BACKGROUND: Knee osteoarthritis (OA) is still challenging to prevent or treat. Enhanced endoplasmic reticulum (ER) stress and increased pyroptosis in chondrocytes may be responsible for cartilage degeneration. This study aims to investigate the effect of ER stress on chondrocyte pyroptosis and the upstream regulatory mechanisms, which have rarely been reported. METHODS: The expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2), microRNA-142-3p (miR-142-3p), and high mobility group box 1 (HMGB1) and the levels of ER stress, pyroptosis, and metabolic markers in normal and OA chondrocytes were investigated by western blotting, quantitative polymerase chain reaction, immunohistochemistry, fluorescence in situ hybridization, fluorescein amidite-tyrosine-valine-alanine-aspartic acid-fluoromethyl ketone (FAM-YVAD-FMK)/Hoechst 33342/propidium iodide (PI) staining, lactate dehydrogenase (LDH) release assays, and cell viability assessments. The effects of EZH2, miR-142-3p, and HMGB1 on ER stress and pyroptosis and the hierarchical regulatory relationship between them were analyzed by chromatin immunoprecipitation, luciferase reporters, gain/loss-of-function assays, and rescue assays in interleukin (IL)-1β-induced OA chondrocytes. The mechanistic contribution of EZH2, miR-142-3p, and HMGB1 to chondrocyte ER stress and pyroptosis and therapeutic prospects were validated radiologically, histologically, and immunohistochemically in surgically induced OA rats. RESULTS: Increased EZH2 and HMGB1, decreased miR-142-3p, enhanced ER stress, and activated pyroptosis in chondrocytes were associated with OA occurrence and progression. EZH2 and HMGB1 exacerbated and miR-142-3p alleviated ER stress and pyroptosis in OA chondrocytes. EZH2 transcriptionally silenced miR-142-3p via H3K27 trimethylation, and miR-142-3p posttranscriptionally silenced HMGB1 by targeting the 3'-UTR of the HMGB1 gene. Moreover, ER stress mediated the effects of EZH2, miR-142-3p, and HMGB1 on chondrocyte pyroptosis. In vivo experiments mechanistically validated the hierarchical regulatory relationship between EZH2, miR-142-3p, and HMGB1 and their effects on chondrocyte ER stress and pyroptosis. CONCLUSIONS A novel EZH2/miR-142-3p/HMGB1 axis mediates chondrocyte pyroptosis and cartilage degeneration by regulating ER stress in OA, contributing novel mechanistic insights into OA pathogenesis and providing potential targets for future therapeutic research.
Enhancer of Zeste Homolog 2 Protein/genetics* ; Osteoarthritis, Knee/pathology* ; Chondrocytes/metabolism* ; Pyroptosis/physiology* ; HMGB1 Protein/genetics* ; MicroRNAs/metabolism* ; Endoplasmic Reticulum Stress/genetics* ; Humans ; Animals ; Rats ; Male ; Rats, Sprague-Dawley ; Middle Aged

Atherosclerosis (AS) is a prevalent clinical vascular condition and serves as a pivotal pathological foundation for cardiovascular diseases. Understanding the pathogenesis of AS has significant clinical and societal implications, aiding in the development of targeted drugs. Neutrophils, the most abundant leukocytes in circulation, assume a central role during inflammatory responses and closely interact with AS, which is a chronic inflammatory vascular disease. Neutrophil extracellular traps (NETs) are substantial reticular formations discharged by neutrophils that serve as an immune defense mechanism. These structures play a crucial role in inducing dysfunction of the vascular barrier following endothelial cell injury. Components released by NETs pose a threat to the integrity of vascular endothelium, which is essential as it acts as the primary barrier to maintain vascular wall integrity. Endothelial damage constitutes the initial stage in the onset of AS. Recent investigations have explored the intricate involvement of NETs in AS progression. The underlying structures of NETs and their active ingredients, including histone, myeloperoxidase (MPO), cathepsin G, neutrophil elastase (NE), matrix metalloproteinases (MMPs), antimicrobial peptide LL-37, alpha-defensin 1-3, and high mobility group protein B1 have diverse and complex effects on AS through various mechanisms. This review aims to comprehensively examine the interplay between NETs and AS while providing insights into their mechanistic underpinnings of NETs in this condition. By shedding light on this intricate relationship, this exploration paves the way for future investigations into NETs while guiding clinical translation efforts and charting new paths for therapeutic interventions.
Extracellular Traps/physiology* ; Humans ; Atherosclerosis/immunology* ; Neutrophils/physiology* ; Leukocyte Elastase/metabolism* ; Peroxidase/physiology* ; Matrix Metalloproteinases/physiology* ; Cathepsin G/metabolism* ; Cathelicidins ; HMGB1 Protein/physiology* ; Histones ; Animals ; Endothelium, Vascular

This study aims to integrate data mining techniques of single cell transcriptomics and spatial transcriptomics, along with animal experiment validation, so as to systematically characterize the protective effects of Jianpi Tongluo Formula(JTF) on the cartilage in knee osteoarthritis(KOA) and elucidate the underlying molecular mechanisms. Single cell transcriptomics and spatial transcriptomics datasets(GSE254844 and GSE255460) of the cartilage tissue obtained from KOA patients were analyzed to map the single cell-spatial heterogeneity and identify key pathogenic factors. After that, a KOA rat model was established via knee joint injection of papain. The intervention effects of JTF on the expression features of these key factors were assessed through real-time quantitative polymerase chain reaction(PCR), Western blot, and immunohistochemical staining. As a result, the integrated single cell and spatial transcriptomics data identified distinct cell subsets with different pathological changes in different regions of the inflamed cartilage tissue in KOA, and their differentiation trajectories were closely related to the inflammatory fibrosis-like pathological changes of chondrocytes. Accordingly, the expression levels of the two key effect targets, namely nuclear receptor coactivator 4(NCOA4) and high mobility group box 1(HMGB1) were significantly reduced in the articular surface and superficial zone of the inflamed joints when JTF effectively alleviated various pathological changes in KOA rats, thus reversing the abnormal chondrocyte autophagy level, relieving the inflammatory responses and fibrosis-like pathological changes, and promoting the repair of chondrocyte function. Collectively, this study revealed the heterogeneous characteristics and dynamic changes of inflamed cartilage tissue in different regions and different cell subsets in KOA patients. It is worth noting that NCOA4 and HMGB1 were crucial in regulating chondrocyte autophagy and inflammatory reaction, while JTF could reverse the regulation of NCOA4 and HMGB1 and correct the abnormal molecular signal axis in the target cells of the inflamed joints. The research can provide a new research idea and scientific basis for developing a personalized therapeutic schedule targeting the spatiotemporal heterogeneity characteristics of KOA.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Osteoarthritis, Knee/pathology* ; Humans ; Male ; Cartilage, Articular/metabolism* ; Chondrocytes/metabolism* ; Rats, Sprague-Dawley ; Female ; Protective Agents/administration & dosage* ; Single-Cell Analysis ; Middle Aged ; HMGB1 Protein/metabolism*

Objective This study investigated the regulatory effect of high mobility group protein B1 (HMGB1) in the peripheral blood of patients with primary immune thrombocytopenia (ITP) on myeloid dendritic cells (mDC) and Th17/regulatory T cells (Treg) balance. Methods The study enrolled 30 newly diagnosed ITP patients and 30 healthy controls.Flow cytometry was used to measure the proportion of mDC, Th17, and Treg cells in the peripheral blood of ITP patients and healthy controls. ELISA was conducted to quantify the serum levels of HMGB1, interleukin 6 (IL-6), IL-23, IL-17, and transforming growth factor β(TGF-β). The mRNA levels of retinoic acid-related orphan receptor γt(RORγt) and forehead box P3(FOXP3) were detected by real-time PCR. The correlation between the abovementioned cells, cytokines, and platelet count was assessed using Pearson linear correlation analysis. Results The proportion of Th17 cells and the expression levels of HMGB1, IL-6, IL-23, IL-17 and the level of RORγt mRNA in the peripheral blood of ITP patients were higher than those in healthy controls. However, the Treg cell proportion and TGF-β level were lower in ITP patients than those in healthy controls. In patients with ITP, the proportion of mDC and the level of FOXP3 mRNA did not show significant changes. The proportion of mDC cells was significantly correlated with the expression of IL-6 and IL-23. Moreover, the expression of HMGB1 showed a significant correlation with the expression of mDC, IL-6, IL-23, RORγt mRNA, and IL-17. Notably, both the proportion of mDC cells and the expression of HMGB1 were negatively correlated with platelet count. Conclusion The high expression of HMGB1 in peripheral blood of ITP patients may induce Th17/Treg imbalance by promoting the maturation of mDC and affecting the secretion of cytokines, thereby potentially playing a role in the immunological mechanism of ITP.
Humans ; Th17 Cells/cytology* ; HMGB1 Protein/genetics* ; T-Lymphocytes, Regulatory/cytology* ; Female ; Male ; Dendritic Cells/metabolism* ; Adult ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic/genetics* ; Nuclear Receptor Subfamily 1, Group F, Member 3/genetics* ; Young Adult ; Interleukin-23/blood* ; Interleukin-17/blood* ; Interleukin-6/blood* ; Forkhead Transcription Factors/genetics* ; Myeloid Cells/cytology* ; Aged

OBJECTIVES: To explore the mechanism of Pingchuanning Formula (PCN) for inhibiting airway inflammation in rats with asthmatic cold syndrome. METHODS: A total of 105 SD rats were randomized equally into 7 groups, including a control group, an asthmatic cold syndrome model group, 3 PCN treatment groups at high, medium and low doses, a Guilong Kechuanning (GLCKN) treatment group, and a dexamethasone (DEX) treatment group. In all but the control rats, asthma cold syndrome models were established and daily gavage of saline, PCN, GLCKN or DEX was administered 29 days after the start of modeling. The changes in general condition, lung function and lung histopathology of the rats were observed, and inflammatory factors in the alveolar lavage fluid (BALF), oxidative stress, lung tissue ultrastructure, cytokine levels, and expressions of the genes related to the HMGB1/Beclin-1 axis and autophagy were analyzed. RESULTS: The rat models had obvious manifestations of asthmatic cold syndrome with significantly decreased body mass, food intake, and water intake, reduced FEV_0.3, FVC, and FEV_0.3/FVC, obvious inflammatory cell infiltration in the lung tissue, and increased alveolar inflammation score and counts of neutrophils, eosinophils, lymphocytes, macrophages, and leukocytes in the BALF. The rat models also had significantly increased MDA level and decreased SOD level and exhibited obvious ultrastructural changes in the lung tissues, where the expressions of HMGB1, Beclin-1, ATG5, TNF-α, IL-6,IL-1β, and IL-13 and the LC3II/I ratio were increased, while the levels of Bcl-2 and IFN-γ were decreased. PCN treatment significantly improved these pathological changes in the rat models, and its therapeutic effect was better than that of GLKCN and similar to that of DEX. CONCLUSIONS PCN can effectively alleviate airway inflammation in rat models of asthmatic cold syndrome possibly by modulating the HMGB1/Beclin-1 signaling axis to suppress cell autophagy, thereby attenuating airway inflammatory damages.
Animals ; Rats ; Autophagy/drug effects* ; Rats, Sprague-Dawley ; Asthma/pathology* ; Beclin-1 ; HMGB1 Protein/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Disease Models, Animal ; Male ; Lung/pathology* ; Inflammation

High mobility group box 1 (HMGB1), when released extracellularly, plays a pivotal role in the development of spinal cord synapses and exacerbates autoimmune diseases within the central nervous system. In experimental autoimmune encephalomyelitis (EAE), a condition that models multiple sclerosis, the levels of extracellular HMGB1 and interleukin-33 (IL-33) have been found to be inversely correlated. However, the mechanism by which IL-33 deficiency enhances HMGB1 release during EAE remains elusive. Our study elucidates a potential signaling pathway whereby the absence of IL-33 leads to increased binding of P300/CBP-associated factor with HMGB1 in the nuclei of astrocytes, upregulating HMGB1 acetylation and promoting its release from astrocyte nuclei in the spinal cord of EAE mice. Conversely, the addition of IL-33 counteracts the TNF-α-induced increase in HMGB1 and acetylated HMGB1 levels in primary astrocytes. These findings underscore the potential of IL-33-associated signaling pathways as a therapeutic target for EAE treatment.
Animals ; Encephalomyelitis, Autoimmune, Experimental/metabolism* ; Astrocytes/metabolism* ; Interleukin-33/metabolism* ; HMGB1 Protein/metabolism* ; Acetylation ; Mice, Knockout ; Mice, Inbred C57BL ; p300-CBP Transcription Factors/metabolism* ; Mice ; Spinal Cord/metabolism* ; Cells, Cultured ; Female ; Signal Transduction

Infiltration and activation of peripheral immune cells are critical in the progression of multiple sclerosis and its experimental animal model, experimental autoimmune encephalomyelitis (EAE). This study investigates the role of high mobility group box 1 (HMGB1) in oligodendrocyte precursor cells (OPCs) in modulating pathogenic T cells infiltrating the central nervous system through the blood-brain barrier (BBB) by using OPC-specific HMGB1 knockout (KO) mice. We found that HMGB1 released from OPCs promotes BBB disruption, subsequently allowing increased immune cell infiltration. The migration of CD4+ T cells isolated from EAE-induced mice was enhanced when co-cultured with OPCs compared to oligodendrocytes (OLs). OPC-specific HMGB1 KO mice exhibited lower BBB permeability and reduced immune cell infiltration into the CNS, leading to less damage to the myelin sheath and mitigated EAE progression. CD4+ T cell migration was also reduced when co-cultured with HMGB1 knock-out OPCs. Our findings reveal that HMGB1 secretion from OPCs is crucial for regulating immune cell infiltration and provides insights into the immunomodulatory function of OPCs in autoimmune diseases.
Animals ; Encephalomyelitis, Autoimmune, Experimental/metabolism* ; HMGB1 Protein/deficiency* ; Mice, Knockout ; Oligodendrocyte Precursor Cells/immunology* ; Mice, Inbred C57BL ; CD4-Positive T-Lymphocytes/immunology* ; Cell Movement ; Blood-Brain Barrier/immunology* ; Mice ; Myelin Sheath/pathology* ; Disease Models, Animal ; Coculture Techniques ; Oligodendroglia/metabolism* ; Female ; Cells, Cultured

The aim of this study was to investigate the underlying mechanism of chrysophanol(Chr) in reducing inflammation and foam cell formation induced by oxidized low-density lipoprotein(ox-LDL) and to investigate the targets and pathways related to effects of Chr on coronary atherosclerosis, providing a theoretical basis for the development of new clinical drugs. RAW264.7 macrophages were cultured in vitro, and after determining the appropriate concentrations of Chr and ox-LDL for treating RAW264.7 macrophages using a cell counting kit-8(CCK-8), the macrophages were treated with different concentrations of Chr(10, 15 μmol·L~(-1)) and ox-LDL(with or without 80 mg·mL~(-1)) for 24 h. RAW264.7 macrophages were divided into four groups: control group, model group(80 mg·mL~(-1) ox-LDL), treatment group(80 mg·mL~(-1) ox-LDL+10 μmol·L~(-1) Chr), and treatment group(80 mg·mL~(-1) ox-LDL+15 μmol·L~(-1) Chr). Lipid accumulation in each group was detected by oil red O staining. CD36 expression was analyzed by flow cytometry. Western blot was used to detect the expression of scavenger receptor class A1(SR-A1), scavenger receptor class B type Ⅰ(SR-B1), autophagy-related protein 5(Atg5), Beclin-1, autophagy adaptor protein p62(P62), the ratio of microtubule-associated protein light chain 3(LC3)Ⅱ to LC3Ⅰ(LC3Ⅱ/LC3Ⅰ), nuclear factor kappa B P65(NF-κB P65), inhibitor of κB kinase β(IKKβ), nuclear factor of κB inhibitor(IκB), high mobility group box protein 1(HMGB1), phosphatidylinositol 3-kinase(PI3K), protein kinase B(Akt), and phosphorylated mammalian target of rapamycin(mTOR). Real-time quantitative polymerase chain reaction(RT-qPCR) was used to detect the mRNA expression levels of ATP-binding cassette transporter A1(ABCA1), ATP-binding cassette transporter G1(ABCG1), interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), HMGB1, inducible nitric oxide synthase(iNOS), arginase 1(Arg1), macrophage galactose-type lectin-1(Mgl-1), and NF-κB P65. Immunofluorescence analysis was performed to determine the localization of HMGB1 in RAW264.7 cells in each group. The autophagy inhibitor 3-methyladenine(3-MA) was added as a control for reverse validation, and the RAW264.7 macrophages were divided into four groups again: control group, model group(80 mg·mL~(-1) ox-LDL), treatment group(80 mg·mL~(-1) ox-LDL + 15 μmol·L~(-1) Chr), and inhibitor group(80 mg·mL~(-1) ox-LDL+15 μmol·L~(-1) Chr+3-MA). The results showed that Chr effectively reduced foam cell formation by regulating the expression levels of SR-A1, ABCA1, ABCG1, the LC3Ⅱ/LC3Ⅰ ratio, Atg5, Beclin-1, and p62, and inhibited the NF-κB/HMGB1-PI3K/Akt/mTOR signaling pathway. Moreover, the inhibitory effects of Chr on autophagy and the NF-κB/HMGB1-PI3K/Akt/mTOR pathway were reversed by the autophagy inhibitor 3-MA. In conclusion, Chr exhibits therapeutic potential for the treatment of atherosclerosis by inducing autophagy and modulating the NF-κB/HMGB1 and PI3K/Akt/mTOR pathways to inhibit the formation of macrophage inflammatory foam cells.
Animals ; Lipoproteins, LDL/metabolism* ; Mice ; TOR Serine-Threonine Kinases/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Macrophages/cytology* ; RAW 264.7 Cells ; Proto-Oncogene Proteins c-akt/genetics* ; Signal Transduction/drug effects* ; NF-kappa B/genetics* ; Anthraquinones/pharmacology* ; Foam Cells/cytology* ; HMGB1 Protein/genetics* ; Humans

Objective To investigate the efficacy and safety of Xihuang capsules as an adjuvant treatment for metastatic colorectal cancer and their impact on immune function. Methods A retrospective analysis was conducted on clinical data from 112 patients diagnosed with metastatic colorectal cancer. The patients were categorized into two groups: a control group (n=56) that did not take Xihuang capsules and an observation group (n=56) that did. The efficacy, improvement of quality of life, toxic and side effects and immune function of the two groups were analyzed and compared. Results After treatment, the disease control rate (DCR) and the rate of improvement in quality of life were significantly higher in the observation group compared to the control group. Additionally, levels of carcinoembryonic antigen (CEA) and the incidence of adverse reactions, including bone marrow suppression and liver and kidney function damage, were significantly lower in the observation group. Furthermore, the percentages of CD4⁺ and CD8⁺ T cells, the CD8⁺/CD4⁺ T cells ratio, as well as serum levels of high mobility group box-1 (HMGB1) and interleukin 2 (IL-2) in observation group were significantly elevated compared to pre-treatment levels. Subgroup analysis revealed that patients with a Karnofsky Performance Status (KPS) score ≤80, a high CD8⁺/CD4⁺ T cells ratio, and elevated HMGB1 levels experienced a significantly higher objective response rate (ORR) in the observation group. Conversely, patients with stage IVB disease, who had KPS score ≤80, a low CD8⁺/CD4⁺ T cells ratio and high CEA and IL-2 levels demonstrated a more pronounced DCR in the observation group. Conclusion Xihuang capsules exhibit promising clinical efficacy as an adjuvant treatment for advanced colorectal cancer. They not only enhance patients' quality of life and reduce the toxic and adverse effects of chemotherapy, but also improve immune function. These benefits are particularly significant in patients with a high tumor burden, indicating that Xihuang capsules are worthy of clinical application.
Humans ; Colorectal Neoplasms/immunology* ; Male ; Female ; Middle Aged ; Drugs, Chinese Herbal/adverse effects* ; Capsules ; Aged ; Carcinoembryonic Antigen/blood* ; Retrospective Studies ; Quality of Life ; Adult ; Neoplasm Metastasis ; Interleukin-2/blood* ; HMGB1 Protein/blood* ; Chemotherapy, Adjuvant

OBJECTIVES: To explore the mechanism by which Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) regulates lipopolysaccharide (LPS)-induced mitochondrial metabolic abnormalities and inflammatory responses in macrophages. METHODS: Macrophage cell lines with overexpressed WAVE1 (mouse BMDM and human THP1 cells) were prepared. The macrophages were treated with LPS (500 ng/mL) to simulate sepsis-induced inflammatory responses. The experiment consisted of two parts. The first part included control, LPS, vector (LPS+oe-NC), WAVE1 overexpression (LPS+oe-WAVE1) groups. The second part included LPS, LPS+oe-NC, LPS+oe-WAVE1 and exogenous high mobility group box-1 (HMGB1) intervention (LPS+oe-WAVE1+HMGB1) groups. RT-PCR was used to measure mitochondrial DNA content, and RT-qPCR was used to detect the mRNA expression levels of WAVE1, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. Western blot was performed to measure the protein expression of WAVE1, hexokinase 2, and pyruvate kinase M2. ELISA was utilized to detect the levels of TNF-α, IL-1β, IL-6, and HMGB1. JC-1 staining was used to assess mitochondrial membrane potential. Seahorse XP96 was used to evaluate oxygen consumption rate and extracellular acidification rate. MitoSOX probe was employed to measure mitochondrial reactive oxygen species levels, and 2-NBDG method was used to assess glucose uptake. Kits were used to measure pyruvate kinase activity, lactate, adenosine triphosphate (ATP), and HMGB1 levels. RESULTS: Compared with the control group, the LPS group showed lower levels of WAVE1 protein and mRNA expression, mitochondrial membrane potential, oxygen consumption rate, and mitochondrial DNA content (P<0.05), while TNF-α, IL-1β, IL-6 levels and mRNA expression, mitochondrial reactive oxygen species, glucose uptake, lactate, ATP, hexokinase 2, and pyruvate kinase M2 protein expression levels as well as extracellular acidification rate, pyruvate kinase activity, and HMGB1 release were significantly increased (P<0.05). Compared with the LPS+oe-NC group, the LPS+oe-WAVE1 group showed increased WAVE1 protein and mRNA expression, mitochondrial membrane potential, oxygen consumption rate, and mitochondrial DNA content (P<0.05), while TNF-α, IL-1β, IL-6 levels and mRNA expression, mitochondrial reactive oxygen species, glucose uptake, lactate, ATP, hexokinase 2, and pyruvate kinase M2 protein expressions, as well as extracellular acidification rate, pyruvate kinase activity, and HMGB1 release were decreased (P<0.05). Compared with the LPS+oe-WAVE1 group, the LPS+oe-WAVE1+HMGB1 group exhibited increased glucose uptake, lactate, ATP levels, and extracellular acidification rate (P<0.05). CONCLUSIONS WAVE1 participates in the regulation of LPS-induced inflammatory responses in macrophages by modulating the release of inflammatory factors, mitochondrial metabolism, and HMGB1 release.
Lipopolysaccharides ; Humans ; Mitochondria/metabolism* ; Animals ; Macrophages/metabolism* ; Mice ; Hexokinase/genetics* ; Wiskott-Aldrich Syndrome Protein Family/metabolism* ; HMGB1 Protein/physiology* ; Inflammation/metabolism* ; DNA, Mitochondrial ; Pyruvate Kinase/metabolism*