OBJECTIVES: To investigate the effect of high glucose on macrophage polarization and the role of immune-responsive gene 1 (IRG1) in mediating its effect. METHODS: RAW264.7 cells were transfected with IRG1-overexpressing plasmid or IRG1 siRNA via electroporation and cultured in either normal or high glucose for 72 h to observe the changes in cell viability and morphology using CCK-8 assay and phase contrast microscopy. The protein levels of IRG1, iNOS, Arg-1, IL-1β and IL-10 in the treated cells were detected with Western blotting, and the fluorescence intensities of iNOS and Arg-1 were detected using immunofluorescence assay. The protein levels of IL-1β and IL-10 in the culture medium were determined with ELISA. RESULTS: High glucose exposure significantly reduced IRG1 and Arg-1 expressions, increased iNOS and IL-1β expressions and IL-1β secretion, and decreased IL-10 level in RAW264.7 cells. Transfection with the IRG1-overexpressing plasmid provided the cells with obvious resistance to high glucose-induced changes in iNOS, Arg-1, IL-1β and IL-10, whereas IRG1 knockdown further enhanced the effects of high glucose exposure on Arg-1 expression and the expression and secretion of IL-10. CONCLUSIONS High glucose promotes M1 polarization of the macrophages possibly through a mechanism to inhibit the expression of IRG1 protein, thus leading to chronic inflammatory response.
Animals ; Mice ; Macrophages/drug effects* ; Glucose/pharmacology* ; Interleukin-10/metabolism* ; Nitric Oxide Synthase Type II/metabolism* ; RAW 264.7 Cells ; Interleukin-1beta/metabolism* ; Arginase/metabolism* ; RNA, Small Interfering/genetics* ; Transfection ; Inflammation

OBJECTIVES: To explore the active components that mediate the therapeutic effect of Centella asiatica on psoriasis and their therapeutic mechanisms. METHODS: TCMSP, TCMIP, PharmMapper, Swiss Target Prediction, GeneCards, OMIM and TTD databases were searched for the compounds in Centella asiatica and their targets and the disease targets of psoriasis. A drug-active component-target network and the protein-protein interaction network were constructed, and DAVID database was used for pathway enrichment analysis. In a RAW264.7 macrophage model of LPS-induced inflammation, the anti-inflammatory effect of 7.5, 15, 30, and 60 μmol/L quercetin, asiaticoside, and asiatic acid, which were identified as the main active components in Centella asiatica, were tested by measuring cellular production of NO, TNF‑α and IL-6 using Griess method and ELISA and by detecting mRNA expressions of IL-23, IL-17A, TNF-α and IL-6 and protein expressions of p-STAT3 (Tyr705) and p-STAT3 (Ser727) with RT-qPCR and Western blotting. RESULTS: A total of 139 targets of Centella asiatica and 4604 targets of psoriasis were obtained, and among them CASP3, EGFR, PTGS2, and ESR1 were identified as the core targets. KEGG analysis suggested that quercetin, asiaticoside, and asiatic acid in Centella asiatica were involved in cancer and IL-17 and MAPK signaling pathways. In the RAW264.7 macrophage model of inflammation, treatment with quercetin significantly reduced cellular production of NO, TNF‑α and IL-6, and lowered mRNA expressions of IL-23, IL-17A, TNF‑α and IL-6 and protein expressions of p-STAT3 (Tyr705) and p-STAT3 (Ser727). CONCLUSIONS Quercetin, asiaticoside and asiatic acid are the main active components in Centella asiatica to mediate the therapeutic effect against psoriasis, and quercetin in particular is capable of suppressing cellular production of NO, TNF‑α and IL-6 and regulating the IL-23/IL-17A inflammatory axis by mediating STAT3 phosphorylation to inhibit inflammatory response.
Quercetin/pharmacology* ; Psoriasis/metabolism* ; STAT3 Transcription Factor/metabolism* ; Mice ; Animals ; Centella/chemistry* ; Triterpenes/pharmacology* ; Phosphorylation ; Interleukin-17/metabolism* ; Interleukin-23/metabolism* ; RAW 264.7 Cells ; Pentacyclic Triterpenes/pharmacology* ; Macrophages/drug effects* ; Signal Transduction ; Plant Extracts

OBJECTIVES: To investigate the effect of amentoflavone (AF) for alleviating lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and inhibiting NLRP3/ASC/Caspase-1 axis-mediated pyroptosis. METHODS: Female BALB/c mice were randomly divided into control group, LPS group, and AF treatment groups at low, moderate and high doses (n=12). ALI models were established by tracheal LPS instillation, and in AF treatment groups, AF was administered by gavage 30 min before LPS instillation. Six hours after LPS instillation, the mice were euthanized for examining lung tissue histopathological changes, protein levels in BALF, and MPO levels in the lung tissue. In the in vitro experiment, RAW264.7 cells were pretreated with AF, AC (a pyroptosis inhibitor), or their combination for 2 h before stimulation with LPS and ATP. The changes in cell proliferation and viability were detected using CCK-8 assay, and IL-1β, IL-6, IL-18, and TNF-α levels were determined with ELISA. Immunohistochemistry, immunofluorescence assay, and immunoblotting were used to detect the protein levels of NLRP3, ASC, cleaved caspase-1, and GSDMD N in rat lung tissues and the treated cells. RESULTS: In mice with LPS exposure, AF treatment significantly improved lung pathologies and edema, reduced protein levels in BALF and pulmonary MPO level, inhibited the high expression of NLRP3/ASC/Aspase-1 axis, reduced the expression of GSDMD N, and lowered the release of IL-1β, IL-6, IL-18, and TNF‑α. In RAW264.7 cells with LPS and ATP stimulation, AF pretreatment effectively reduced cell death, inhibited activation of the NLRP3/ASC/Aspase-1 axis, and reduced GSDMD N expression and the inflammatory factors. The pyroptosis inhibitor showed a similar effect to AF, and their combination produced more pronounced effects in RAW264.7 cells. CONCLUSIONS Amentoflavone can alleviate ALI in mice possibly by inhibiting NLRP3/ASC/Caspase-1 axis-mediated cell pyroptosis.
Animals ; Pyroptosis/drug effects* ; Acute Lung Injury/pathology* ; Mice ; Mice, Inbred BALB C ; Female ; Lipopolysaccharides ; Biflavonoids/pharmacology* ; RAW 264.7 Cells ; NLR Family, Pyrin Domain-Containing 3 Protein ; Caspase 1/metabolism* ; Lung

OBJECTIVES: To observe the therapeutic effect of spermine in neonatal mouse models of severe hand, foot and mouth disease (HFMD) caused by enterovirus 71 (EV71) infection and explore its therapeutic mechanism in light of regulation of macrophage GBP5/NLRP3 inflammasome pathway. METHODS: Neonatal BALB/c mice (3-5 days old) were divided into control group, EV71 infection group and Spermine treatment group. The mice in the latter two groups received an intraperitoneal injection of 50 μL EV71 suspension (1×10⁶ TCID50 of EV71), followed 3 days later by intraperitoneal injection of 50 μL PBS or 100 μmol/L spermine. GBP5, NLRP3, CXCL10, and TNFSF10 expressions in heart, liver, lung and kidney tissues of the mice were detected using Western blotting and qPCR, and tissue pathologies and macrophage infiltration were assessed with HE staining and immunohistochemistry. In cultured THP-1 and RAW264.7 cells, the effects of EV71 infection, GBP5 siRNA transfection and treatment with spermine or eflornithine on GBP5, NLRP3, CXCL10, and TNFSF10 mRNA expressions were investigated using qPCR. RESULTS: In the neonatal mice, EV71 infection resulted in multiple organ damage, macrophage infiltration and activation of the GBP5/NLRP3 pathway, and spermine treatment significantly improved tissue injuries, reduced macrophage infiltration, and down-regulated the expressions of GBP5, NLRP3 and the inflammatory factors in the infected mice. In THP-1 and RAW264.7 cells, EV71 infection caused significant upregulation of GBP5, NLRP3, CXCL10, and TNFSF10 expressions, which were obviously lowered by spermine treatment. In THP-1 cells, treatment with eflornithine significantly suppressed the reduction of GBP5, NLRP3, CXCL10, and TNFSF10 expressions induced by GBP5 siRNA transfection. CONCLUSIONS Spermine suppressed EV71 infection-induced inflammatory responses by inhibiting GBP5-mediated NLRP3 inflammasome activation, suggesting a new strategy for treatment of severe HFMD.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein ; Mice ; Macrophages/metabolism* ; Enterovirus A, Human ; Mice, Inbred BALB C ; Inflammasomes/metabolism* ; Spermine/therapeutic use* ; Animals, Newborn ; Humans ; Enterovirus Infections ; Hand, Foot and Mouth Disease/drug therapy* ; RAW 264.7 Cells ; Chemokine CXCL10/metabolism*

OBJECTIVES: To compare the anti-inflammatory, antibacterial and analgesic effects of Yinqiao Powder (YQS) formulated granules and decoction. METHODS: We first evaluated the anti-inflammatory effects of the two dosage forms of YQS in a LPS-induced RAW 264.7 cell model using RT-qPCR and Western blotting. We further constructed zebrafish models of inflammation by copper sulfate exposure, caudal fin transection, or LPS and Poly (I:C) microinjection, and evaluated anti-inflammatory effects of YQS granules and decoction by examining neutrophil aggregation and HE staining findings. In a mouse model of acute lung injury (ALI) induced by intratracheal LPS instillation, the effects of YQS gavage at 10, 15, and 20 g/kg on lung pathologies were evaluated by calculating lung wet-dry weight ratio and using HE staining, ELISA and Western blotting. The microbroth dilution method was used to evaluate the antibacterial effect of YQS. Mouse pain models established by hot plate and intraperitoneal injection of glacial acetic acid were used to evaluate the analgesic effects of YQS at 10, 15, and 20 g/kg. RESULTS: Both YQS granules and decoction significantly reduced TNF-α, IL-6, and IL-1β expressions and p-STAT3 (Tyr 705) phosphorylation level in LPS-induced RAW 264.7 cells, and obviously inhibited neutrophil aggregation in the zebrafish models. In ALI mice, YQS granules and decoction effectively ameliorated lung injury, lowered lung wet-dry weight ratio, and reduced p-STAT3 (Tyr 705) expression and TNF-α and IL-6 levels. YQS produced obvious antibacterial effect at the doses of 15.63 and 31.25 mg/mL, and significantly reduced body torsion and increased pain threshold in the mouse pain models. CONCLUSIONS The two dosage forms of TQS have similar anti-inflammatory, antibacterial and analgesic effects with only differences in their inhibitory effect on TNF-α, IL-6 and IL-1β mRNA expressions in LPS-induced RAW 264.7 cells.
Animals ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Anti-Inflammatory Agents/pharmacology* ; Analgesics/pharmacology* ; RAW 264.7 Cells ; Zebrafish ; Anti-Bacterial Agents/pharmacology* ; Powders ; Tumor Necrosis Factor-alpha/metabolism* ; Acute Lung Injury/drug therapy* ; Interleukin-6/metabolism* ; Lipopolysaccharides

OBJECTIVES: To investigate the effect of ecliptasaponin A (ESA) for alleviating dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) in mice and the underlying mechanism. METHODS: Twenty-four male C57BL/6 mice (8-10 weeks old) were equally randomized into control group, DSS-induced IBD model group, and DSS+ESA (50 mg/kg) treatment group. Disease activity index (DAI), colon length and spleen index of the mice were measured, and intestinal pathology was examined with HE staining. The expressions of inflammatory mediators (TNF-α, IL-6, and iNOS) in the colon mucosa were detected using ELISA and RT-qPCR, and intestinal barrier integrity was assessed using AB-PAS staining and by detecting ZO-1 and claudin-1 expressions using immunofluorescence staining and Western blotting. In cultured RAW264.7 macrophages, the effects of treatment with 50 μmol/L ESA, alone or in combination with 20 μmol/L RO8191 (a JAK2/STAT3 pathway activator), on M1 polarization of the cells induced by LPS and IFN-γ stimulation and expressions of JAK2/STAT3 pathway proteins were analyzed using flow cytometry and Western blotting. RESULTS: In the mouse models of DSS-induced IBD, ESA treatment significantly alleviated body weight loss and colon shortening, reduced DAI, spleen index and histological scores, and ameliorated inflammatory cell infiltration in the colon tissue. ESA treatment also suppressed TNF‑α, IL-6 and iNOS expressions, protected the goblet cells and the integrity of the mucus and mechanical barriers, and upregulated the expressions of ZO-1 and claudin-1. ESA treatment obviously decreased CD86⁺ M1 polarization in the mesenteric lymph nodes of IBD mice and in LPS and IFN-γ-induced RAW264.7 cells, and significantly reduced p-JAK2 and p-STAT3 expressions in both the mouse models and RAW264.7 cells. Treatment with RO8191 caused reactivation of JAK2/STAT3 and strongly attenuated the inhibitory effect of ESA on CD86⁺ polarization in RAW264.7 cells. CONCLUSIONS ESA alleviates DSS-induced colitis in mice by suppressing JAK2/STAT3-mediated M1 macrophage polarization and mitigating inflammation-driven intestinal barrier damage.
Animals ; Mice ; Janus Kinase 2/metabolism* ; STAT3 Transcription Factor/metabolism* ; Mice, Inbred C57BL ; Male ; Dextran Sulfate ; Macrophages/cytology* ; Colitis/metabolism* ; Saponins/pharmacology* ; Signal Transduction/drug effects* ; RAW 264.7 Cells ; Triterpenes/pharmacology* ; Interleukin-6/metabolism*

OBJECTIVES: To investigate the effect of Qianggu Kangshu Formula (QGKSF) for alleviating osteoclast differentiation in rheumatoid arthritis and the underlying mechanism. METHODS: RAW264.7 cells cultured under hypoxic conditions were treated with RANKL to induce osteoclast differentiation and incubated with normal rat serum or sera from rats medicated with methotrexate (MTX) or QGKSF at low and high doses. Cell viability, TRAP-positive multinucleated cells and F-actin ring formation in the treated cells were assessed with CCK-8 assay, TRAP staining and Phalloidin staining, respectively. Autophagy and autophagosomes in the cells were observed with MDC staining and transmission electron microscopy. ELISA was used to measure IL-6 and TNF-α levels in the culture supernatant, and the expressions of HIF-1α, BNIP3, Bcl-2, Beclin1, LC3-I, LC3-II, P62 and TRAP mRNAs and proteins were analyzed using RT-qPCR and Western blotting. RESULTS: In hypoxia- and RANKL-induced RAW264.7 cells treated with normal rat serum, significant increments of TRAP-positive cells and F-actin ring formation were observed with an enhanced autophagic fluorescence intensity and increased autophagosomes. Treatment of the induced cells with rat sera medicated with MTX and low- and high-dose QGKSF obviously reduced the TRAP-positive cells, F-actin rings and autophagosomes as well as the autophagic fluorescence intensity. RANKL treatment significantly increased IL-6 and TNF-α levels in RAW264.7 cells, which were obviously decreased by treatment with MTX- and QGKSF-medicated sera. RANKL also significantly increased the mRNA and protein expression levels of HIF-1α, BNIP3, Bcl-2, Beclin1, LC3 and TRAP and lowered P62 expressions, and these changes were effectively reversed by treatment with MTX- and QGKSF-medicated sera. CONCLUSIONS QGKSF attenuates RANKL-induced osteoclast differentiation in hypoxic RAW264.7 cells by inhibiting the HIF-1α/BNIP3 autophagy signaling pathway, suggesting its potential for treatment of bone destruction in rheumatoid arthritis.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Osteoclasts/drug effects* ; Autophagy/drug effects* ; Mice ; Signal Transduction/drug effects* ; Rats ; Cell Differentiation/drug effects* ; Arthritis, Rheumatoid/pathology* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; RAW 264.7 Cells ; Membrane Proteins/metabolism* ; Mitochondrial Proteins

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*

OBJECTIVE: The present study investigated the cytoprotective effects of a Pogonatherum paniceum extract prepared with 80% ethanol (PPE) using synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy and determined its phytochemical profile. METHODS: The volatile and polyphenolic compounds in PPE were characterized using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, respectively. The antioxidant capacity of PPE was evaluated using chemical and cell-based assays. The SR-FTIR microspectroscopy was performed to evaluate the cytoprotective effect of PPE by identifying changes in macromolecule composition in tert-butyl hydroperoxide (tBuOOH)-induced oxidative damage in RAW264.7 cells. RESULTS: A total of 48 volatile compounds and 28 polyphenol components were found in PPE. PPE exhibited a high potential for antioxidant activity by scavenging the intracellular reactive oxygen species in tBuOOH-induced oxidative damage in RAW264.7 cells. PPE treatment also significantly protected RAW264.7 cells against tBuOOH-induced toxicity and restored cell viability. The SR-FTIR analysis revealed that tBuOOH increased the lipid and ester lipid content in RAW264.7 cells. The PPE exerted a cytoprotective effect by decreasing the levels of lipid and ester lipid compounds that had been elevated by tBuOOH in RAW264.7 cells. These findings indicate that PPE has cytoprotective potential due to its ability to inhibit endogenous reactive oxygen species. CONCLUSION This study extends the current knowledge on the phytochemistry of PPE and its antioxidant and cytoprotective effects. These findings support the use of SR-FTIR microspectroscopy to determine the cytoprotective effects of natural products. PPE extract may be a candidate compound for new therapeutics and nutraceuticals that target the prevention of oxidative stress-associated diseases. Please cite this article as: Dunkhunthod B, Thumanu K, Teethaisong Y, Sittisart P, Sittisart P. Cytoprotective activity of Pogonatherum paniceum (Lam.) Hack. ethanolic extract evaluated by synchrotron radiation-based Fourier transform infrared microspectroscopy. J Integr Med. 2025; 23(2): 182-194.
Animals ; Mice ; Spectroscopy, Fourier Transform Infrared/methods* ; Plant Extracts/chemistry* ; RAW 264.7 Cells ; Synchrotrons ; Oxidative Stress/drug effects* ; Antioxidants/pharmacology* ; Ethanol/chemistry* ; Poaceae/chemistry* ; Cell Survival/drug effects* ; Cytoprotection/drug effects* ; Reactive Oxygen Species/metabolism* ; tert-Butylhydroperoxide