ; Flavones/administration & dosage* ; Male ; Depression/genetics* ; Glycerophospholipids/metabolism* ; Humans ; Drugs, Chinese Herbal/administration & dosage* ; Mice, Inbred C57BL

This study investigated the protective effect of arbutin(Arb) in yam on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in a mouse model and revealed its possible mechanism of action by metabolomics technology, providing a theoretical basis for clinical treatment of ALI. SPF BALB/c mice were randomly divided into normal control group, model group, resveratrol(Rv)-positive control group, Arb low-dose(15 mg·kg~(-1)) group, and Arb high-dose(30 mg·kg~(-1)) group. The LPS-induced ALI model was established in all groups except the normal control group. Hematoxylin-eosin(HE) staining, TUNEL staining, and WBP whole-body non-invasive pulmonary function testing were used to evaluate the degree of lung tissue damage and lung function changes. Enzyme-linked immunosorbent assay(ELISA) was used to detect the level of inflammatory factors in lung tissue. Flow cytometry was used to analyze the M1/M2 polarization status of macrophages in lung tissue. Western blot was used to detect the expression levels of the TLR4 signaling pathway and related apoptotic proteins. Liquid chromatograph-mass spectrometer(LC-MS) metabolomics was used to analyze the changes in serum metabolic profile after Arb intervention. The results showed that Arb pretreatment significantly alleviated LPS-induced lung tissue injury, improved lung function, reduced the levels of pro-inflammatory factors(IL-6, TNF-α, IL-18, and IL-1β), and regulated the polarization status of M1/M2 macrophages. In addition, Arb inhibited the activation of the TLR4 signaling pathway, reduced the expression of pro-apoptotic proteins such as Bax, caspase-3, and caspase-9, up-regulated the level of Bcl-2 protein, and inhibited apoptosis of lung cells. Metabolomic analysis showed that Arb significantly improved LPS-induced metabolic abnormalities, mainly involving key pathways such as galactose metabolism, phenylalanine metabolism, and lipid metabolism. In summary, Arb can significantly reduce LPS-induced ALI by regulating the release of inflammatory factors, inhibiting the activation of the TLR4 signaling pathway, improving metabolic disorders, and regulating macrophage polarization, indicating that Arb has potential clinical application value.
Animals ; Acute Lung Injury/chemically induced* ; Mice ; Mice, Inbred BALB C ; Arbutin/administration & dosage* ; Male ; Toll-Like Receptor 4/immunology* ; Apoptosis/drug effects* ; Lung/metabolism* ; Signal Transduction/drug effects* ; Protective Agents/administration & dosage* ; Humans ; Macrophages/immunology* ; Drugs, Chinese Herbal/administration & dosage*

BACKGROUND: Macrophage polarization anomalies and dysfunction play a crucial role in the pathogenesis of immune thrombocytopenia (ITP). Itaconate is a Krebs cycle-derived immunometabolite synthesized by myeloid cells to modulate cellular metabolism and inflammatory responses. This study aimed to evaluate the immunoregulatory effects of an itaconate derivative on macrophages in patients with ITP. METHODS: Peripheral blood-derived macrophages from patients with ITP and healthy controls were treated with 4-octyl itaconate (4-OI), a derivative of itaconate that can penetrate the cell membrane. Macrophage polarization, antigen-presenting functions, and phagocytic capability were measured via flow cytometry and enzyme-linked immunosorbent assay (ELISA). Macrophage glycolysis in patients with ITP and the metabolic regulatory effect of 4-OI were detected using a Seahorse XFe96 Analyzer. An active murine model of ITP was used to evaluate the therapeutic effects of 4-OI in vivo . RESULTS: 4-OI reduced the levels of CD80 and CD86 in M1 macrophages and suppressed the release of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 pro-inflammatory cytokines, suggesting that 4-OI could hinder the polarization of macrophages toward an M1 phenotype. We found that 4-OI pretreated M1 macrophages reduced the proliferation of CD4 + T cells and promoted the differentiation of regulatory T cells. In addition, after 4-OI treatment, the phagocytic capacity of M1 macrophages toward antibody-coated platelets decreased significantly in patients with ITP. In addition, the glycolytic function of M1 macrophages was elevated in individuals with ITP compared to those in healthy controls. 4-OI treatment downregulated glycolysis in M1 macrophages. The glycolysis inhibitor 2-deoxy-d-glucose (2-DG) also inhibited the polarization of M1 macrophages and restored their functions. In vivo , 4-OI treatment significantly increased platelet counts in the active ITP murine model. CONCLUSIONS Itaconate derivative 4-OI inhibited M1 macrophage polarization and restored impaired functions through metabolic reprogramming. This study provides a novel therapeutic option for ITP.
Macrophages/metabolism* ; Humans ; Animals ; Succinates/pharmacology* ; Mice ; Male ; Female ; Adult ; Middle Aged ; Flow Cytometry ; Tumor Necrosis Factor-alpha/metabolism* ; Enzyme-Linked Immunosorbent Assay ; Purpura, Thrombocytopenic, Idiopathic/metabolism* ; Glycolysis/drug effects* ; Metabolic Reprogramming

OBJECTIVE: Aloin, the main active component in Aloe vera (L.) Burm. f., has shown promising anti-tumor effects. This study investigated the impact of aloin in lung squamous cell carcinoma (LUSC) and explored its functional mechanism. METHODS: We analyzed the viability, migration, invasion, proliferation, and apoptosis of two LUSC cell lines after treatment with aloin. Target molecules of aloin and downstream target transcripts of nuclear receptor subfamily 3 group C member 2 (NR3C2) were predicted by bioinformatics. The biological functions of NR3C2 and metallothionein 1 M (MT1M) in the malignant properties of LUSC cells were determined. A co-culture system of LUSC cells with monocyte-derived macrophages was constructed. Mouse xenograft tumor models were generated to analyze the functions of aloin and NR3C2 in the tumorigenic activity of LUSC cells and macrophage polarization in vivo. RESULTS: Aloin suppressed malignant properties of LUSC cells in vitro. However, these effects were negated by the silencing of NR3C2. NR3C2 was found to activate MT1M transcription by binding to its promoter. Additional upregulation of MT1M suppressed the malignant behavior of LUSC cells augmented by NR3C2 silencing. Analysis of the M1 and M2 markers/cytokines in the macrophages or the culture supernatant revealed that aloin treatment or MT1M overexpression in LUSC cells enhanced M1 polarization while suppressing M2 polarization of macrophages, whereas NR3C2 silencing led to reverse trends. Consistent findings were reproduced in vivo. CONCLUSION This study demonstrated that aloin activates the NR3C2/MT1M axis to suppress the malignant behavior of LUSC cells and M2 macrophage polarization. Please cite this article as: Chen YN, Lu JY, Gao CF, Fang ZR, Zhou Y. Aloin blocks the malignant behavior of lung squamous cell carcinoma cells and M2 macrophage polarization by modulating the NR3C2/MT1M axis. J Integr Med. 2025; 23(2): 195-208.
Lung Neoplasms/metabolism* ; Humans ; Animals ; Cell Line, Tumor ; Carcinoma, Squamous Cell/metabolism* ; Mice ; Macrophages/drug effects* ; Emodin/analogs & derivatives* ; Metallothionein/genetics* ; Cell Proliferation/drug effects* ; Cell Movement/drug effects* ; Apoptosis/drug effects* ; Receptors, Glucocorticoid/genetics*

Our previous research demonstrated that the Wenxia Changfu Formula (WCF), as a neoadjuvant therapy, inhibits M2 macrophage infiltration in the tumor microenvironment and prevents lung cancer metastasis. Given tumor-associated macrophages (TAMs) in epithelial-mesenchymal transition (EMT), this study investigated whether WCF impedes lung cancer metastasis by attenuating TAM-induced EMT in non-small cell lung cancer (NSCLC) cells. Utilizing a co-culture model treated with or without WCF, we observed that WCF downregulated cluster of differentiation 163 (CD163) expression in macrophages, reduced CCL18 levels in the conditioned medium, and inhibited the growth, invasion, and EMT of NSCLC cells induced by macrophage co-culture. Manipulation of CCL18 levels and Src overexpression in NSCLC cells revealed that WCF's effects are mediated through CCL18 and Src signaling. In vivo, WCF inhibited recombinant CCL18 (rCCL18)-induced tumor metastasis in nude mice by blocking Src signaling. These findings indicate that WCF inhibits NSCLC metastasis by impeding TAM-induced EMT via antagonistic modulation of CCL18, providing evidence for its potential development and clinical application in NSCLC patients.
Epithelial-Mesenchymal Transition/drug effects* ; Carcinoma, Non-Small-Cell Lung/metabolism* ; Humans ; Animals ; Lung Neoplasms/metabolism* ; Chemokines, CC/antagonists & inhibitors* ; Mice ; Mice, Nude ; Drugs, Chinese Herbal/administration & dosage* ; Cell Line, Tumor ; Neoplasm Metastasis ; Tumor-Associated Macrophages/drug effects* ; Mice, Inbred BALB C ; Signal Transduction/drug effects*

This study identified six novel azaphilones, isochromophilones G-L (1-6), and three novel biosynthetically related congeners (7-9) from Diaporthe sp. SCSIO 41011. The structures and absolute configurations were elucidated through comprehensive spectroscopic analyses combined with experimental and calculated electronic circular dichroism (ECD) spectra. Significantly, three highly oxygenated azaphilones contain an acetyl group at the terminal chain (4) or linear conjugated polyenoid moieties (5 and 6), which occur infrequently in the azaphilone family. Additionally, several compounds demonstrated inhibition of lipopolysaccharide (LPS)-induced nuclear factor kappa-B (NF-κB) activation in RAW 264.7 macrophages at 20 μmol·L^-1. The novel compound (1) effectively inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation without exhibiting cytotoxicity in bone marrow and RAW 264.7 macrophages, indicating its potential as a promising lead compound for osteolytic disease treatment. This research presents the first documented evidence of azaphilone derivatives as inhibitors of RANKL-induced osteoclastogenesis.
Animals ; Mice ; RANK Ligand/genetics* ; RAW 264.7 Cells ; Osteoclasts/metabolism* ; Benzopyrans/isolation & purification* ; Osteogenesis/drug effects* ; Macrophages/metabolism* ; Molecular Structure ; Pigments, Biological/isolation & purification* ; Ascomycota/chemistry* ; NF-kappa B/genetics* ; Cell Differentiation/drug effects*