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 observe the evolution of intrahepatic macrophage polarization in mice with liver fibrosis induced by iron overload. METHODS: Thirty-two C57BL/6 mice (6-8 weeks) were randomized into control group (n=8) and liver fibrosis model group (n=24) induced by aidly intraperitoneal injection of iron dextran. At the 3rd, 5th, and 7th weeks of modeling, 8 mice in the model group were sacrificed for observing liver fibrosis using Masson, Sirius Red and immunohistochemical staining and detecting serum levels of ALT, AST and the levels of serum iron, ferritin, liver total Fe and ferrous Fe. iNOS⁺/F4/80⁺ cells and CD206⁺/F4/80⁺ cells were detected by double immunofluorescence assay to observe the proportion and distribution of M1 and M2 macrophages. The hepatic expressions of Arg-1, iNOS, IL-6, IL-10, and TNF‑α proteins were detected using Western blotting or ELISA, and the expression of CD206 mRNA was detected using RT-PCR. RESULTS: The mice in the model group showed gradual increase of fibrous tissue hyperplasia in the portal area over time, structural destruction of the hepatic lobules and formation of pseudolobules. With the passage of time during modeling, the rat models showed significantly increased hepatic expressions of α-SMA and COL-1, elevated serum levels of ALT, AST, Fe, ferritin, and increased liver total Fe and ferrous Fe levels. The expressions of M1 polarization markers IL-6, TNF‑α, and iNOS all increased with time and reached their peak levels at the 3rd week; The expressions of M2 polarization markers (IL-10 and Arg-1 proteins and CD206 mRNA) significantly increased in the 3rd week and but decreased in the 5th and 7th weeks. CONCLUSIONS Iron overload promotes M1 polarization of macrophages in mice. Liver fibrosis in the early stage promotes M2 polarization of macrophages but negatively regulate M2 polarization at later stages.
Animals ; Mice ; Mice, Inbred C57BL ; Iron Overload/pathology* ; Macrophages/metabolism* ; Male ; Liver Cirrhosis/etiology* ; Nitric Oxide Synthase Type II/metabolism* ; Interleukin-10/metabolism* ; Liver/pathology* ; Interleukin-6/metabolism* ; Mannose Receptor ; Tumor Necrosis Factor-alpha/metabolism* ; Mannose-Binding Lectins/metabolism* ; Arginase

The angiogenic response is essential for the repair of ischemic brain tissue. Integrin α6 (Itga6) expression has been shown to increase under hypoxic conditions and is expressed exclusively in vascular structures; however, its role in post-ischemic angiogenesis remains poorly understood. In this study, we demonstrate that mice with endothelial cell-specific knockout of Itga6 exhibit reduced neovascularization, reduced pericyte coverage on microvessels, and accelerated breakdown of microvascular integrity in the peri-infarct area. In vitro, endothelial cells with ITGA6 knockdown display reduced proliferation, migration, and tube-formation. Mechanistically, we demonstrated that ITGA6 regulates post-stroke angiogenesis through the PI3K/Akt-eNOS-VEGFA axis. Importantly, the specific overexpression of Itga6 in endothelial cells significantly enhanced neovascularization and enhanced the integrity of microvessels, leading to improved functional recovery. Our results suggest that endothelial cell Itga6 plays a crucial role in key steps of post-stroke angiogenesis, and may represent a promising therapeutic target for promoting recovery after stroke.
Animals ; Nitric Oxide Synthase Type III/metabolism* ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; Integrin alpha6/genetics* ; Endothelial Cells/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Stroke/pathology* ; Vascular Remodeling/physiology* ; Vascular Endothelial Growth Factor A/metabolism* ; Mice, Knockout ; Signal Transduction/physiology* ; Mice, Inbred C57BL ; Male ; Neovascularization, Physiologic/physiology*

Guided by molecular networking, nine novel curvularin derivatives (1-9) and 16 known analogs (10-25) were isolated from the hydrothermal vent sediment fungus Penicillium sp. HL-50. Notably, compounds 5-7 represented a hybrid of curvularin and purine. The structures and absolute configurations of compounds 1-9 were elucidated via nuclear magnetic resonance (NMR) spectroscopy, X-ray diffraction, electronic circular dichroism (ECD) calculations, ¹³C NMR calculation, modified Mosher's method, and chemical derivatization. Investigation of anti-inflammatory activities revealed that compounds 7-9, 11, 12, 14, 15, and 18 exhibited significant suppressive effects against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine macrophage RAW264.7 cells, with IC₅₀ values ranging from 0.44 to 4.40 μmol·L^-1. Furthermore, these bioactive compounds were found to suppress the expression of inflammation-related proteins, including inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), NLR family pyrin domain-containing protein 3 (NLRP3), and nuclear factor kappa-B (NF-κB). Additional studies demonstrated that the novel compound 7 possessed potent anti-inflammatory activity by inhibiting the transcription of inflammation-related genes, downregulating the expression of inflammation-related proteins, and inhibiting the release of inflammatory cytokines, indicating its potential application in the treatment of inflammatory diseases.
Penicillium/chemistry* ; Mice ; Animals ; Anti-Inflammatory Agents/isolation & purification* ; RAW 264.7 Cells ; Nitric Oxide/metabolism* ; Hydrothermal Vents/microbiology* ; Macrophages/immunology* ; Molecular Structure ; Nitric Oxide Synthase Type II/immunology* ; Cyclooxygenase 2/immunology* ; Geologic Sediments/microbiology* ; NF-kappa B/immunology* ; NLR Family, Pyrin Domain-Containing 3 Protein/immunology*

Ten novel xanthones, garpedunxanthones A-G (1-5, 6a/6b, 7a/7b) and nujiangxanthone Q (8), along with sixteen known analogs (9-24), were isolated from Garcinia pedunculata and G. nujiangensis. Their structures were elucidated through high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data, comprehensive nuclear magnetic resonance (NMR) spectroscopic analyses, and electronic circular dichroism (ECD) calculations. All compounds without cytotoxicity were assessed for anti-inflammatory properties by measuring the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 cells. Structure-activity relationships are also discussed. Compounds 7b, 19, and 21 exhibited significant anti-inflammatory activity with IC₅₀ values of 16.44 ± 0.69, 14.28 ± 0.78, and 10.67 ± 3.28 μmol·L^-1, respectively. Enzyme-linked immunosorbent assay (ELISA) demonstrated that compounds 7b, 19, and 21 inhibited the expression of pro-inflammatory cytokines TNF-α and IL-6 in a dose-dependent manner. The inhibitory effect of compound 21 on IL-6 at 20 μmol·L^-1 was comparable to that of the positive control. In network pharmacology studies, potential targets of compounds and inflammation were identified from PharmMapper and GeneCards databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the overlapped targets were intricately associated with major pathogenic processes linked to inflammation, including positive regulation of mitogen-activated protein kinase (MAPK) cascade, protein kinase activity, NO synthase regulator activity, MAPK signaling pathway, and EGFR tyrosine kinase inhibitor resistance.
Xanthones/therapeutic use* ; Garcinia ; Anti-Inflammatory Agents/therapeutic use* ; Plant Preparations/therapeutic use* ; Structure-Activity Relationship ; Nitric Oxide/metabolism* ; RAW 264.7 Cells ; Animals ; Mice ; Enzyme-Linked Immunosorbent Assay ; Mitogen-Activated Protein Kinase Kinases/metabolism* ; Circular Dichroism

The anti-inflammatory phytochemical investigation of the leaves of Illicium dunnianum (I. dunnianum) resulted in the isolation of five pairs of new lignans (1-5), and 7 known analogs (6-12). The separation of enantiomer mixtures 1-5 to 1a/1b-5a/5b was achieved using a chiral column with acetonitrile-water mixtures as eluents. The planar structures of 1-2 were previously undescribed, and the chiral separation and absolute configurations of 3-5 were reported for the first time. Their structures were determined through comprehensive spectroscopic data analysis [nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass (HR-ESI-MS), infrared (IR), and ultraviolet (UV)] and quantum chemistry calculations (ECD). The new isolates were evaluated by measuring their inhibitory effect on NO in lipopolysaccharide (LPS)-stimulated BV-2 cells. Compounds 1a, 3a, 3b, and 5a demonstrated partial inhibition of NO production in a concentration-dependent manner. Western blot and real-time polymerase chain reaction (PCR) assays revealed that 1a down-regulated the messenger ribonucleic acid (mRNA) levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), COX-2, and iNOS and the protein expressions of COX-2 and iNOS. This research provides guidance and evidence for the further development and utilization of I. dunnianum.
Lignans/isolation & purification* ; Plant Leaves/chemistry* ; Anti-Inflammatory Agents/isolation & purification* ; Mice ; Animals ; Molecular Structure ; Plant Extracts/pharmacology* ; Illicium/chemistry* ; Cyclooxygenase 2/immunology* ; Interleukin-6/immunology* ; Nitric Oxide/metabolism* ; Cell Line ; Tumor Necrosis Factor-alpha/immunology* ; Nitric Oxide Synthase Type II/immunology* ; Lipopolysaccharides

The chemical constituents of Commiphora myrrha was investigated by column chromatography on silica gel, ODS, Sephadex LH-20, and semi-preparative HPLC. Their structures were elucidated by comprehensive spectroscopic methods including UV, IR, MS, NMR, as well as ECD calculation. Seven compounds were isolated from the dichloromethane-soluble fraction of C. myrrha and their structures were identified as(1S,2R,4S,5R,8S)-guaiane-2-hydroxy-7(11),10(15)-dien-6-oxo-12,8-olide(1), commipholide E(2), myrrhterpenoid H(3), myrrhterpenoid I(4), myrrhterpenoid E(5), 2α-methoxy-8α-hydroxy-6-oxogermacra-1(10),7(11)-dien-8,12-olide(6), 8,12-epoxy-1α,9α-hydroxy-eudesma-7,11-diene-6-dione(7). Compound 1 was a new compound and named myrrhterpenoid P. Compound 7 was isolated from Commiphora genus for the first time. Compounds 2, 5, and 6 significantly inhibited nitric oxide(NO) production in LPS-stimulated RAW264.7 cells, with IC_(50) values of(49.67±4.16),(40.80±1.27),(47.22±0.87) μmol·L~(-1), respectively [indomethacin as the positive control, with IC_(50) value of(63.92±2.60) μmol·L~(-1)].
Commiphora/chemistry* ; Animals ; Mice ; Resins, Plant/chemistry* ; Sesquiterpenes/isolation & purification* ; Molecular Structure ; Nitric Oxide ; Macrophages/metabolism* ; RAW 264.7 Cells ; Drugs, Chinese Herbal/pharmacology*

This paper aims to evaluate the pharmacodynamic effect and mechanism of Zhifuxin in the prevention and treatment of vascular dementia(VD), providing a theoretical basis for later development. Bilateral common carotid artery ligation in male Wistar rats was conducted to replicate the long-term hypoperfused VD model, and the drug was given to groups after one month. The rats were fed daily with nimodipine of 20 mg·kg~(-1), Zhifuxin of 50, 100, and 200 mg·kg~(-1), or the same volume of solvent for four weeks. 24 hours after the last dose, Morris water maze experiments were performed to detect the learning and memory abilities of rats. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in the brain tissue of rats; the immunohistochemical method was used to detect the expression of muscarinic acetylcholine receptors M1 and M4 in rats and determine the content of acetyl choline(Ach), acetylcholin esterase(AchE), malondialdehyde(MDA), choline acetyl transferase(ChAT), and dimethyl arginine hydrolase 1(DDAH1) in the cerebral cortex of rats. Western blot was employed to detect protein expression of endothelial nitric oxide synthase(eNOS), caveolin-1, monoamine oxidase A(MAO-A), and monoamine oxidase B(MAO-B). RT-qPCR was utilized to detect mRNA expression of eNOS, caveolin-1, MAO-A, and MAO-B. The results showed that compared with the model group, the different doses of Zhifuxin were able to shorten the latency of VD rats in the water maze positioning navigation test, increase the number of crossing platforms in the space exploration test, and alleviate cone cell contracture in the hippocampus of VD rats. The expression of biochemical indicators related to the cholinergic system in the cerebral cortex: M1 and M4 receptors increased, as well as ChAT activity, and AchE activity significantly decreased. The protein and mRNA expression of indicators related to the eNOS/NO pathway: DDAH1 content, eNOS, and caveolin-1 increased, and that of indicators related to monoamine oxidase(MAO): MAO-A and MAO-B significantly decreased. The results show that Zhifuxin can improve cognition ability in long-term hypoperfused VD rats, and its mechanism of action may be related to its ability to modulate the cholinergic system and the eNOS/NO pathway and inhibit MAO expression.
Animals ; Dementia, Vascular/metabolism* ; Male ; Rats, Wistar ; Rats ; Drugs, Chinese Herbal/administration & dosage* ; Maze Learning/drug effects* ; Nitric Oxide Synthase Type III/genetics* ; Acetylcholinesterase/metabolism* ; Humans ; Choline O-Acetyltransferase/genetics* ; Disease Models, Animal

This study aims to reveal the correlation between the pharmacokinetics(PK) and pharmacodynamics(PD) of multiple components in Epimedii Folium-Chuanxiong Rhizoma and clarify the pharmacodynamic material basis and mechanism of this herb pair in treating osteoarthritis. The Hulth method was used to establish the rat model of osteoarthritis and plasma was collected at various time points after drug administration. The plasma concentrations of multiple components were measured. Enzyme-linked immunosorbent assay(ELISA) was used to measure the plasma concentrations of matrix metalloproteinase(MMP)-3, MMP-13, interleukin-1β(IL-1β), nitric oxide(NO), and tumor necrosis factor-α(TNF-α) as pharmacodynamic indicators. Self-defined weighting coefficients were used to calculate the PK and PD data, and a Sigmoid E_(max) fitting model was used to evaluate the synergistic effect of the compatibility of Epimedii Folium-Chuanxiong Rhizoma. The PK-PD models for Epimedii Folium, Chuanxiong Rhizoma, and Epimedii Folium-Chuanxiong Rhizoma were E=(1.926×C~(2.652))/(0.136 6~(2.652)+C~(2.652)), E=(1.618×C~(345.2))/(0.118 4~(345.2)+C~(345.2)), and E=(2.305×C~(2.786))/(0.240 3~(2.786)+C~(2.786)), respectively. The E_(max) of Epimedii Folium-Chuanxiong Rhizoma was larger than those of the two herbal medicines alone. The EC_(50) of the herb pair was lower than the sum of Epimedii Folium and Chuanxiong Rhizoma alone. The concentrations of MMP-3, MMP-13, IL-1β, NO, and TNF-α were correlated with mass concentrations of multiple components in Epimedii Folium and Chuanxiong Rhizoma, and the compatibility was better than single use. Epimedii Folium, Chuanxiong Rhizoma, and Epimedii Folium-Chuanxiong Rhizoma may play a role in the treatment of osteoarthritis by inhibiting MMP-3, MMP-13, IL-1β, NO, and TNF-α.
Animals ; Rats ; Drugs, Chinese Herbal/pharmacology* ; Male ; Rats, Sprague-Dawley ; Osteoarthritis/metabolism* ; Epimedium/chemistry* ; Interleukin-1beta/blood* ; Tumor Necrosis Factor-alpha/blood* ; Disease Models, Animal ; Nitric Oxide/blood* ; Humans ; Rhizome/chemistry*

The chemical constituents were systematically separated from the roots of Berberis polyantha by various chromatographic methods, including silica gel column chromatography, HP20 column chromatography, polyamide column chromatography, reversed-phase C_(18) column chromatography, and preparative high-performance liquid chromatography. The structures of the compounds were identified by physicochemical properties and spectroscopic techniques(1D NMR, 2D NMR, UV, MS, and CD). Four phenylpropanoids were isolated from the methanol extract of the roots of B. polyantha, and they were identified as(2R)-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone-O-β-D-glucopyranoside(1), methyl 4-hydroxy-3,5-dimethoxybenzoate(2),(+)-syringaresinol(3), and syringaresinol-4-O-β-D-glucopyranoside(4). Compound 1 was a new compound, and other compounds were isolated from this plant for the first time. The anti-inflammatory activity of these compounds was evaluated based on the release of nitric oxide(NO) in the culture of lipopolysaccharide(LPS)-induced RAW264.7 macrophages. At a concentration of 10 μmol·L~(-1), all the four compounds inhibited the LPS-induced release of NO in RAW264.7 cells, demonstrating potential anti-inflammatory properties.
Plant Roots/chemistry* ; Animals ; Mice ; Berberis/chemistry* ; RAW 264.7 Cells ; Macrophages/immunology* ; Drugs, Chinese Herbal/isolation & purification* ; Nitric Oxide/metabolism* ; Molecular Structure ; Anti-Inflammatory Agents/isolation & purification*