High Mobility Group Box 1 Knockdown Research Articles

HMGB1 derived from lung epithelial cells after cobalt nanoparticle exposure promotes the activation of lung fibroblasts

We have previously demonstrated that exposure to cobalt nanoparticles (Nano-Co) caused extensive interstitial fibrosis and inflammatory cell infiltration in mouse lungs. However, the underlying mechanisms of Nano-Co-induced pulmonary fibrosis remain unclear. In this study, we investigated the role of high-mobility group box 1 (HMGB1) in the epithelial cell-fibroblast crosstalk in Nano-Co-induced pulmonary fibrosis. Our results showed that Nano-Co exposure caused remarkable production and release of HMGB1, as well as nuclear accumulation of HIF-1α in human bronchial epithelial cells (BEAS-2B) in a dose- and a time-dependent manner. Pretreatment with CAY10585, an inhibitor against HIF-1α, significantly blocked the overexpression of HMGB1 in cell lysate and the release of HMGB1 in the supernatant of BEAS-2B cells induced by Nano-Co exposure, indicating that Nano-Co exposure induces HIF-1α-dependent HMGB1 overexpression and release. In addition, treatment of lung fibroblasts (MRC-5) with conditioned media from Nano-Co-exposed BEAS-2B cells caused increased RAGE expression, MAPK signaling activation, and enhanced expression of fibrosis-associated proteins, such as fibronectin, collagen 1, and α-SMA. However, conditioned media from Nano-Co-exposed BEAS-2B cells with HMGB1 knockdown had no effects on the activation of MRC-5 fibroblasts. Finally, inhibition of ERK1/2, p38, and JNK all abolished MRC-5 activation induced by conditioned media from Nano-Co-exposed BEAS-2B cells, suggesting that MAPK signaling might be a key downstream signal of HMGB1/RAGE to promote MRC-5 fibroblast activation. These findings have important implications for understanding the pro-fibrotic potential of Nano-Co.

High mobility group box 1 knockdown inhibits EV71 replication and attenuates cell pyroptosis through TLR4/NF-κB/NLRP3 axis.

Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD) in children. Nowadays, there are still no effective antiviral drugs for EV71 infection. High mobility group box 1 (HMGB1) is reported to be highly expressed in HFMD patients. However, therole and underlying mechanism of HMGB1 in EV71-associated HFMD are still unclear. HMGB1 expression was detected using RT-qPCR and western blot assays. Loss- and gain-function experiments were performed to evaluate the effects of HMGB1 on EV71-infected cells. The virus titer was examined by TCID50. CCK-8 and flow cytometry assays were applied to detect the cell viability and cell cycle. Oxidative stress was determined by relative commercial kits. HMGB1 level was elevated in the serum of EV71-infected patients with HFMD and EV71-induced RD cells. EV71 infection induced the transfer of HMGB1 from the nucleus into the cytoplasm. HMGB1 knockdown inhibited virus replication,viral protein (VP1) expression and promoted antiviral factor expression. In addition, the inhibition of HMGB1 improved cell viability, protected against S phase arrest, and inhibited EV71-induced cell injury and oxidative stress, whereasHMGB1 overexpression showed the opposite effects. In terms of mechanism, HMGB1 overexpression activated the TLR4/NF-κB/NLRP3 signaling pathway and promoted cell pyroptosis. The inhibition of TLR4 and NF-κB reversed the effects of HMGB1 overexpression on virus replication, oxidative stress, and pyroptosis. In conclusion, HMGB1 knockdown inhibits EV71 replication and attenuates pyroptosis through TLR4/NF-κB/NLRP3 axis.

Restoring glucose balance: Conditional HMGB1 knockdown mitigates hyperglycemia in a Streptozotocin induced mouse model

Diabetes mellitus (DM) poses a significant global health burden, with hyperglycemia being a primary contributor to complications and high morbidity associated with this disorder. Existing glucose management strategies have shown suboptimal effectiveness, necessitating alternative approaches. In this study, we explored the role of high mobility group box 1 (HMGB1) in hyperglycemia, a protein implicated in initiating inflammation and strongly correlated with DM onset and progression. We hypothesized that HMGB1 knockdown will mitigate hyperglycemia severity and enhance glucose tolerance. To test this hypothesis, we utilized a novel inducible HMGB1 knockout (iHMGB1 KO) mouse model exhibiting systemic HMGB1 knockdown. Hyperglycemic phenotype was induced using low dose streptozotocin (STZ) injections, followed by longitudinal glucose measurements and oral glucose tolerance tests to evaluate the effect of HMGB1 knockdown on glucose metabolism. Our findings showed a substantial reduction in glucose levels and enhanced glucose tolerance in HMGB1 knockdown mice. Additionally, we performed RNA sequencing analyses, which identified potential alternations in genes and molecular pathways within the liver and skeletal muscle tissue that may account for the in vivo phenotypic changes observed in hyperglycemic mice following HMGB1 knockdown. In conclusion, our present study delivers the first direct evidence of a causal relationship between systemic HMGB1 knockdown and hyperglycemia in vivo, an association that had remained unexamined prior to this research. This discovery positions HMGB1 knockdown as a potentially efficacious therapeutic target for addressing hyperglycemia and, by extension, the DM epidemic. Furthermore, we have revealed potential underlying mechanisms, establishing the essential groundwork for subsequent in-depth mechanistic investigations focused on further elucidating and harnessing the promising therapeutic potential of HMGB1 in DM management.

HMGB1-activated tumor-associated macrophages promote migration and invasion via NF-κB/IL-6 signaling in oral squamous cell carcinoma

Tumor-associated macrophages (TAMs) are a highly abundant cell population within the tumor microenvironment of oral squamous cell carcinomas (OSCC). Recent studies have identified an intricate cross-talk between cancer cells and macrophages in the tumor microenvironment. However, the underlying mechanism remains unclear. High-mobility group box 1 (HMGB1) was linked to metastasis and an unfavorable prognosis in head and neck squamous cell carcinoma. Furthermore, it was significantly upregulated in moderately differentiated OSCC tissues and the OSCC cell lines CAL27 and SCC9. HMGB1 knockdown impedes the ability of TAMs to induce invasion and migration of OSCC cells. Phenotypic changes in macrophages were measured after incubation of supernatant from OSCC cells transfected with HMGB1 siRNA or supplemented with recombinant HMGB1. HMGB1 induced M1 polarization of macrophages and the secretion of IL-6 via the NF-κB pathway, contributing to the OSCC malignant migration. HMGB1 originating from OSCC cells, along with its downstream signaling pathways, holds promise as a potential therapeutic target for mitigating metastasis and improving the survival rate of OSCC.

Knockdown of HMGB1 inhibits the crosstalk between oral squamous cell carcinoma cells and tumor-associated macrophages.

Tumor-associated macrophages (TAMs), the major component of the tumor microenvironment (TME), play distinctly different roles in different tumors. High mobility group box 1 (HMGB1), a nonhistone protein in the nucleus, can perform functions during inflammation and cancers. However, the role of HMGB1 in the crosstalk between oral squamous cell carcinoma (OSCC) cells and TAMs remains unclear. Here, we established a coculture system of TAMs and OSCC cells to explore the bidirectional effect and potential mechanism of HMGB1 in OSCC cell-TAM interactions. Our results showed that HMGB1 was significantly upregulated in OSCC tissues and positively associated with tumor progression, immune cell infiltration and macrophage polarization. Then, knocking down HMGB1 in OSCC cells inhibited the recruitment and polarization of cocultured TAMs. Moreover, the knockdown of HMGB1 in macrophages not only suppressed polarization, but also inhibited cocultured OSCC cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, macrophages secreted higher levels of HMGB1 than OSCC cells, and dampening endogenous HMGB1 reduced HMGB1 secretion. Both OSCC cell-generated and macrophage-endogenous HMGB1 may regulate TAM polarization by promoting receptor TLR4 expression and NF-κB/p65 activation and enhancing IL-10/TGF-β expression. HMGB1 in OSCC cells may regulate macrophage recruitment via IL-6/STAT3. In addition, TAM-derived HMGB1 may affect aggressive phenotypes of cocultured OSCC cells by regulating the immunosuppressive microenvironment through the IL-6/STAT3/PD-L1 and IL-6/NF-κB/MMP-9 pathways. In conclusion, HMGB1 may regulate the crosstalk between OSCC cells and TAMs, including modulating macrophage polarization and attraction, enhancing cytokine secretion, and remodeling and creating an immunosuppressive TME to further affect OSCC progression.

Sp1-mediated miR-193b suppresses atopic dermatitis by regulating HMGB1.

Atopic dermatitis (AD) is a chronic and recurrent inflammatory skin disease. Keratinocyte dysfunction plays a central role in AD development. MicroRNA is a novel player in many inflammatory and immune skin diseases. In this study, we investigated the potential function and regulatory mechanism of miR-193b in AD. Inflamed human keratinocytes (HaCaT) were established by tumor necrosis factor (TNF)-α/interferon (IFN)-γ stimulation. Cell viability was measured using MTT assay, while the cell cycle was analyzed using flow cytometry. The cytokine levels were examined by enzyme-linked immunosorbent assay. The interaction between Sp1, miR-193b, and HMGB1 was analyzed using dual luciferase reporter and/or chromatin immunoprecipitation (ChIP) assays. Our results revealed that miR-193b upregulation enhanced the proliferation of TNF-α/IFN-γ-treated keratinocytes and repressed inflammatory injury. miR-193b negatively regulated high mobility group box 1 (HMGB1) expression by directly targeting HMGB1. Furthermore, HMGB1 knockdown promoted keratinocyte proliferation and inhibited inflammatory injury by repressing nuclear factor kappa-B (NF-κB) activation. During AD progression, HMGB1 overexpression abrogated increase of keratinocyte proliferation and repression of inflammatory injury caused by miR-193b overexpression. Moreover, transcription factor Sp1 was identified as the biological partner of the miR-193b promoter in promoting miR-193b expression. Therefore, Sp1 upregulation promotes keratinocyte proliferation and represses inflammatory injury during AD development via promoting miR-193b expression and repressing HMGB1/NF-κB activation.

Glycyrrhizin, an inhibitor of HMGB1 induces autolysosomal degradation function and inhibits Helicobacter pylori infection

BackgroundHelicobacter pylori is a key agent for causing gastric complications linked with gastric disorders. In response to infection, host cells stimulate autophagy to maintain cellular homeostasis. However, H. pylori have evolved the ability to usurp the host’s autophagic machinery. High mobility group box1 (HMGB1), an alarmin molecule is a regulator of autophagy and its expression is augmented during infection and gastric cancer. Therefore, this study aims to explore the role of glycyrrhizin (a known inhibitor of HMGB1) in autophagy during H. pylori infection.Main methodsHuman gastric cancer (AGS) cells were infected with the H. pylori SS1 strain and further treatment was done with glycyrrhizin. Western blot was used to examine the expression of autophagy proteins. Autophagy and lysosomal activity were monitored by fluorescence assays. A knockdown of HMGB1 was performed to verify the effect of glycyrrhizin. H. pylori infection in in vivo mice model was established and the effect of glycyrrhizin treatment was studied.ResultsThe autophagy-lysosomal pathway was impaired due to an increase in lysosomal membrane permeabilization during H. pylori infection in AGS cells. Subsequently, glycyrrhizin treatment restored the lysosomal membrane integrity. The recovered lysosomal function enhanced autolysosome formation and concomitantly attenuated the intracellular H. pylori growth by eliminating the pathogenic niche. Additionally, glycyrrhizin treatment inhibited inflammation and improved gastric tissue damage in mice.ConclusionThis study showed that inhibiting HMGB1 restored lysosomal activity to ameliorate H. pylori infection. It also demonstrated the potential of glycyrrhizin as an antibacterial agent to address the problem of antimicrobial resistance.

Etanercept ameliorates psoriasis progression through regulating high mobility group box 1 pathway.

As a common skin disease, psoriasis is related to inflammation and immune response. Due to the frequent recurrence of psoriasis, the treatment of psoriasis remains a clinical challenge. As an effective tumor necrosis factor-alpha (TNF-α) inhibitor, etanercept has been used for the treatment of psoriasis. However, some patients with psoriasis have no response to etanercept or discontinue treatment. To improve the therapeutic effect of etanercept, searching the potential biomarkers and investigating the related mechanisms of etanercept in the treatment of psoriasis are vital. We stimulated HaCaT cells with lipopolysaccharide (LPS) to generate cellular psoriatic changes and established an imiquimod (IMQ)-induced psoriasis-like mouse model, and then used an etanercept to treat cell and mouse model. Etanercept alleviated IMQ-induced pathological changes and inflammation, and it also decreased the protein expression of high mobility group box 1 (HMGB1), receptor for advanced glycation end-products, and toll-like receptor 4. Moreover, the results of in vitro experiments showed that etanercept inhibited proliferation and inflammation, and promoted cell cycle arrest and apoptosis in LPS-treated HaCaT cells. Knockdown of HMGB1 further enhanced the inhibitory effects of etanercept on LPS-treated HaCaT cell viability and inflammation, while overexpression of HMGB1 notably reversed the inhibitory effects of etanercept on LPS-induced HaCaT cell viability and inflammation. Etanercept inhibited proliferation and inflammation and promoted cell cycle arrest and apoptosis in LPS-induced HaCaT cells, and etanercept ameliorated inflammation in a psoriasis-like mouse model.

Irradiation-induced exosomal HMGB1 to confer radioresistance via the PI3K/AKT/FOXO3A signaling pathway in ESCC

BackgroundRadioresistance is a major cause of treatment failure in esophageal squamous cell carcinoma (ESCC) radiotherapy, and the underlying mechanisms of radioresistance are still unclear. Irradiation (IR) stimulates changes in tumor-derived exosome contents, which can be taken up by recipient cells, playing an important role in the proliferation, cell cycle and apoptosis of recipient cells. This study investigated the effect of IR-induced exosomal high mobility group box 1 (HMGB1) on radioresistance in ESCC cells.MethodsPlasma exosomes were isolated from 21 ESCC patients and 24 healthy volunteers, and the expression of HMGB1 was examined. Then, the therapeutic effect of radiotherapy was analyzed according to the different expression levels of plasma exosomal HMGB1 in ESCC patients. The uptake of exosomes by recipient cells was verified by immunofluorescence staining, and the localization of exosomes and HMGB1 in cells before and after IR was evaluated. The effects of IR-induced exosomes on cell proliferation, invasion, apoptosis, cell cycle distribution and radioresistance after HMGB1 knockdown were verified. Moreover, western blotting was used to measure changes in the expression of cyclin B1, CDK1, Bax, Bcl2, phosphorylated histone H2AX and the PI3K/AKT/FOXO3A pathway in the HMGB1-knockdown exosome group and the negative control group.ResultsThe expression of HMGB1 in ESCC plasma exosomes was significantly increased compared with that in healthy volunteers, and high expression of HMGB1 in plasma exosomes was associated with radioresistance (P = 0.016). IR-induced the release of exosomal HMGB1 and promoted proliferation and radioresistance in recipient cells, with a sensitization enhancement ratio (SER) of 0.906 and 0.919, respectively. In addition, IR-induced exosomal HMGB1 promotes G2/M phase arrest by regulating the proteins cyclin B1 and CDK1, cooperating with the proteins Bax and Bcl2 to reduce the apoptosis rate through the PI3K/AKT/FOXO3A signaling pathway, and participated in IR-induced DNA damage repair through γH2AX.ConclusionThese findings indicate that high expression of plasma exosomal HMGB1 is associated with an adverse radiotherapy response. IR-induced exosomal HMGB1 enhances the radioresistance of ESCC cells.

LPS preconditioning of MSC-CM improves protection against hypoxia/reoxygenation-induced damage in H9c2 cells partly via HMGB1/Bach1 signalling.

Mesenchymal stem cell-derived conditioned medium (MSC-CM) improves cardiac function after myocardial infarction; however, this cardioprotective effect is moderate and transient. Lipopolysaccharide (LPS) pretreatment partially improves MSC-CM-mediated cardioprotective effects owing to the presence of paracrine factors. However, the mechanism underlying these improved effects remains unknown. To study the effect of LPS-pretreated MSC-CM on hypoxia/reoxygenation (H/R)-induced injury, MSCs were treated with or without LPS (400 ng/mL) for 48 h, and the supernatant was collected (MSC-CM). Subsequently, H9c2 cells were co-cultured with Nor-CM (CM derived from LPS-untreated MSCs) and LPS-CM (CM derived from LPS-pretreated MSCs) for 24 h and subjected to H/R. MSC-CM inhibited the progression of H/R-induced injury in H9c2 cells, and this protective effect was enhanced via LPS pretreatment as evidenced by the improved apoptosis assessment index (i.e. caspase-3 and B-cell lymphoma-2 [Bcl-2] expression) and decreased levels of lactic dehydrogenase (LDH) and cardiac troponin (cTn). In addition, the results of haematoxylin-eosin staining (H&E), transmission electron microscopy (TEM) and TdT-mediated dUTP nick-end labelling (TUNEL) validated that MSC-CM inhibited H/R-induced injury in H9c2 cardiomyocytes. LPS pretreatment downregulated the expression of high mobility group box-1 (HMGB1) and BTB and CNC homology-1 (Bach1) proteins in MSCs but upregulated the expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and insulin-like growth factor (IGF). HMGB1 knockdown (MSC/siHMGB1-CM) significantly decreased the expression of Bach1 and increased the expression of VEGF, HGF and IGF. Bach1 knockdown (MSC/siBach1-CM) did not alter the production of HMGB1 but increased the expression of VEGF and IGF. LPS pretreatment did not alter the expression of the paracrine factors VEGF and HGF in the MSC/siHMGB1 group but increased their expression in the MSC/siBach1 group. The myocyte anti-apoptotic effects of MSCs/siBach1-CM were similar to those of untreated MSCs, which were not enhanced by LPS. LPS-pretreated MSC-CM protects H9c2 cells against H/R-induced injury partly through the HMGB1/Bach1 signalling pathway.

Cytosolic HMGB1 Mediates LPS-Induced Autophagy in Microglia by Interacting with NOD2 and Suppresses Its Proinflammatory Function.

The high mobility group box 1 (HMGB1), a well-known danger-associated molecule pattern (DAMP) molecule, is a non-histone chromosomal protein localized in the nucleus under normal physiological conditions. HMGB1 exhibits diverse functions depending on its subcellular location. In the present study, we investigated the role of HMGB1-induced autophagy in the lipopolysaccharide (LPS)-treated BV2 microglial cell line in mediating the transition between the inflammatory and autophagic function of the nucleotide-binding oligomerization domain-containing 2 (NOD2), a cytoplasmic pattern-recognition receptor. The induction of the microtubule-associated protein 1 light chain 3 (LC3), an autophagy biomarker, was detected slowly in BV2 cells after the LPS treatment, and peak induction was detected at 12 h. Under these conditions, NOD2 level was significantly increased and the binding between HMGB1 and NOD2 and between HMGB1 and ATG16L1 was markedly enhanced and the temporal profiles of the LC3II induction and HMGB1-NOD2 and HMGB1-ATG16L1 complex formation coincided with the cytosolic accumulation of HMGB1. The LPS-mediated autophagy induction was significantly suppressed in BV2 cells after HMGB1 or NOD2 knock-down (KD), indicating that HMGB1 contributes to NOD2-mediated autophagy induction in microglia. Moreover, NOD2-RIP2 interaction-mediated pro-inflammatory cytokine induction and NF-κB activity were significantly enhanced in BV2 cells after HMGB1 KD, indicating that HMGB1 plays a critical role in the modulation of NOD2 function between pro-inflammation and pro-autophagy in microglia. The effects of the cell-autonomous pro-autophagic pathway operated by cytoplasmic HMGB1 may be beneficial, whereas those from the paracrine pro-inflammatory pathway executed by extracellularly secreted HMGB1 can be detrimental. Thus, the overall functional significance of HMGB1-induced autophagy is different, depending on its temporal activity.

Hypoxia-induced HMGB1 promotes glioma stem cells self-renewal and tumorigenicity via RAGE

SummaryGlioma stem cells (GSCs) in the hypoxic niches contribute to tumor initiation, progression, and recurrence in glioblastoma (GBM). Hypoxia induces release of high-mobility group box 1 (HMGB1) from tumor cells, promoting the development of tumor. Here, we report that HMGB1 is overexpressed in human GBM specimens. Hypoxia promotes the expression and secretion of HMGB1 in GSCs. Furthermore, silencing HMGB1 results in the loss of stem cell markers and a reduction in self-renewal ability of GSCs. Additionally, HMGB1 knockdown inhibits the activation of RAGE-dependent ERK1/2 signaling pathway and arrests the cell cycle in GSCs. Consistently, FPS-ZM1, an inhibitor of RAGE, downregulates HMGB1 expression and the phosphorylation of ERK1/2, leading to a reduction in the proliferation of GSCs. In xenograft mice of GBM, HMGB1 knockdown inhibits tumor growth and promotes mouse survival. Collectively, these findings uncover a vital function for HMGB1 in regulating GSC self-renewal potential and tumorigenicity.

1303-P: Decreased Hepatic HMGB1 in Type 2 Diabetes Increases Mitochondrial Glucose Uptake by Regulating Insulin/AKT Signaling

Introduction: High Mobility Group Box-1 (HMGB1) is strongly associated with Type 2 Diabetes (T2D) and hepatocyte function. HMGB1 can regulate mitochondrial function under high glucose conditions. Our group has shown that knockdown of HMGB1 reduces hyperglycemia with decrease insulin activity. We want to further understand the role of HMGB1 in hepatocyte mitochondrial function and the insulin/AKT signaling, a vital pathway in glucose hemostasis. We hypothesize that by decreasing HMGB1, hepatocytes increase glucose uptake by regulating mitochondrial function with more effective Insulin/AKT signaling. Methods: Primary hepatocytes and liver lysates were isolated from inducible HMGB1 KO (iHMGB1 KO) and HMGB1 Flox (wildtype) mice after Tamoxifen and Streptozotocin IP injections for HMGB1 knockout and T2D-10-weeks development. HMGB1 knockdown and insulin/AKT signaling were assessed by RT-PCR and immunoblot. Flow cytometry was performed on primary hepatocyte cell suspension for mitochondrial function/mass (TMRE, Mitotracker) and glucose uptake (NDBG) cellular markers. Results: Liver lysates showed significant decreased expression/activity of HMGB1, Insulin Receptor-B, phosphorylated AKT, PDK and SIRT3 in iHMGB1 KO T2D mice. A counter regulator of metabolic and glucose activity, phosphorylated PDH, was found increased in iHMGB1 KO mice. Flow cytometry of isolated hepatocytes showed that iHMGB1 KO T2D animals had significantly less mitochondrial mass and functioning mitochondria, however showed increased glucose uptake. Conclusion: We found a potential mechanism by which HMGB1 regulates glucose uptake by regulating mitochondrial function and mass potentially by regulating the effectiveness of the Insulin/Akt signaling in T2D. Future studies will be directed to test glucose transport/metabolism in HMGB1 KO hepatocytes guided by more effective Insulin activity in the mediation of mitochondria function at the different stages of T2D progression. Disclosure Z.Liu: None. C.Kaltenmeier: None. R.I.Mota alvidrez: None. Funding This work was funded by department of surgery support for RIMA

1513-PUB: HMGB1 Knockdown Increases Glucose Sensitivity, Decreases Hyperglycemia and Insulin Resistance in Type 2 Diabetes

Introduction: Type-2 diabetes (T2D) characterizes by chronic hyperglycemia and insulin resistance. Hyperglycemia causes tissue damage and serious complications. High Mobility Group Box-1 (HMGB1) mediates the onset and progression of T2D, but the exact mechanism of HMGB1 glucose regulation in T2D is poorly understood. Global knockout of HMGB1 is fatal in neonatal mice primarily due to hypoglycemia. Therefore, using an inducible HMGB1 Knockout (iHMGB1 KO) model we evaluated the role of HMGB1 in adult mice progression of T2D (3) . We hypothesize that iHMGB1 KO mice will have decreased longitudinal hyperglycemia with decreased insulin resistance in a Streptozotocin (STZ) induced model of T2D. Methods: Male 8-week adult iHMGB1 KO mice were injected with Tamoxifen IP for days (1mg/kg) to induce HMGB1 knockdown. HMGB1 Flox (wildtype) were used as control. Then, mice were injected with STZ IP for 5 days (25mg/kg) , diabetic phenotype developed for weeks. HMGB1 knockdown was characterized via immunoblot and RT-PCR. Mice were placed on normal or high fat diet to evaluate T2D progression via metabolic cage analysis (MCA) . Glucose and insulin tolerance tests (GTT and ITT) were performed. Results: HMGB1 was significantly knocked down in iHMGB1 KO mice. MCA showed that STZ + normal diet had the appropriate respiratory quotient for translational T2D. GTT and ITT demonstrated higher glucose sensitivity and insulin tolerance in iHMGB1 KO T2D mice. Longitudinal glycemia measurement showed that iHMGB1 KO T2D mice have a progressively lower glycemia. Conclusion: With an effective knockdown of HMGB1, we evidenced increased glucose sensitivity, decreased hyperglycemia and insulin resistance in STZ induced T2D mice. Findings show that decreasing HMGB1 allows T2D adult mice to regulate glucose metabolism and insulin sensitivity better. Further studies will be directed to investigate the mechanisms that govern the role of HMGB1 in T2D. Disclosure Z.Liu: None. R.I.Mota alvidrez: None. Funding This work was funded by department of surgery support for RIMA

Ethanol potentiates mirtazapine-induced cardiotoxicity by inducing dysfunctional autophagy via HMGB1-dependent Akt/mTOR signaling pathway

Previous surveys have revealed that mirtazapine (MIR), one of the most commonly prescribed antidepressants, is associated with a higher risk of adverse cardiac events compared with other newer antidepressants. Chronic ethanol (EtOH) abuse could also lead to myocardial injuries. Concerning the common comorbidity of major depression and alcohol dependence, combined consumption of MIR and EtOH might be prevalent in patients with depression, resulting in an additive or synergistic cardiotoxic effect. To this end, the present study evaluated cardiotoxicity induced by MIR-plus-EtOH in vivo (male C57BL/6J mice) and in vitro (H9c2 cardiomyoblasts), Further research on the role of autophagy and underlying signaling pathway were carried out in H9c2 cells. We found that EtOH exacerbated MIR-induced cardiotoxicity both in vivo and in vitro. Furthermore, EtOH significantly potentiated MIR-induced dysfunctional autophagy as reflected by upregulated protein levels of LC3-II, p62, Beclin1 and LAMP-1. Pharmacological inhibition of autophagy by 3-methyladenine alleviated MIR-plus-EtOH-induced myocardial injury. High mobility group box 1 (HMGB1) is a positive regulator of autophagy. In our work, HMGB1 knockdown decreased autophagosome accumulation and boosted viability in H9c2 cells. Additionally, HMGB1 blockage markedly upregulated p-Akt/Akt and p-mTOR/mTOR levels which were suppressed in MIR-plus-EtOH treated cells. In general, the present study demonstrates that EtOH potentiates MIR-induced cardiotoxicity which might be attributed to dysfunctional autophagy via inhibiting Akt/mTOR signaling pathway, while HMGB1 knockdown might contribute to improve autophagy flux.

Helicobacter hepaticus Infection Promotes the Progression of Liver Preneoplasia in BALB/c Mice via the Activation and Accumulation of High-Mobility Group Box-1

It has been documented that Helicobacter hepaticus (H. hepaticus) infection is linked to chronic hepatitis and fibrosis in male BALB/c mice. However, the mechanism underlying the mice model of H. hepaticus–induced hepatocellular carcinoma is not fully known. In this study, male BALB/c mice were infected with H. hepaticus for 3, 6, 12, and 18 months. H. hepaticus colonization, histopathology, expression of proinflammatory cytokines, key signaling pathways, and protein downstream high-mobility group box-1 (HMGB1) in the liver were examined. Our data suggested that the H. hepaticus colonization level in the colon and liver progressively increased over the duration of the infection. H. hepaticus–induced hepatic inflammation and fibrosis were aggravated during the infection, and hepatic preneoplasia developed in the liver of infected mice at 12 and 18 months post-inoculation (MPI). H. hepaticus infection increased the levels of alanine aminotransferase and aspartate aminotransferase in the infected mice. In addition, the mRNA levels of IL-6, Tnf-α, Tgf-β, and HMGB1 were significantly elevated in the liver of H. hepaticus–infected mice from 3 to 18 MPI as compared to the controls. In addition, Ki67 was increased throughout the duration of the infection. Furthermore, HMGB1 protein was activated and translocated from the nucleus to the cytoplasm in the hepatocytes and activated the proteins of signal transducers and activators of transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) [extracellular regulated protein kinases 1/2 (Erk1/2) and mitogen-activated protein kinase p38 (p38)] upon H. hepaticus infection. In conclusions, these data demonstrated that male BALB/c mice infected with H. hepaticus are prone to suffering hepatitis and developing into hepatic preneoplasia. To verify the effect of HMGB1 in the progression of liver preneoplasia, mice were infected by H. hepaticus for 2 months before additional HMGB1 recombinant adenovirus treatment. All mice were sacrificed at 4 MPI, and the sera and liver tissues from all of the mice were collected. Immunology and histopathology evaluation showed that HMGB1 knockdown attenuated the H. hepaticus–induced hepatic and fibrosis at 4 MPI. Therefore, we showed that H. hepaticus–induced liver preneoplasia is closely correlated with the activation and accumulation of HMGB1.

Release of HMGB1 in Podocytes Exacerbates Lipopolysaccharide-Induced Acute Kidney Injury.

Objective Acute kidney injury (AKI) usually occurs during sepsis. Inflammation factors, such as high-mobility group box 1 (HMGB1), are dramatically upregulated under septic conditions. In our current work, the functions of HMGB1 in AKI were explored. Methods An AKI model was induced by the lipopolysaccharide (LPS) challenge in C57 mice. Podocytes were challenged by LPS for different durations. Subsequently, podocytes transfected with HMGB1 siRNA were exposed to LPS for 24 h. The expressions of supernatant HMGB1 and cellular active caspase-3 were examined by Western blotting analysis. To explore the effect of HMGB1 on tubular epithelial cells (TECs), HK-2 cells were exposed to HMGB1 at various concentrations for 24 h. Epithelial-mesenchymal transition (EMT) of HK-2 cells was evaluated by Western blotting analysis. Mitochondrial division and apoptosis of HK-2 cells were assessed by MitoTracker Red and Western blotting analysis, respectively. Results Compared with the sham control group, the expression of HMGB1 was increased in the kidney of AKI mice. Moreover, the expression of supernatant HMGB1 was increased in LPS-challenged podocytes compared with the control group. Knockdown of HMGB1 attenuated LPS-induced podocyte injury. Besides, EMT in TECs was triggered by HMGB1. Mitochondrial damage and apoptosis of HK-2 cells exposed to HMGB1 were markedly elevated compared with the control group. Conclusions Collectively, HMGB1 release in podocytes was induced by LPS, subsequently leading to exacerbated AKI.

Japanese encephalitis virus restricts HMGB1 expression to maintain MAPK pathway activation for viral replication

Japanese encephalitis virus (JEV) is a typical insect-borne flavivirus and an important zoonotic pathogen that causes human viral encephalitis and reproductive failure in pigs. Various strategies were utilized by JEV to facilitate its replication. It is important to identify key molecules that mediate JEV infection, as well as to investigate their underlying mechanism. In this study, the critical role of high-mobility group box 1 (HMGB1), a non-histone, DNA-binding protein, was assessed in JEV propagation. Upon JEV infection, the HMGB1 mRNA and protein levels were down-regulated at late infection in Huh7 cells. JEV replication was significantly enhanced with HMGB1 knock-down by siRNA and knock-out by the CRISPR/Cas9 system, whereas JEV growth was restricted in HMGB1-over-expressed Huh7 cells. Further investigation showed that HMGB1 suppressed MAPK pathway, and demonstrated that the weakening of MAPK pathway negatively regulated JEV infection. Together, these results suggested that JEV restricted HMGB1 expression to maintain MAPK pathway activation for viral replication. Our data showed that HMGB1 played a key role in JEV infection, providing the potential for the development of a novel drug to combat JEV infection.

Protective Effects of Dexazoxane on Rat Ferroptosis in Doxorubicin-Induced Cardiomyopathy Through Regulating HMGB1.

Dexrazoxane (DXZ) reduces cytotoxicity caused by Doxorubicin (DOX). However, the mechanism of DXZ in ferroptosis and cardiomyopathy remains unclear. This research, therefore, explores the role and mechanism of DXZ in DOX-induced ferroptosis and cardiomyopathy in rats. Kaplan–Meier survival analysis was performed in rats treated by DOX in combination with ferroptosis inhibitor (FER-1) or other cell death–associated inhibitors. The ferroptosis, cardiotoxicity, and expression of high mobility group box 1 (HMGB1) in rats treated by DOX in combination with FER-1 or with DXZ were determined by hematoxylin and eosin staining, echocardiographic analysis, and quantitative real-time PCR. The ferroptosis in DOX-treated rats that received HMGB1 knockdown or overexpression was further detected using molecular experiments. Finally, the viability, level of malondialdehyde (MDA), and expressions of ferroptosis-related markers (PTGS2, GPX4, and FTH1) of rat cardiomyocyte H9c2 exposed to DOX combined with FER-1, zVAD (an apoptosis inhibitor), DXZ, or not were detected by performing molecular experiments. FER-1 increased the survival of the rats induced by DOX. The DOX-induced ferroptosis and cardiotoxicity could be reversed by FER-1 or DXZ. HMGB1 was induced by DOX but was inhibited by DXZ or FER-1. Overexpression of HMGB1 promoted the ferroptosis and cardiotoxicity induced by DOX in the rats although silencing of HMGB1 showed opposite effects. The data indicate that DOX suppressed the viability and increased the MDA level in H9c2 cells in a dose-dependent manner. Moreover, DOX-induced increase of PTGS2 and decrease of GPX4 and FTH1 in H9c2 cells was reversed by DXZ or FER-1. Therefore, DXZ has protective effects on ferroptosis and cardiomyopathy in rats through regulating HMGB1.

HMGB1 knockdown increases the radiosensitivity of esophageal squamous cell carcinoma by regulating the expression of molecules involved in DNA repair.

Radiotherapy is an effective therapeutic strategy in esophageal squamous cell carcinoma (ESCC). However, acquired radioresistance of cancer cells leads to radiotherapy failure. The present study aimed to investigate the mechanisms of the effect of high mobility group box 1 (HMGB1) on the radiation sensitivity of ESCC. Small interfering RNA (si) transfection was used to generate three groups of TE-1 cells (TE-1, negative control and TE-1+siHMGB1), and the protein expression levels of HMGB1 in TE-1 cells were detected by western blotting. These groups of TE-1 cells were irradiated with different doses (0, 2, 4, 6 and 8 Gy) of X-rays after transfection. Subsequently, the viability of TE-1 cells was detected using an MTT assay, and the survival fraction of TE-1 cells was observed using a colony formation assay. The apoptotic rate, reactive oxygen species (ROS) content and levels of phosphorylated (p)-histone H2AX at S139 (p-γH2AX) of the cells were detected by flow cytometry. The alterations in mRNA expression levels of nicotinamide adenine nucleotide phosphate oxidase (NOX)1 and NOX5 were detected by reverse transcription-quantitative PCR, while the changes in protein levels of caspase-3, poly(ADP-ribose) polymerase, p-p38, p-ERK1/2 and p-JNK were detected by western blotting. The results revealed that HMGB1 knockdown significantly decreased cell viability, and the apoptosis rate of TE-1 cells transfected with siHMGB1 combined with radiation treatment was increased compared with that in cells with either siHMGB1 transfection or radiation treatment alone. HMGB1 knockdown increased nicotinamide adenine nucleotide phosphate oxidase-mediated ROS production and induced DNA damage via the MAPK signaling pathway, which may promote apoptosis and radiosensitivity after radiation in TE-1 cells. In conclusion, targeting HMGB1 may represent a promising strategy to increase the efficacy of radiation therapy for ESCC.