HMGB1 Expression Research Articles

The effect of the HMGB1/RAGE/TLR4/NF-κB signalling pathway in patients with idiopathic epilepsy and its relationship with toxoplasmosis.

This study aims to investigate the relationship between toxoplasmosis and this pathway, which may be effective in the formation of epilepsy by acting through the HMGB1/RAGE/TLR4/NF-κB signalling pathway in patients with idiopathic epilepsy. In the study, four different experimental groups were formed by selecting Toxoplasma gondii IgG positive and negative patients with idiopathic epilepsy and healthy controls. Experimental groups were as follows: Group 1: Epilepsy+/Toxo- (E+, T-) (n = 10), Group 2: Epilepsy-/Toxo- (E-, T-) (n = 10), Group 3: Epilepsy-/Toxo+ (E-, T+) (n = 10), Group 4: Epilepsy+/Toxo+ (E+, T+) (n = 10). HMGB1, RAGE, TLR4, TLR1, TLR2, TLR3, IRAK1, IRAK2, IKBKB, IKBKG, BCL3, IL1β, IL10, 1 L8 and TNFα mRNA expression levels in the HMGB/RAGE/TLR4/NF-κB signalling pathway were determined by quantitative simultaneous PCR (qRT-PCR) after collecting blood samples from all patients in the groups. Statistical analysis was performed by one-way ANOVA followed by LSD post-hoc tests, and p < 0.05 was considered to denote statistical significance. The gene expression levels of HMGB1, TLR4, IL10, IL1B, IL8, and TLR2 were significantly higher in the G1 group than in the other groups (p < 0.05). In the G3 group, RAGE and BCL3 gene expression levels were significantly higher than in the other groups (p < 0.05). In the G4 group, however, IRAK2, IKBKB, and IKBKG gene expression levels were significantly higher than in the other groups (p < 0.05). HMGB1, TLR4, IRAK2, IKBKB, IL10, IL1B, IL1B, and IL8 in this signalling pathway are highly expressed in epilepsy patients in G1 and seizures occur with the stimulation of excitatory mechanisms by acting through this pathway. The signalling pathway in epilepsy may be activated by HMGB1, TLR4, and TLR2, which are considered to increase the level of proinflammatory cytokines. In T. gondii, this pathway is activated by RAGE and BCL3.

Aconitate decarboxylase 1 (Acod1) suppresses inflammatory stimuli-induced immune responses in Japanese flounder (Paralichthys olivaceus)

Aconitate decarboxylase 1 (ACOD1, formerly termed as immunoresponsive gene 1) is an enzyme responsible for generation of anti-inflammatory metabolite itaconate through decarboxylation of cis-aconitate and plays a key role in inflammation regulation and anti-microbial host defense in mammals. However, the immune functions of Acod1 in fish remain largely undetermined. Here, we characterize a Acod1 homolog that is highly induced by LPS, poly(I:C) and extracellular ATP (eATP) challenges in the Japanese flounder Paralichthys olivaceus head kidney macrophages (HKMs). We find that Acod1 syntenic genes in P. olivaceus are similar with those in mammalian species and the deduced Japanese flounder ACOD1 protein shares similar protein domains and 3-D structures with its counterparts in mammals. We next show that itaconate derivatives dimethyl itaconate (DMI) and citraconate (Cit) can markedly down-regulate inflammatory stimuli, including LPS-, poly(I:C)- and eATP-induced inflammatory mediator nitric oxide (NO) production and gene expression of proinflammatory cytokines (IL-1β and TNF-α), chemokines (CXCL-8, CXCL-9 and CXCL-13), HMGB1 and inflammasome components (NLRP3, NLRC3, caspase1, ASC and GSDME), but up-regulate anti-inflammatory gene (IL-10 and SOCS1/3) expression in P. olivaceus HKMs. In addition, DMI and Cit treatment significantly inhibits Edwardsiella piscicida growth in P. olivaceus HKMs. Furthermore, knockdown of the endogenous Acod1 expression by siRNA silencing not only significantly increases LPS-, poly(I:C)- and eATP-induced proinflammatory gene expression but also decreases anti-inflammatory gene expression in the HKMs. In contrast, the LPS-, poly(I:C)- and eATP-indued proinflammatory gene expression and NO production are greatly attenuated in ACOD1 overexpressed Japanese flounder FG-9307 cells. Collectively, our studies demonstrate that the inducible fish Acod1 gene is a novel immune regulator in dampening inflammatory stimuli-induced immune responses and ACOD1-produced itaconate may play an essential role in anti-bacterial immunity in fish macrophages.

The role of HMGB1 on SiC NPs-induced inflammation response in lung epithelial-macrophage co-culture system

In recent years, carbonized silicon nanoparticles (SiC NPs) have found widespread scientific and engineering applications, raising concerns about potential human health risks. SiC NPs may induce pulmonary damage through sustained inflammatory responses and oxidative stress, with unclear toxicity mechanisms. This study uses an in vitro co-culture model of alveolar macrophages (NR8383) and alveolar epithelial cells (RLE-6TN) to simulate the interaction between airway epithelial cells and immune cells, providing initial insights into SiC NP-triggered inflammatory responses. The research reveals that increasing SiC NP exposure prompts NR8383 cells to release high mobility group box 1 protein (HMGB1), which migrates into RLE-6TN cells and activates the receptor for advanced glycation end-products (RAGE) and Toll-like receptor 4 (TLR4). RAGE and TLR4 synergistically activate the MyD88/NF-κB inflammatory pathway, ultimately inducing inflammatory responses and oxidative stress in RLE-6TN cells, characterized by excessive ROS generation and altered cytokine levels. Pretreatment with RAGE and TLR4 inhibitors attenuates SiC-induced HMGB1 expression and downstream pathway proteins, reducing inflammatory responses and oxidative damage. This highlights the pivotal role of RAGE-TLR4 crosstalk in SiC NP-induced pulmonary inflammation, providing insights into SiC NP cytotoxicity and nanomaterial safety guidelines.

Intervention Effect and Mechanism of Regulating MiR-155 on Young Rats with Dysfunction of Blood Coagulation

To investigate the intervention effect and mechanism of regulating miR-155 on young rats with dysfunction of blood coagulation. Twenty-six healthy and clean SD male rats were selected to establish the coagulopathy models. Twenty-four rats successfully established models and were randomly divided into three groups: model group, up-regulated miR-155 group and down-regulated miR-155 group, with 8 rats in each group. The expression of miR-155 was detected by real-time fluorescence quantitative polymerase chain reaction. The changes of coagulation factors and coagulation indicators were observed. Liver pathological tissues were observed by HE staining. The expressions of HMGB1-RAGE/TLRs-NF-κB signaling pathway related proteins were detected by Western blot. Compared with model group, the expressions of HMGB1, RAGE, TLR2, TLR4 and NF-κB were significantly increased in up-regulated miR-155 group (all P < 0.05), while decreased in down-regulated miR-155 group (all P < 0.05). Compared with model group, the expressions of coagulation factor Ⅱ, Ⅶ, Ⅸ, and Ⅹ were significantly decreased in up-regulated miR-155 group (all P < 0.05), while increased in down-regulated miR-155 group (P < 0.05). There was no significant difference in the expression of coagulation factor Ⅺ among the three groups (P >0.05). Compared with model group, the levels of prothrombin time (PT) and activated partial thromboplastin time (APTT) were lower and fibrinogen (FIB) was higher in up-regulated miR-155 group (all P < 0.05), while in the down-regulated miR-155 group they were opposite. Down-regulation of miR-155 can effectively improve coagulation factors and coagulation indexes and inhibit inflammation in young rats with dysfunction of blood coagulopathy, and the mechanism may be related to HMGB1-RAGE/TLRs-NF-κB signaling pathway.

Effect of UVRAG Gene on Ferroptosis Induced by Sorafenib in K562 Cells

To explore the effect of UV radiation resistance-associated gene (UVRAG) on ferroptosis induced by sorafenib in leukemia K562 cells. K562 cells were treated with 0, 0.625, 1.25, 2.5, 5, 10, and 20 μmol/L sorafenib for 24 or 48 hours, and the cell viability was detected by CCK-8 assay. Flow cytometry technology was used to detect the changes of reactive oxygen species (ROS) in K562 cells treated with 0, 5, and 10 μmol/L sorafenib for 24 hours. Western blot was used to detect the protein expression of GPX4 in K562 cells treated with 0, 5, and 10 μmol/L sorafenib and pretreatment with ferroptosis inhibitor. A recombinant lentiviral vector was used to construct UVRAG overexpression cell line in K562 cells. qPCR and Western blot were used to verify UVRAG gene overexpression, and Western blot detected the effect of UVRAG on the protein expression of GPX4 and HMGB1 after treatment with sorafenib. Different concentrations of sorafenib could significantly inhibit the proliferation of K562 cells, and the cell viability gradually decreased with the increase of concentration (r 24 h=-0.9841, r 48 h=-0.9970). The level of ROS was increased (When the concentration was 10 μmol/L, P <0.001), while the expression of GPX4 protein was decreased in the process of 0, 5, 10 μmol/L sorafenib-induced K562 cell death (P <0.05), and the decrease in GPX4 protein could be partially reversed by pretreatment with ferroptosis inhibitor (P <0.05). Compared with NC group and NC-Sorafenib group, the expression of GPX4 protein was significantly decreased (both P <0.05), while HMGB1 protein was significantly increased (both P <0.05). Sorafenib can induce ferroptosis in K562 cells, and this process can be promoted by UVRAG.

Crocin Suppresses Inflammatory Response in LPS-Induced Acute Lung Injury (ALI) Via Regulation of HMGB1/TLR4 Inflammation Pathway

Background and Purpose: The most significant pathogen hypothesized to be causing the formation of Acute lung injury (ALI) in sepsis is thought to be lipopolysaccharide (LPS), a key endotoxin component of gram-negative bacteria. The main objective of this study is to determine possible anti-inflammatory effects of crocin (CRO) which has many biological properties such as anti-inflammatory, antioxidant, and anti-apoptotic in LPS-induced ALI. Methods: 40 Wistar albino rats were divided into four groups: Control (no treatment), CRO (given 50 mg/kg crocin for 9 days), LPS (given 30 mg/kg LPS at 9th day), LPS+CRO (given 50 mg/kg crocin for 9 days and 30 mg/kg LPS at 9th day). After experimental, rats were sacrificed and lungs were extracted. Histological examinations were performed in the lung tissue and the changes in the HMGB1 and TLR4 expressions were determined via immunohistochemical staining. Results and Conclusion: Hemorrhage, mononuclear cell infiltration and HMGB1 and TLR4 expressions significantly increased in the LPS group. However, CRO administrations exerted a strong protective effect on the lungs in terms of these parameters in LPS+CRO group. According to our results, we suggest that CRO can be considered as a protective agent against LPS induced ALI via inhibition of HMGB1/TLR4 pathway-mediated inflammatory response.

SHARPIN contributes to sevoflurane-induced neonatal neurotoxicity through up-regulating HMGB1 to repress M2 like-macrophage polarization.

Sevoflurane exposure can result in neurotoxicity especially among children, which remains an important complication after surgery. However, its related mechanisms remain unclear. Here, we investigated the biological roles of SHARPIN in sevoflurane-induced neurotoxicity. As detected by qPCR, Western blotting and immunohistochemical staining, SHARPIN and HMGB1 expression was elevated in sevoflurane-stimulated mice as compared with the control mice. SHARPIN depletion attenuated hippocampus injury, repressed the expression of HMGB1 and M1-like macrophage markers (iNOS, TNF-α, IL-1β, IL-6), but enhanced the expression of M2-like macrophage markers (ARG-1, IL-10). GST pull-down and Co-IP assays demonstrated that SHARPIN directly interacted with HMGB1 to enhance HMGB1 expression in SH-SY5Y cells. The inhibitory effects of SHARPIN silencing on inflammatory reaction and M1-like macrophages were counteracted by HMGB1 overexpression. Finally, SHARPIN-HMGB1 pathway affected neuroinflammation triggered by sevoflurane via modulating macrophage polarization. Collectively, our data suggested that SHARPIN stimulated sevoflurane-induced neurotoxicity via converting M2-like macrophages to M1-like macrophages by enhancing HMGB1 expression. SHARPIN intervention may be a promising therapeutic method to relieve sevoflurane-induced neurotoxicity.

Pomegranate polyphenol punicalin ameliorates lipopolysaccharide-induced memory impairment, behavioral disorders, oxidative stress, and neuroinflammation via inhibition of TLR4-NF-кB pathway.

Neuroinflammation may play an important role in the development of Alzheimer's disease (AD). Previous studies have reported that lipopolysaccharide (LPS)-induced neuroinflammation causes memory impairments and behavioral disorders. We investigated the potential preventive effects of punicalin (PUN), a polyphenolic component of pomegranate, on LPS-induced memory deficiency and anxiety- and depression-like behaviors, along with the underlying mechanisms. LPS-treated cultured microglial BV2 cells and BV2 cell/Neuro-2a (N2a) cell coculture system were investigated for anti-neuroinflammatory effects of PUN in vitro. The in vivo experiments involved mice administered a 4-week course of oral gavage with 1500 mg/kg/d PUN before intraperitoneal LPS (250 mg/kg daily 7 times) injections. The in vitro results demonstrated that PUN inhibited the LPS-induced inflammatory cytokine (IL-18, IL-1β, TNF-ɑ, and IL-6) production in BV2 cells and protected N2a cells from synaptic damage mediated by BV2 microglia-induced neuroinflammation. In in vivo studies, it was observed that PUN improved memory impairment and anxiety- and depression-like behaviors caused by LPS and reduced the expression of inflammatory proteins such as iNOS, COX-2, IL-1β, IL-2, IL-6, and TNF-α. Furthermore, PUN inhibited the LPS-induced production of MDA; increased the activities of CAT, SOD, and GSH-Px, and inhibited LPS-induced Aβ1-42 generation through down-regulation of APP and BACE1 expression. Moreover, PUN also suppressed the expression of TLR4, IRAK4, TRAF6, IKK-β, NF-κB, p65, and HMGB1 in LPS-treated mouse brain and cultured microglial BV-2 cells. These results suggest that PUN inhibits LPS-induced memory impairment via anti-inflammatory and anti-amylogenic mechanisms through inhibition of TLR4-NF-kB activation.

Novel Role of Endothelial CD47 in the Regulation of Pathogenesis of Coronary Artery Disease

Background: The anti-phagocytic molecule CD47 is upregulated in atherosclerotic plaques of patients with CVD. However, the role of endothelial cell-specific expression of CD47 in the pathogenesis and progression of atherosclerosis has not yet been fully defined. Kojima et.al. described atheroprotective effect of anti-CD47 antibody by enhancing efferocytosis in atherosclerosis. In this study, we determined whether endothelial cell-specific CD47 regulates efferocytosis in atherosclerosis. Methods: Endothelial cell-specific, tamoxifen-inducible CD47knockout (CD47iECKO) mice were injected with a single dose of AAV-mutant PCSK9 via the tail vein. Susceptibility to atherosclerosis was evaluated in cross sections of the aortic sinus in mice fed Western Diet for 12 weeks. Using single-cell RNA sequencing and in vitro efferocytosis assays, we investigated the role of CD47 in regulating efferocytosis in an atherosclerotic mouse model. Results: Endothelial cell-specific deletion of CD47 led to a significant plaque reduction in the aortic sinus of athero-CD47iECKO mice. Cluster analysis and gene expression profiles of 20,000 aortic endothelial cells from athero-control and athero-CD47iECKO mice identified 10 major cell types from the integrated data: endothelial cells, vascular smooth muscle cells, fibroblasts, immune cells (B cells, T cells, macrophage), erythrocytes, oligodendrocytes cells, Schwann cells, and pericytes. After Western Diet intake, in the CD47iECKO group, a subpopulation of endothelial cells as well as fibroblasts, T-cells, B-cells, resident aortic macrophages, erythrocytes, oligodendrocytes, and pericytes were increased, while vascular smooth muscle cell numbers decreased. Using an in vitro efferocytosis assay on isolated primary endothelial cells from control and CD47iECKO mice, we observed CD47 deletion induced engulfment of apoptotic Jurkat cells, which had been exposed to BAY11-7082 (NF-κB inhibitor) to stimulate the apoptotic attributes. Quantitative PCR analyses showed that CD47 deletion increases the expression of efferocytosis receptor FasL, CX3CL1, Gpr132, MerTK, Scarb1, Sirpa, TSP1, and HMGB1. Conclusion: Our study indicates that genetic deletion of CD47 in endothelial cells attenuates atheroma progression by enhancing efferocytosis. This study also reveals phenotypic heterogenicity of endothelial cells at the single cell level during atherogenesis and provides a deeper understanding of endothelial cell biology in atherosclerosis that may facilitate the development of novel therapeutic interventions for this cardiovascular disease. NIH, AHA. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Celastrol Elicits Antitumor Effects through Inducing Immunogenic Cell Death and Downregulating PD-L1 in ccRCC.

Targeting immunogenic cell death (ICD) is considered a promising therapeutic strategy for cancer. However, the commonly identified ICD inducers promote the expression of programmed cell death ligand 1 (PD-L1) in tumor cells, thus aiding them to evade the recognition and killing by the immune system. Therefore, the finding of novel ICD inducers to avoid enhanced PD-L1 expression is of vital significance for cancer therapy. Celastrol (CeT), a triterpene isolated from Tripterygium wilfordii Hook. F induces various forms of cell death to exert anti-cancer effects, which may make celastrol an attractive candidate as an inducer of ICD. In the present study, bioinformatics analysis was combined with experimental validation to explore the underlying mechanism by which CeT induces ICD and regulates PD-L1 expression in clear cell renal cell carcinoma (ccRCC). The results showed that EGFR, IKBKB, PRKCQ and MAPK1 were the crucial targets for CeT-induced ICD, and only MAPK1 was an independent prognostic factor for the overall survival (OS) of ccRCC patients. In addition, CeT triggered autophagy and up-regulated the expressions of HMGB1 and CRT to induce ICD in 786-O cells in vitro. Importantly, CeT can down-regulate PD-L1 expression through activating autophagy. At the molecular level, CeT suppressed PD-L1 via the inhibition of MAPK1 expression. Immunologically, the core target of celastrol, MAPK1, was tightly correlated with CD8+ T cells and CD4+ T cells in ccRCC. These findings indicate that CeT not only induces ICD but also suppresses PD-L1 by down-regulating MAPK1 expression, which will provide an attractive strategy for ccRCC immunotherapy.

HMGB1 Expression Levels Correlate with Response to Immunotherapy in Non-Small Cell Lung Cancer.

High-mobility group box 1 protein (HMGB1) is subject to exportin 1 (XPO1)-dependent nuclear export, and it is involved in functions implicated in resistance to immunotherapy. We investigated whether HMGB1 mRNA expression was associated with response to immune checkpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC). RNA was isolated from pretreatment biopsies of patients with advanced NSCLC treated with ICI. Gene expression analysis of several genes, including HMGB1, was conducted using the NanoString Counter analysis system (PanCancer Immune Profiling Panel). Western blotting analysis and cell viability assays in EGFR and KRAS mutant cell lines were carried out. Evaluation of the antitumoral effect of ICI in combination with XPO1 blocker (selinexor) and trametinib was determined in a murine Lewis lung carcinoma model. HMGB1 mRNA levels in NSCLC patients treated with ICI correlated with progression-free survival (PFS) (median PFS 9.0 versus 18.0 months, P=0.008, hazard ratio=0.30 in high versus low HMGB1). After TNF-α stimulation, HMGB1 accumulates in the cytoplasm of PC9 cells, but this accumulation can be prevented by using selinexor or antiretroviral drugs. Erlotinib or osimertinib with selinexor in EGFR-mutant cells and trametinib plus selinexor in KRAS mutant abolish tumor cell proliferation. Selinexor with a PD-1 inhibitor with or without trametinib abrogates the tumor growth in the murine Lewis lung cancer model. An in-depth exploration of the functions of HMGB1 mRNA and protein is expected to uncover new potential targets and provide a basis for treating metastatic NSCLC in combination with ICI.

Gfi-1 modulates HMGB1-Mediated autophagy to overcome oxaliplatin resistance in colorectal cancer

BackgroundResistance to oxaliplatin (L-OHP) is a major barrier in the treatment of colorectal cancer (CRC). Autophagy is the main cause of L-OHP tolerance in CRC cells. MethodThe human colon cancer cell lines HCT116 and SW480 were treated with L-OHP to obtain the drug-resistant cell lines HCT116/L-OHP and SW480/L-OHP, respectively. To probe the relationship between autophagy and L-OHP tolerance of growth factor independent 1 (Gfi-1) and high-mobility group protein 1 (HMGB1) in CRC cells, gene knockout or overexpression was performed, and Western blotting was used to determine the levels of drug tolerance interrelated proteins. Transwell and CCK-8 assays were employed to analyze the proliferation of cancer cells. Immunofluorescence detection of LC3 reflected autophagy levels. Finally, the relationship between Gfi-1 and HMGB1 was detected by chromatin immunoprecipitation (ChIP). ResultCompared to normal CRC cells, L-OHP-tolerant CRC cells exhibited greater autophagy (8.2 times greater in HCT116/L-OHP cells and 7.4 times greater in SW480/L-OHP cells). In addition, we detected low levels of Gfi-1 (0.6-fold for HCT116/L-OHP cells and 0.4-fold for SW480/L-OHP cells), and OE-Gfi-1 decreased HMGB1 levels (0.6-fold for HCT116/L-OHP + OE-Gfi-1 cells and 0.5-fold for SW480/L-OHP + OE-Gfi-1 cells). The inhibition of Gfi-1 further enhanced cell viability (1.7 times in HCT116+sh-Gfi-1 cells and 1.2 times in SW480+sh-Gfi-1 cells) and invasion (1.8 times in HCT116+sh-Gfi-1 cells and 2.1 times in SW480+sh-Gfi-1 cells) in CRC cells, thus promoting oxaliplatin resistance in these cells. The autophagy inhibitor 3-MA reversed the above effects. Furthermore, we noted that Gfi-1 can restrain HMGB1 expression by binding to its promoter (0.5 times in HCT116+OE-Gfi-1 cells and 0.5 times in SW480+OE-Gfi-1 cells). The inhibitory influence of 3-MA on HMGB1 reversed the influence of Gfi-1 on autophagy and malignant progression in CRC cells. ConclusionOur study suggested that Gfi-1 inhibited HMGB1 to reduce CRC autophagy levels, increasing CRC sensitivity to L-OHP.

Plumbagin inhibits fungal growth, HMGB1/LOX-1 pathway and inflammatory factors in A. fumigatus keratitis.

To investigate the anti-inflammatory and antifungal effects of plumbagin (PL) in Aspergillus fumigatus (A. fumigatus) keratitis, the minimum inhibitory concentration (MIC), time-killing curve, spore adhesion, crystal violet staining, calcium fluoride white staining, and Propidium Iodide (PI) staining were employed to assess the antifungal activity of PL in vitro against A. fumigatus. The cytotoxicity of PL was assessed using the Cell Counting Kit-8 (CCK8). The impact of PL on the expression of HMGB1, LOX-1, TNF-α, IL-1β, IL-6, IL-10 and ROS in A. fumigatus keratitis was investigated using RT-PCR, ELISA, Western blot, and Reactive oxygen species (ROS) assay. The therapeutic efficacy of PL against A. fumigatus keratitis was assessed through clinical scoring, plate counting, Immunofluorescence and Hematoxylin-Eosin (HE) staining. Finally, we found that PL inhibited the growth, spore adhesion, and biofilm formation of A. fumigatus and disrupted the integrity of its cell membrane and cell wall. PL decreased IL-6, TNF-α, and IL-1β levels while increasing IL-10 expression in fungi-infected mice corneas and peritoneal macrophages. Additionally, PL significantly attenuated the HMGB1/LOX-1 pathway while reversing the promoting effect of Boxb (an HMGB1 agonist) on HMGB1/LOX-1. Moreover, PL decreased the level of ROS. In vivo, clinical scores, neutrophil recruitment, and fungal burden were all significantly reduced in infected corneas treated with PL. In summary, the inflammatory process can be inhibited by PL through the regulation of the HMGB-1/LOX-1 pathway. Simultaneously, PL can exert antifungal effects by limiting fungal spore adhesion and biofilm formation, as well as causing destruction of cell membranes and walls.

HMGB1 Modulates High Glucose-Induced Erroneous Differentiation of Tendon Stem/Progenitor Cells through RAGE/β-Catenin Pathway.

The association of tendinopathy with diabetes has been well recognized. Tendon stem/progenitor cells (TSPCs) play critical roles in tendon repair, regeneration, and homeostasis maintenance. Diabetic TSPCs exhibit enhanced erroneous differentiation and are involved in the pathogenesis of diabetic tendinopathy, whereas the underlying mechanism of the erroneous differentiation of TSPCs remains unclear. Here, we showed that high glucose treatment promoted the erroneous differentiation of TSPCs with increased osteogenic differentiation capacity and decreased tenogenic differentiation ability, and stimulated the expression and further secretion of HMGB1 in TSPCs and. Functionally, exogenous HMGB1 significantly enhanced the erroneous differentiation of TSPCs, while HMGB1 knockdown mitigated high glucose-promoted erroneous differentiation of TSPCs. Mechanistically, the RAGE/β-catenin signaling was activated in TSPCs under high glucose, and HMGB1 knockdown inhibited the activity of RAGE/β-catenin signaling. Inhibition of RAGE/β-catenin signaling could ameliorate high glucose-induced erroneous differentiation of TSPCs. These results indicated that HMGB1 regulated high glucose-induced erroneous differentiation of TSPCs through the RAGE/β-catenin signaling pathway. Collectively, our findings suggest a novel essential mechanism of the erroneous differentiation of TSPCs, which might contribute to the pathogenesis of diabetic tendinopathy and provide a promising therapeutic target and approach for diabetic tendinopathy.

Cartilage protective and anti-edema effects of JTF in osteoarthritis via inhibiting NCOA4-HMGB1-driven ferroptosis and aquaporin dysregulation

BackgroundPreventing joint edema is crucial in halting osteoarthritis (OA) progression. Growing clinical evidence indicate that Jianpi-Tongluo Formula (JTF) may have a promising anti-edema effect. However, the therapeutic properties of JTF and the underlying mechanisms remains unclear. Materials and methodsAn OA rat model was established and employed to evaluate pharmacological effects of JTF in vivo based on dynamic histopathologic assessments and micro-CT observations. Then, OA-related genes and potential targets of JTF were identified through clinical transcriptomic data analysis and "disease gene-drug target" network analysis, which were verified by a series of in vivo experiments. ResultsJTF administration effectively reduced pain and joint edema, inhibited matrix degradation, chondrocyte apoptosis, and aquaporin expression in OA rats. Notably, JTF dose-dependently reversed damage-associated molecular patterns and inflammatory factor upregulation. Mechanically, our "disease gene-drug target" network analysis indicated that the NCOA4-HMGB1-GSK3B-AQPs axis, implicated in ferroptosis and aquaporin dysregulation, may be potentially served as a target of JTF against OA. Accordingly, JTF mitigated NCOA4, HMGB1, and GSK3B expression, oxidative stress, and iron metabolism aberrations in OA rats. Furthermore, JTF treatment significantly attenuated the aberrant upregulation of AQP1, AQP3, and AQP4 proteins observed in cartilage tissues of OA rats. ConclusionOur data reveal for the first time that JTF may exert cartilage protective and anti-edema effects in osteoarthritis therapy by inhibiting NCOA4-HMGB1-driven ferroptosis and aquaporin dysregulation.

Clinical Significance of HMGB1 and Autophagy-Related Genes in Sinonasal Inverted Papilloma.

Sinonasal inverted papilloma (SNIP) is a noncancerous tumor that develops in the mucous membrane of the nasal sinuses. Many malignancies are tightly linked to autophagy, an intracellular self-degradation mechanism. HMGB1 has demonstrated its ability to modulate autophagy in many pathological conditions. This work investigates how HMGB1 and other genes involved in autophagy contribute to SNIP. The study included 45 patients with SNIP and a control group consisting of 28 individuals. In each group, qPCR was employed to examine the mRNA expression levels of genes correlated with autophagy and HMGB1. HMGB1 and genes associated with autophagy were examined for protein expression levels via Western Blot and immunohistochemical staining assays. At the same time, the association between HMGB1 and genes involved in autophagy was discovered through correlation analysis. Furthermore, Krouse staging was utilized for investigating the expression levels of HMGB1 and other autophagy-related genes at various stages in clinically staged SNIP patients. LC3B, ATG5, and Beclin1 autophagy-related genes and HMGB1 were substantially expressed in SNIP. Additionally, there was a positive correlation between HMGB1 and these genes. During various phases of SNIP, the levels of HMGB1 expression and autophagy-related genes were notably elevated at stage T4 compared with stage T2. Clinical staging in SNIP is correlated with HMGB1 expression in conjunction with autophagy-related genes LC3B, ATG5, and Beclin1, suggesting the possibility of novel prognostic indicators. NA Laryngoscope, 134:3941-3946, 2024.

DETERMINATION OF IL-1β mRNA GENE EXPRESSION AND HMGB-1 mRNA GENE EXPRESSION IN WOMEN WITH GENITAL ENDOMETRIOSIS ASSOCIATED WITH INFERTILITY AT THE STAGE OF PREPARATION FOR ASSISTED REPRODUCTIVE TECHNOLOGIES

Despite the large number of studies on the identification and search for noninvasive early diagnostic markers of genital endometriosis, this problem remains relevant today. It is important to introduce early diagnosis of genital endometriosis for the purpose of timely prevention and treatment.Objective: to study the expression levels of IL-1β and HMGB-1 mRNA genes in women with genital endometriosis associated with infertility, and to evaluate the effect of our proposed preparation for assisted reproductive technologies on the level of IL-1β and HMGB-1 mRNA gene expression.Material and methods. To analyze gene expression IL-1β and HMGB-1 and determine relative normalized mRNA expression IL-1β and HMGB-1, the polymerase chain reaction method was used with real-time reverse transcription(RT-PCR. The object of molecular genetic studies using the RT-PCR method was the fraction of mononuclear cells isolated from the whole blood of patients with endometriosis. In this study, we performed a retrospective analysis of the medical records of 30 women with infertility undergoing an assisted reproductive technology program. The main group consisted of 20 women diagnosed with external genital endometriosis who underwent assisted reproductive technologies. Women in the main group, in addition to standard training for assisted reproductive technologies, received a probiotic containing Lactobacilli 1010 manufactured by Unic Biotech Ltd, India. They took one pill twice a day for one month as part of a general treatment before undergoing assisted reproductive technologies. We determined the expression level of IL-1β and HMGB-1 before and after this preparation stage. The control group consisted of 10 women who had tubal infertility due to a previous inflammatory disease, but according to the results of a comprehensive clinical and laboratory examination, they were compared to healthy women. These women, aged 21 to 42 years, with a mean age of 29,75 years, did not receive our proposed preparation with the inclusion of a probiotic. This study was conducted at the Bukovyna State Medical University and the clinic "Yuzko medical center".Results. In the main group, the IL-1β mRNA gene expression level before training was (26,7877±0,01), which was significantly higher than the level after training (0,1610±0,01*). Consequently, the use of the probiotic led to a dramatic reduction in elevated mesothelin levels, underscoring its efficacy and potential application in assisted reproductive technology training programs. Also, a high level of expression of mRNA genes can be noted HMGB-1in whole blood of women of the main group before preparation for assisted reproductive technologies (table 2). Expression level of mRNA genes HMGB-1in whole blood of women of the main group before preparation for assisted reproductive technologies was (11,9198±0,01). After our proposed preparation for assisted reproductive technologies with the inclusion of a probiotic, the mRNA gene expression level HMGB-1in the whole blood of women of the main group was (0,2821±0,01*). Therefore, our proposed preparation for assisted reproductive technologies with the inclusion of a probiotic is quite effective, since the expression levels of IL-1β mRNA genes and HMGB-1sharply decreased.Conclusions. Extremely high expression of mRNA genes IL-1β and HMGB-1 indicates a close connection between the pathogenesis of endometriosis and inflammation. The inclusion of probiotics in the complex scheme of preparation for assisted reproductive technologies leads to a noticeable improvement in the well-being of the patient and a significant decrease in the expression of IL-1β and HMGB-1 mRNA genes. Therefore, we recommend our proposed preparation for assisted reproductive technologies with the inclusion of a probiotic.

CGA mitigates HMGB1 mediated TLR4 activated hepatic cancer in urethane primed mice

BackgroundLiver cancer is fourth leading cause of cancer deaths worldwide. Urethane presents in herbicides, pesticides and food beverages as preservatives upregulate the expression of inflammatory cytokines in liver tissues for the genesis of cancer. It also naturally formed in fermented food products and distilled beverages. Study designBalb/c mice of approx same age and body weight were selected and divided into four groups G-I (PBS treated), G-II (urethane of 500 mg/kgb.w.), G-III (CGA of 50 mg/kg b.w.) and G-IV (urethane & CGA). Treatments were given on alternate day upto eight consecutive weeks and 3-12 month of latency period. Blood sample collected was used for counting of inflammatory immune cells by automated cell counter. Total RNA and protein samples isolated from dissected liver of sacrificed mice were used for molecular analysis at transcriptional and translational level. MethodsGene expression is assessed by RT-PCR at transcriptional level and by western blot at translational level. Immunological effects are evaluated by counting inflammatory immue cells and histopathology is done to examine hepatic tissues injury by carcinogen urethane. Hypothesis and purposeInflammatory cytokines create microenvironment for tumor initiation and development which is mediated through TLR-4 pathway activation via HMGB1 upon carcinogen exposure. Here aimed to find out the role of Chlorogenic acid (CGA) a phenolic compound naturally present in plants as anti-cancerous properties to prevent and cure cancer by acting upon HMGB1 & TLR4 pathway. ResultsAt transcriptional level up-regulated expression of HMGB1, TLR-4 pathway genes, pro-inflammatory cytokines IL-1β, IL-6, TNF-α and IL-18 in urethane treated mice is observed. Western blot results showed up-regulated expression of HMGB1 and NF-κB protein at translational level. CGA treated mice liver showed down-regulated expression of these inflammatory molecules. Increased number of inflammatory immune cells and presence of hepatic tissues injury in histopathological slides is observed in urethane treated mice that is mitigated by CGA. ConclusionCGA is an important anti-cancerous bioactive compound targets inflammatory cytokines and HMGB1 at molecular level to prevent, cure and mitigate liver cancer. Down regulated expression of HMGB1 and TLR-4 activation pathway in cancerous Balb/c mice liver proved its anti-cancerous properties to become a potential future drug.

Abstract 6517: ALDOC as an indicator of HMGB1 blockade combined with checkpoint inhibitor based immunotherapy in GBM patients

Abstract Aldolase is an enzyme that takes part in glycolysis by catalyzing a six-carbon intermediate to yield two three-carbon products, requiring ATP. The aldolase family comprises three members, namely ALDOA, ALDOB, and brain-specific ALDOC. Low-grade gliomas and glioblastoma (GBM) are characterized by shared genetic events, including chromosome 1p/19q codeletion, EGFR amplification, and ATRX loss of function, in which ALDOC plays a role. Additionally, ALDOC is responsive to IDH1 mutations. If patients display IDH1 mutations, it is expected that ALDOC performance will be relatively higher. This finding suggests a more favorable prognosis. The ALDOC promoter region's function is impeded by hypermethylation, which declines as glioma progresses. The regulatory mechanisms and implications remain unclear. We established a multi-omics profile and identified several crucial mechanisms activated upon the loss of ALDOC expression. Among these mechanisms, we found HMGB1 to have a key role in linking the immune response, tumor microenvironment, and epigenetic modifications. We compared the proteomic and secretomic platforms in an ALDOC overexpression/knockdown model, which showed abundant HMGB1 expression following ALDOC inhibition. Importantly, there is an inverse correlation between ALDOC expression and HMGB1 protein levels. Additionally, our findings reveal that upregulation of ALDOC promotes its translocation to the nucleus, thereby enhancing its binding affinity with HMGB1. This, in turn, enables extracellular release of HMGB1 in GBM, which triggers signaling via receptors for advanced glycation end-products (RAGE) and several cytokines. Moreover, HMGB1 remodels the immune microenvironment and upregulates multiple immune checkpoint molecules, including PD-L1, PD-L2, B7-H3, and MHCs. Not all GBM models exhibited significant effects when combined with immune checkpoint blockade using HMGB1 blockade. However, ALDOC knockout mice demonstrated poorer survival and treatment efficacy, suggesting that ALDOC may be crucial for a personalized immunotherapy strategy. Citation Format: Yi-Fang Yang, Ming-Hsien Chan, Chi-Long Chen, Michael Hsiao, Yu-Chan Chang. ALDOC as an indicator of HMGB1 blockade combined with checkpoint inhibitor based immunotherapy in GBM patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6517.

IGF2BP3 prevent HMGB1 mRNA decay in bladder cancer and development

BackgroundIGF2BP3 functions as an RNA-binding protein (RBP) and plays a role in the posttranscriptional control of mRNA localization, stability, and translation. Its dysregulation is frequently associated with tumorigenesis across various cancer types. Nonetheless, our understanding of how the expression of the IGF2BP3 gene is regulated remains limited. The specific functions and underlying mechanisms of IGF2BP3, as well as the potential benefits of targeting it for therapeutic purposes in bladder cancer, are not yet well comprehended.MethodsThe mRNA and protein expression were examined by RT-qPCR and western blotting, respectively. The methylation level of CpG sites was detected by Bisulfite sequencing PCR (BSP). The regulation of IGF2BP3 expression by miR-320a-3p was analyzed by luciferase reporter assay. The functional role of IGF2BP3 was determined through proliferation, colony formation, wound healing, invasion assays, and xenograft mouse model. The regulation of HMGB1 by IGF2BP3 was investigated by RNA immunoprecipitation (RIP) and mRNA stability assays.ResultsWe observed a significant elevation in IGF2BP3 levels within bladder cancer samples, correlating with more advanced stages and grades, as well as an unfavorable prognosis. Subsequent investigations revealed that the upregulation of IGF2BP3 expression is triggered by copy number gain/amplification and promoter hypomethylation in various tumor types, including bladder cancer. Furthermore, miR-320a-3p was identified as another negative regulator in bladder cancer. Functionally, the upregulation of IGF2BP3 expression exacerbated bladder cancer progression, including the proliferation, migration, and invasion of bladder cancer. Conversely, IGF2BP3 silencing produced the opposite effects. Moreover, IGF2BP3 expression positively correlated with inflammation and immune infiltration in bladder cancer. Mechanistically, IGF2BP3 enhanced mRNA stability and promoted the expression of HMGB1 by binding to its mRNA, which is a factor that promotes inflammation and orchestrates tumorigenesis in many cancers. Importantly, pharmacological inhibition of HMGB1 with glycyrrhizin, a specific HMGB1 inhibitor, effectively reversed the cancer-promoting effects of IGF2BP3 overexpression in bladder cancer. Furthermore, the relationship between HMGB1 mRNA and IGF2PB3 is also observed in mammalian embryonic development, with the expression of both genes gradually decreasing as embryonic development progresses.ConclusionsOur present study sheds light on the genetic and epigenetic mechanisms governing IGF2BP3 expression, underscoring the critical involvement of the IGF2BP3-HMGB1 axis in driving bladder cancer progression. Additionally, it advocates for the investigation of inhibiting IGF2BP3-HMGB1 as a viable therapeutic approach for treating bladder cancer.Graphical