OBJECTIVE: To evaluate the differential expression of RNA in blood monocytes in patients with Juvenile idiopathic arthritis (JIA) treated with TNF antagonists (TNFi), and to explore the effect and mechanism of gene expression on the efficacy of JIA. METHODS: A total of 29 children with JIA treated with methotrexate (MTX) and TNFi in Guangzhou Women and Children's Medical Center of Guangzhou Medical University from April 2021 to November 2023 were enrolled. After 6 months, the children were divided into two groups according to the treatment effect, i.e., 13 cases in the ineffective group and 16 cases in the effective group, the peripheral blood of the children was collected, the blood mononuclear cells were isolated for transcriptome sequencing, the differentially expressed genes between the groups were analyzed, the signaling pathways and metabolic pathways related to the efficacy of TNFi were analyzed by GO and KEGG enrichment, and the mechanism related to the efficacy of TNFi was explored. This study was approved by Medical Ethics Committee of the Guangzhou Women and Children's Medical Center of Guangzhou Medical University (Ethics No.: 2023-330B00). RESULTS: There was a statistically significant difference in the gender and age distribution between the two groups of children (P < 0.05), while no statistically significant differences were observed in disease duration, rheumatoid antibody levels, or JIA subtypes (P > 0.05). After sequencing data quality control and comparison of reference genomes, a total of 18 523 protein-coding genes were identified in all children's samples. A total of 705 differentially expressed genes (DEGs) were identified between the effective group and the invalid group through differential analysis, of which 579 were up-regulated in the effective group and 126 in the inactive group. GO function and KEGG pathway enrichment analysis showed that DEG was significantly enriched in 55 GO entries and 32 KEGG metabolic pathways, which were mainly related to IL-1β production and regulation, cytokine production and regulation, cytokine-cytokine receptor interaction, immune response regulation, and Toll-like receptor signaling pathway. CONCLUSION DEG between the effective and ineffective groups of TNFi treatment may be involved in the biological processes such as cytokine production and regulation, cytokine-receptor interaction, and immune response regulation, which will be helpful to predict the efficacy and prognosis of TNFi treatment for JIA.
Humans ; Arthritis, Juvenile/blood* ; Female ; Male ; Child ; Methotrexate/therapeutic use* ; Child, Preschool ; Tumor Necrosis Factor-alpha/antagonists & inhibitors* ; Transcriptome ; Adolescent ; RNA/genetics* ; Signal Transduction ; Gene Expression Profiling

Microglia, the resident immune cells in the central nervous system (CNS), rapidly transition from a resting to an active state in the acute phase of ischemic brain injury. This active state mediates a pro-inflammatory response that can exacerbate the injury. Targeting the pro-inflammatory response of microglia in the semi-dark band during this acute phase may effectively reduce brain injury. Shionone (SH), an active ingredient extracted from the dried roots and rhizomes of the genus Aster (Asteraceae), has been reported to regulate the inflammatory response of macrophages in sepsis-induced acute lung injury. However, its function in post-stroke neuroinflammation, particularly microglia-mediated neuroinflammation, remains uninvestigated. This study found that SH significantly inhibited lipopolysaccharide (LPS)-induced elevation of inflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS), in microglia in vitro. Furthermore, the results demonstrated that SH alleviated infarct volume and improved behavioral performance in middle cerebral artery occlusion (MCAO) mice, which may be attributed to the inhibition of the microglial inflammatory response induced by SH treatment. Mechanistically, SH potently inhibited the phosphorylation of serine-threonine protein kinase B (AKT), mammalian target of rapamycin (mTOR), and signal transducer and activator of transcription 3 (STAT3). These findings suggest that SH may be a potential therapeutic agent for relieving ischemic stroke (IS) by alleviating microglia-associated neuroinflammation.
Animals ; Microglia/immunology* ; Mice ; Male ; Mice, Inbred C57BL ; Brain Ischemia/immunology* ; Neuroinflammatory Diseases/drug therapy* ; Neuroprotective Agents/administration & dosage* ; Interleukin-1beta/genetics* ; STAT3 Transcription Factor/genetics* ; TOR Serine-Threonine Kinases/genetics* ; Tumor Necrosis Factor-alpha/genetics* ; Proto-Oncogene Proteins c-akt/immunology* ; Nitric Oxide Synthase Type II/genetics* ; Lipopolysaccharides

Lirispirolides A-L (1-12), twelve novel sesquiterpene-monoterpene heterodimers featuring distinctive carbon skeletons, were isolated from the branches and leaves of Chinese tulip tree [Liriodendron chinense (L. chinense)], a rare medicinal and ornamental plant endemic to China. The structural elucidation was accomplished through comprehensive spectroscopic analyses, quantum-chemical calculations, and X-ray crystallography. These heterodimers exhibit a characteristic 2-oxaspiro[4.5]decan-1-one structural motif, biosynthetically formed through intermolecular [4 + 2]-cycloaddition between a germacrane-type sesquiterpene and an ocimene-type monoterpene. The majority of the isolated compounds demonstrated significant anti-neuroinflammatory effects in lipopolysaccharide (LPS)-induced BV-2 microglial cells by reducing the production of pro-inflammatory mediators, specifically tumor necrosis factor-α (TNF-α) and nitric oxide (NO). Further investigation revealed that the lirispirolides' inhibition of NO release correlated with decreased messenger ribonucleic acid (mRNA) expression of inducible NO synthase (iNOS).
Sesquiterpenes/isolation & purification* ; Anti-Inflammatory Agents/isolation & purification* ; Animals ; Mice ; Tumor Necrosis Factor-alpha/genetics* ; Nitric Oxide/immunology* ; Microglia/immunology* ; Molecular Structure ; Liriodendron/chemistry* ; Monoterpenes/isolation & purification* ; Plants, Medicinal/chemistry* ; Cell Line ; Lipopolysaccharides ; Nitric Oxide Synthase Type II/immunology* ; Plant Extracts/pharmacology* ; China

In continuation of research aimed at identifying anti-inflammatory agents from natural sesquiterpenoids, an activity-guided fractionation approach utilizing lipopolysaccharide (LPS)-mediated RAW264.7 cells was employed to investigate chemical constituents from Inula Britannica (I. britannica). Seven novel sesquiterpenoid dimers inulabritanoids A-G (1-7) and two novel sesquiterpenoid monomers inulabritanoids H (8) and I (9) were isolated from I. britannica together with eighteen known compounds (10-27). The structural elucidation was accomplished through comprehensive analysis of 1D and 2D nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HR-MS), and electronic circular dichroism (ECD) spectra, complemented by quantum chemical calculations. Compounds 1, 2, 12, 16, 19, and 26 demonstrated inhibitory effects on NO production, with IC₅₀ values of 3.65, 5.48, 3.29, 6.91, 3.12, and 5.67 μmol·L^-1, respectively. Mechanistic studies revealed that compound 1 inhibited IκB kinase β (IKKβ) phosphorylation, thereby blocking nuclear factor κB (NF-κB) nuclear translocation, and activated the kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signal pathway, leading to decreased expression of NADPH oxidase 2 (NOX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), IL-1β, and IL-1α and increased expression of NAD(P)H: quinone oxidoreductase 1 (NQO-1) and heme oxygenase-1 (HO-1), thus exhibiting anti-inflammatory effects in vitro. These results indicate that dimeric sesquiterpenoids may serve as promising candidates for anti-inflammatory drug development.
Mice ; Animals ; Sesquiterpenes/isolation & purification* ; Anti-Inflammatory Agents/isolation & purification* ; Inula/chemistry* ; RAW 264.7 Cells ; Nitric Oxide ; Molecular Structure ; NF-kappa B/immunology* ; NF-E2-Related Factor 2/immunology* ; Macrophages/immunology* ; Nitric Oxide Synthase Type II/immunology* ; Plant Extracts/pharmacology* ; Lipopolysaccharides ; Tumor Necrosis Factor-alpha/immunology* ; I-kappa B Kinase/genetics*

Acute lung injury (ALI) is a severe disease caused by viral infection that triggers an uncontrolled inflammatory response. This study investigated the capacity of jasurolignoside (JO), a natural compound, to bind to Toll-like receptor 4 (TLR4) and treat ALI. The anti-inflammatory properties of JO were evaluated in vitro through Western blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, and co-immunoprecipitation. The investigation utilized a lipopolysaccharide (LPS)-induced ALI animal model to examine the therapeutic efficacy and mechanism of JO in vivo. JO attenuated inflammatory symptoms in infected cells and tissues by modulating the NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome and the nuclear factor κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathway. Molecular docking simulations revealed JO binding to TLR4 active sites, confirmed by cellular thermal shift assay. Surface plasmon resonance (SPR) demonstrated direct interaction between JO and TLR4 with a Kd value of 35.1 μmol·L^-1. Moreover, JO inhibited tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6 secretion and reduced leukocyte, neutrophil, lymphocyte, and macrophage infiltration in ALI-affected mice. JO also enhanced lung function and reduced ALI-related mortality. Immunohistochemical staining demonstrated JO's ability to suppress TLR4 expression in ALI-affected mouse lung tissue. This study establishes that JO can bind to TLR4 and effectively treat ALI, indicating its potential as a therapeutic agent for clinical applications.
Toll-Like Receptor 4/chemistry* ; Animals ; Acute Lung Injury/chemically induced* ; Mice ; Humans ; Ilex/chemistry* ; Molecular Docking Simulation ; Male ; NF-kappa B/immunology* ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein/immunology* ; Tumor Necrosis Factor-alpha/genetics* ; Interleukin-1beta/genetics* ; RAW 264.7 Cells ; Disease Models, Animal

Gentianopsis barbata (G. barbata) represents a significant plant species with considerable ornamental and medicinal value in China. This investigation sought to elucidate the primary constituents within the plant and investigate their pharmacological properties. Fifty triterpenoids (1-50), including nine previously undescribed compounds (1, 2, 7, 10, 20, 28, 29, 37, and 41) were isolated and characterized from the whole plants of G. barbata. Notably, compounds 1 and 2 exhibited the novel 3,4;9,10-diseco-24-homo-cycloartane triterpenoid skeleton. The isolated triterpenoids demonstrated substantial anti-inflammatory activity through inhibition of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) cytokine secretion in LPS-induced RAW264.7 macrophages, and hepatoprotective effects by preventing tert-butyl hydroperoxide (t-BHP)-induced oxidative injury in HepG2 cells. These results demonstrate both the presence of diverse triterpenoids in G. barbata and their therapeutic potential for inflammatory and hepatic conditions, providing scientific evidence supporting the clinical application of this traditional Mongolian medicinal plant.
Triterpenes/isolation & purification* ; Mice ; Anti-Inflammatory Agents/isolation & purification* ; Animals ; Humans ; RAW 264.7 Cells ; Hep G2 Cells ; Interleukin-6/genetics* ; Tumor Necrosis Factor-alpha/genetics* ; Medicine, Mongolian Traditional ; Macrophages/immunology* ; Protective Agents/isolation & purification* ; Liver/drug effects* ; Gentianaceae/chemistry* ; Plant Extracts/chemistry* ; Molecular Structure

Objective To explore the effect of miR-582-5p on Mycobacterium tuberculosis (Mtb)-infected macrophages by regulating dual specificity phosphatase 1 (DUSP1). Methods THP-1 macrophages were divided into six groups: control group, Mtb group, inhibitor-NC group, miR-582-5p inhibitor group, miR-582-5p inhibitor+si-NC group, and miR-582-5p inhibitor+si-DUSP1 group. QRT-PCR was applied to detect the gene expression of miR-582-5p and DUSP1 in cells. ELISA kit was used to detect the levels of interferon γ (IFN-γ), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β). CCK-8 method was applied to detect cell proliferation. Flow cytometry was applied to detect cell apoptosis rate. Western blot analysis was used to measure the protein expression levels of B-cell lymphoma 2 (Bcl2), Bcl2-associated X (BAX), and cleaved-caspase 3 (c-caspase-3) in cells. In addition, the target relationship between miR-582-5p and DUSP1 was verified. Results Compared with the control group, the expression of miR-582-5p, levels of IFN-γ, IL-6, TNF-α, IL-1β, bacterial load and OD450 values (24 h, 48 h), and the protein expression of Bcl2 in macrophages were higher in the Mtb group, while the mRNA expression of DUSP1, apoptosis rate, and the protein expression levels of c-caspase-3, BAX and DUSP1 were lower. Compared with the Mtb group and the inhibitor-NC group, the above-mentioned indicators in the miR-582-5p inhibitor group were partially reversed. Down-regulation of DUSP1 expression partially reversed the inhibitory effect of down-regulation of miR-582-5p expression on Mtb-infected macrophages. Conclusion Inhibiting the expression of miR-582-5p can up-regulate DUSP1, thereby inhibiting the proliferation and inflammatory response of Mtb-infected macrophages and promoting cell apoptosis.
Humans ; Macrophages/metabolism* ; Dual Specificity Phosphatase 1/metabolism* ; MicroRNAs/metabolism* ; Mycobacterium tuberculosis/physiology* ; Tuberculosis/microbiology* ; Apoptosis/genetics* ; THP-1 Cells ; Cell Proliferation/genetics* ; Interferon-gamma/genetics* ; Tumor Necrosis Factor-alpha/genetics* ; Interleukin-1beta/genetics*

Objective To investigate the effects of hyaluronic acid and proteoglycan-linked protein 1 (HAPLN1) secreted by synovial fibroblasts (FLS) on the polarization of macrophages (Mϕ) in rheumatoid arthritis (RA). Methods Human monocytic leukemia cells (THP-1) were differentiated into Mϕ, which were subsequently exposed to recombinant HAPLN1 (rHAPLN1). RA-FLS were transfected separately with HAPLN1 overexpression plasmid (HAPLN1^OE) or small interfering RNA targeting HAPLN1 (si-HAPLN1), and then co-cultured with Mϕ to establish a co-culture model. The viability of Mϕ was assessed using the CCK-8 assay, and the proportions of pro-inflammatory M1-type and anti-inflammatory M2-type Mϕ were analyzed by flow cytometry. Additionally, the expression levels of inflammatory markers, including interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS), were quantified using quantitative real-time PCR and Western blot analysis. Results The viability of Mϕ was increased in the rHAPLN1 group compared to the control group. Furthermore, both the M1/Mϕ ratio and inflammatory factor levels were elevated in the rHAPLN1 and HAPLN1^OE groups. In contrast, the si-HAPLN1 group exhibited a decrease in the M1/Mϕ ratio and inflammatory factor expression. Notably, the introduction of rHAPLN1 in rescue experiments further promoted Mϕ polarization towards the M1 phenotype. Conclusion HAPLN1, secreted by RA fibroblast-like synoviocytes (RA-FLS), enhances Mϕ polarization towards the M1 phenotype.
Humans ; Arthritis, Rheumatoid/genetics* ; Macrophages/immunology* ; Fibroblasts/metabolism* ; Phenotype ; Extracellular Matrix Proteins/genetics* ; Proteoglycans/genetics* ; Synovial Membrane/cytology* ; Tumor Necrosis Factor-alpha/genetics* ; Interleukin-1beta/genetics* ; Nitric Oxide Synthase Type II/genetics* ; Cell Differentiation ; Coculture Techniques ; THP-1 Cells

Objective This study aims to investigate the effect of berberine (Ber) on foam cell formation induced by oxidized low-density lipoprotein (ox-LDL) in macrophages and to explore the mechanism's association with the ACE2-Ang(1-7)-Mas axis. Methods They were randomly divided into blank group, model group (RAW264.7 cells induced with 60 μg/mL ox-LDL), and berberine group (the model treated with berberine interventions at 2.5, 5, and 10 μmol/L concentrations). Lipid accumulation within the cells was assessed by Oil Red O staining, and the content of lipid droplets in each group was quantitatively analyzed by enzymatic method. The content of total cholesterol (TC) and free cholesterol (FC) in foam cells were detected by enzymatic method. The levels of oxidative stress factors (malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH)), inflammatory factors such as tumor necrosis factor α(TNF-α), and nitric oxide (NO) were measured using corresponding relevant reagent kits. The mRNA and protein expressions of ACE2 and Mas were evaluated through quantitative real-time PCR and Western blot analysis, respectively. The levels of AngII and Ang(1-7) were detected by ELISA. Results Compared with the model group, the berberine groups exhibited reduced lipid droplet accumulation and a dose-dependent decrease in intracellular lipid content. Berberine significantly lowered TC and FC levels in foam cells and reduced the CE/TC ratio. The levels of the oxidative factor MDA were significantly reduced, while the levels of the antioxidant factors SOD and GSH were markedly increased. Inflammatory factors TNF-α and NO were significantly decreased. The expression of the ACE2-Ang(1-7)-Mas signaling pathway was significantly activated, and the effect was more pronounced in the Ber group with high-concentration compared to the group with low-concentration, demonstrating a dose-dependent response. Conclusion Berberine can inhibit macrophage foam cell formation, potentially through upregulation of the ACE2-Ang(1-7)-Mas signaling pathway, thereby contributing to the alleviation of atherosclerosis.
Berberine/pharmacology* ; Foam Cells/cytology* ; Animals ; Signal Transduction/drug effects* ; Mice ; Angiotensin-Converting Enzyme 2 ; Angiotensin I/genetics* ; Peptidyl-Dipeptidase A/genetics* ; Peptide Fragments/genetics* ; Receptors, G-Protein-Coupled/genetics* ; RAW 264.7 Cells ; Proto-Oncogene Proteins/genetics* ; Proto-Oncogene Mas ; Lipoproteins, LDL/pharmacology* ; Nitric Oxide/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

Objective To investigate the effect and mechanism of baicalin on blood lipid metabolism and immune function in rats with gestational diabetes mellitus (GDM). Methods Female rats fed with high-fat and high-sugar diet and male rats fed with ordinary diet were caged together to prepare pregnant rats, and the GDM rat model was established by intraperitoneal injection of streptozotocin (35 mg/kg). GDM rats were randomly divided into a model group, a fasudil (FA) (RhoA/RocK inhibitor) group (10 mg/kg), low-dose (100 mg/kg) and high-dose (200 mg/kg) baicalin groups, and a high-dose baicalin combined with LPA (RhoA/RocK activator) group (200 mg/kg baicalin+1 mg/kg LPA ), with 12 rats in each group. Another 12 pregnant rats fed with high-fat and high-sugar diet were selected as the control group. After 2 weeks of corresponding drug intervention in each group, the level of fasting blood glucose (FBG) was detected by blood glucose meter. The level of fasting insulin (FINS) in serum was detected by ELISA, and the insulin resistance index (HOMA-IR) was calculated. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) in serum, and the levels of immunomodulator tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-10 in peripheral blood were detected by the kit. The histopathological changes of liver were observed by HE staining. The proportion of T lymphocyte subsets in peripheral blood was detected by flow cytometry. The mRNA and protein expressions of Ras homolog gene family member A (RhoA), Rho associated coiled-coil forming protein kinase 1 (ROCK1), and ROCK2 in liver tissue were detected by real-time quantitative PCR and Western blot. Results Compared with the control group, the levels of FBG, FINS, HOMA-IR, ALT, AST, TG, TC, and LDL-C in serum, the levels of TNF-α, IL-6, the percentage of CD8⁺T cell in peripheral blood, and the mRNA and protein expression of RhoA, ROCK1, and ROCK2 in liver tissue in the model group were higher; the level of HDL-C in serum, the percentage of IL-10 levels, CD3⁺T cells, CD4⁺T cell, and CD4⁺T/CD8⁺T ratio in peripheral blood were lower. Compared with the model group, the levels of FBG, FINS, HOMA-IR, ALT, AST, TG, TC, and LDL-C in serum, the levels of TNF-α, IL-6, the percentage of CD8⁺T cell in peripheral blood, and the mRNA and protein expression of RhoA, ROCK1, and ROCK2 in liver tissue in the the FA group and low-dose and high-dose baicalin groups were lower; the level of HDL-C in serum, IL-10 level, the percentage of CD3⁺T cells, CD4⁺T cell, and CD4⁺T/CD8⁺T ratio in peripheral blood were higher. LPA could obviously weaken the improvement effects of baicalin on blood lipid metabolism and immune function in GDM rats. Conclusion Baicalin may improve blood lipid metabolism and immune function in GDM rats by inhibiting the RhoA/ROCK pathway.
Animals ; Female ; Diabetes, Gestational/metabolism* ; Pregnancy ; rho-Associated Kinases/genetics* ; Flavonoids/pharmacology* ; Rats ; rhoA GTP-Binding Protein/genetics* ; Lipid Metabolism/drug effects* ; Male ; Signal Transduction/drug effects* ; Rats, Sprague-Dawley ; Blood Glucose/metabolism* ; Lipids/blood* ; Tumor Necrosis Factor-alpha/blood* ; rho GTP-Binding Proteins