MSC-derived exosomal miR-140-3p improves cognitive dysfunction in sepsis-associated encephalopathy by HMGB1 and S-lactoylglutathione metabolism.

MiRNAs in mesenchymal stem cells (MSCs)-derived exosome (MSCs-exo) play an important role in the treatment of sepsis. We explored the mechanism through which MSCs-exo influences cognitive impairment in sepsis-associated encephalopathy (SAE). Here, we show that miR-140-3p targeted Hmgb1. MSCs-exo plus miR-140-3p mimic (Exo) and antibiotic imipenem/cilastatin (ABX) improve survival, weight, and cognitive impairment in cecal ligation and puncture (CLP) mice. Exo and ABX inhibit high mobility group box 1 (HMGB1), IBA-1, interleukin (IL)-1ß, IL-6, iNOS, TNF-α, p65/p-p65, NLRP3, Caspase 1, and GSDMD-N levels. In addition, Exo upregulates S-lactoylglutathione levels in the hippocampus of CLP mice. Our data further demonstrates that Exo and S-lactoylglutathione increase GSH levels in LPS-induced HMC3 cells and decrease LD and GLO2 levels, inhibiting inflammatory responses and pyroptosis. These findings suggest that MSCs-exo-mediated delivery of miR-140-3p ameliorates cognitive impairment in mice with SAE by HMGB1 and S-lactoylglutathione metabolism, providing potential therapeutic targets for the clinical treatment of SAE.

Role of CD61+ low-density neutrophils in promoting hepatocellular carcinoma metastasis through CCDC25 upregulation.

BACKGROUND: A subset of neutrophils isolated from the peripheral blood mononuclear cells (PBMC) layer has recently been described in cancer patients. METHODS: Double-gradient centrifugation was used to separate the neutrophil subsets. Western blotting and immunohistochemical assays were performed to assess CCDC25 expression levels. RESULTS: In this study, we found that low-density neutrophils (LDNs) were more highly enriched in metastatic hepatocellular carcinoma (HCC) patients than in non-metastatic HCC patients. We then showed a CD61+ LDNs subset, which displayed distinct functions and gene expression, when compared with high-density neutrophils (HDNs) and CD61- LDNs. Transcriptomic analysis revealed that the CD61+ LDNs were predominantly enhanced in the transcription of glycolysis and angiogenesis associated gene, HMGB1 associated gene and granulation protein gene. These CD61+ LDNs displayed a prominent ability to trigger metastasis, compared with HDNs and CD61- LDNs. Specifically, CD61+ LDN-derived HMGB1 protein increased the invasion of HCC cells by upregulating CCDC25. Mechanistically, the CD61+ LDN-derived HMGB1 protein enhanced the invasiveness of HCC cells and triggered their metastatic potential, which was mediated by TLR9-NF-κB-CCDC25 signaling. Blocking this signaling pathway reversed the invasion of the CD61+ LDN-induced HCC cells. In vivo, we consistently showed that CD61+ LDN-derived HMGB1 enhances HCC metastasis to the lungs. CONCLUSIONS: Overall, our findings showed that a subset of CD61+ LDNs has pro-metastatic effects on HCC, and may be used to target HCC in the clinical setting.

Berberine Improves Cancer-Derived Myocardial Impairment in Experimental Cachexia Models by Targeting High-Mobility Group Box-1.

Cardiac disorders in cancer patients pose significant challenges to disease prognosis. While it has been established that these disorders are linked to cancer cells, the precise underlying mechanisms remain elusive. In this study, we investigated the impact of cancerous ascites from the rat colonic carcinoma cell line RCN9 on H9c2 cardiomyoblast cells. We found that the ascites reduced mitochondrial volume, increased oxidative stress, and decreased membrane potential in the cardiomyoblast cells, leading to apoptosis and autophagy. Although the ascites fluid contained a substantial amount of high-mobility group box-1 (HMGB1), we observed that neutralizing HMGB1 with a specific antibody mitigated the damage inflicted on myocardial cells. Our mechanistic investigations revealed that HMGB1 activated both nuclear factor κB and phosphoinositide 3-kinases-AKT signals through HMGB1 receptors, namely the receptor for advanced glycation end products and toll-like receptor-4, thereby promoting apoptosis and autophagy. In contrast, treatment with berberine (BBR) induced the expression of miR-181c-5p and miR-340-5p while suppressing HMGB1 expression in RCN9 cells. Furthermore, BBR reduced HMGB1 receptor expression in cardiomyocytes, consequently mitigating HMGB1-induced damage. We validated the myocardial protective effects of BBR in a cachectic rat model. These findings underscore the strong association between HMGB1 and cancer cachexia, highlighting BBR as a promising therapeutic agent for myocardial protection through HMGB1 suppression and modulation of the signaling system.

Stretch Causes cffDNA and HMGB1-Mediated Inflammation and Cellular Stress in Human Fetal Membranes.

Danger-associated molecular patterns (DAMPs) are elevated within the amniotic cavity, and their increases correlate with advancing gestational age, chorioamnionitis, and labor. Although the specific triggers for their release in utero remain unclear, it is thought that they may contribute to the initiation of parturition by influencing cellular stress mechanisms that make the fetal membranes (FMs) more susceptible to rupture. DAMPs induce inflammation in many different tissue types. Indeed, they precipitate the subsequent release of several proinflammatory cytokines that are known to be key for the weakening of FMs. Previously, we have shown that in vitro stretch of human amnion epithelial cells (hAECs) induces a cellular stress response that increases high-mobility group box-1 (HMGB1) secretion. We have also shown that cell-free fetal DNA (cffDNA) induces a cytokine response in FM explants that is fetal sex-specific. Therefore, the aim of this work was to further investigate the link between stretch and the DAMPs HMGB1 and cffDNA in the FM. These data show that stretch increases the level of cffDNA released from hAECs. It also confirms the importance of the sex of the fetus by demonstrating that female cffDNA induced more cellular stress than male fetuses. Our data treating hAECs and human amnion mesenchymal cells with HMGB1 show that it has a differential effect on the ability of the cells of the amnion to upregulate the proinflammatory cytokines and propagate a proinflammatory signal through the FM that may weaken it. Finally, our data show that sulforaphane (SFN), a potent activator of Nrf2, is able to mitigate the proinflammatory effects of stretch by decreasing the levels of HMGB1 release and ROS generation after stretch and modulating the increase of key cytokines after cell stress. HMGB1 and cffDNA are two of the few DAMPs that are known to induce cytokine release and matrix metalloproteinase (MMP) activation in the FMs; thus, these data support the general thesis that they can function as potential central players in the normal mechanisms of FM weakening during the normal distension of this tissue at the end of a normal pregnancy.

Targeting BMAL1 reverses drug resistance of acute myeloid leukemia cells and promotes ferroptosis through HMGB1-GPX4 signaling pathway.

PURPOSE: Acute myeloid leukemia (AML) is a refractory hematologic malignancy that poses a serious threat to human health. Exploring alternative therapeutic strategies capable of inducing alternative modes of cell death, such as ferroptosis, holds great promise as a viable and effective intervention. METHODS: We analyzed online database data and collected clinical samples to verify the expression and function of BMAL1 in AML. We conducted experiments on AML cell proliferation, cell cycle, ferroptosis, and chemotherapy resistance by overexpressing/knocking down BMAL1 and using assays such as MDA detection and BODIPY 581/591 C11 staining. We validated the transcriptional regulation of HMGB1 by BMAL1 through ChIP assay, luciferase assay, RNA level detection, and western blotting. Finally, we confirmed the results of our cell experiments at the animal level. RESULTS: BMAL1 up-regulation is an observed phenomenon in AML patients. Furthermore, there existed a strong correlation between elevated levels of BMAL1 expression and inferior prognosis in individuals with AML. We found that knocking down BMAL1 inhibited AML cell growth by blocking the cell cycle. Conversely, overexpressing BMAL1 promoted AML cell proliferation. Moreover, our research results revealed that BMAL1 inhibited ferroptosis in AML cells through BMAL1-HMGB1-GPX4 pathway. Finally, knocking down BMAL1 can enhance the efficacy of certain first-line cancer therapeutic drugs, including venetoclax, dasatinib, and sorafenib. CONCLUSION: Our research results suggest that BMAL1 plays a crucial regulatory role in AML cell proliferation, drug resistance, and ferroptosis. BMAL1 could be a potential important therapeutic target for AML.

Tumor cells express and maintain HMGB1 in the reduced isoform to enhance CXCR4-mediated migration.

During inflammation and tissue regeneration, the alarmin High Mobility Group Box 1 (HMGB1), in its reduced isoform, enhances the activity of the chemokine CXCL12, forming a heterocomplex that acts via the chemokine receptor CXCR4. Despite the established roles of both HMGB1 and CXCL12 in tumor progression and metastatic spread to distal sites, the role of the CXCL12/HMGB1 heterocomplex in cancer has never been investigated. By employing a newly established mass spectrometry protocol that allows an unambiguous distinction between reduced (red-HMGB1) and oxidized (ox-HMGB1) HMGB1 isoforms in cell lysates, we demonstrate that human epithelial cells derived from breast (MCF-7 and MDA-MB-231) and prostate (PC-3) cancer predominantly express red-HMGB1, while primary CD3+ T lymphocytes from peripheral blood express both HMGB1 isoforms. All these cancer cells release HMGB1 in the extracellular microenvironment together with varying concentrations of thioredoxin and thioredoxin reductase. The CXCL12/HMGB1 heterocomplex enhances, via CXCR4, the directional migration and invasiveness of cancer cells characterized by high metastatic potential that possess a fully active thioredoxin system, contributing to maintain red-HMGB1. On the contrary, cancer cells with low metastatic potential, lack thioredoxin reductase, promptly uptake CXCL12 and fail to respond to the heterocomplex. Our study demonstrates that the responsiveness of cancer cells to the CXCL12/HMGB1 heterocomplex, resulting in enhanced cell migration and invasiveness, depends on the maintenance of HMGB1 in its reduced isoform, and suggests disruption of the heterocomplex as a potential therapeutic target to inhibit invasion and metastatic spread in cancer therapies.

[Ligusticum cycloprolactam inhibits IL-1ß-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway].

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1ß(IL-1ß) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1ß, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1ß-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.

HMGB1 prefers to interact with structural RNAs and regulates rRNA methylation modification and translation in HeLa cells.

BACKGROUND: High-mobility group B1 (HMGB1) is both a DNA binding nuclear factor modulating transcription and a crucial cytokine that mediates the response to both infectious and noninfectious inflammation such as autoimmunity, cancer, trauma, and ischemia reperfusion injury. HMGB1 has been proposed to control ribosome biogenesis, similar as the other members of a class of HMGB proteins. RESULTS: Here, we report that HMGB1 selectively promotes transcription of genes involved in the regulation of transcription, osteoclast differentiation and apoptotic process. Improved RNA immunoprecipitation by UV cross-linking and deep sequencing (iRIP-seq) experiment revealed that HMGB1 selectively bound to mRNAs functioning not only in signal transduction and gene expression, but also in axon guidance, focal adhesion, and extracellular matrix organization. Importantly, HMGB1-bound reads were strongly enriched in specific structured RNAs, including the domain II of 28S rRNA, H/ACA box snoRNAs including snoRNA63 and scaRNAs. RTL-P experiment showed that overexpression of HMGB1 led to a decreased methylation modification of 28S rRNA at position Am2388, Cm2409, and Gm2411. We further showed that HMGB1 overexpression increased ribosome RNA expression levels and enhanced protein synthesis. CONCLUSION: Taken together, our results support a model in which HMGB1 binds to multiple RNA species in human cancer cells, which could at least partially contribute to HMGB1-modulated rRNA modification, protein synthesis function of ribosomes, and differential gene expression including rRNA genes. These findings provide additional mechanistic clues to HMGB1 functions in cancers and cell differentiation.

IL6/adiponectin/HMGB1 feedback loop mediates adipocyte and macrophage crosstalk and M2 polarization after myocardial infarction.

Background: Differences in border zone contribute to different outcomes post-infarction, such as left ventricular aneurysm (LVA) and myocardial infarction (MI). LVA usually forms within 24 h of the onset of MI and may cause heart rupture; however, LVA surgery is best performed 3 months after MI. Few studies have investigated the LVA model, the differences in border zones between LVA and MI, and the mechanism in the border zone. Methods: The LVA, MI, and SHAM mouse models were used. Echocardiography, Masson's trichrome staining, and immunofluorescence staining were performed, and RNA sequencing of the border zone was conducted. The adipocyte-conditioned medium-treated hypoxic macrophage cell line and LVA and MI mouse models were employed to determine the effects of the hub gene, adiponectin (ADPN), on macrophages. Quantitative polymerase chain reaction (qPCR), Western blot analysis, transmission electron microscopy, and chromatin immunoprecipitation (ChIP) assays were conducted to elucidate the mechanism in the border zone. Human subepicardial adipose tissue and blood samples were collected to validate the effects of ADPN. Results: A novel, simple, consistent, and low-cost LVA mouse model was constructed. LVA caused a greater reduction in contractile functions than MI owing to reduced wall thickness and edema in the border zone. ADPN impeded cardiac edema and promoted lymphangiogenesis by increasing macrophage infiltration post-infarction. Adipocyte-derived ADPN promoted M2 polarization and sustained mitochondrial quality via the ADPN/AdipoR2/HMGB1 axis. Mechanistically, ADPN impeded macrophage HMGB1 inflammation and decreased interleukin-6 (IL6) and HMGB1 secretion. The secretion of IL6 and HMGB1 increased ADPN expression via STAT3 and the co-transcription factor, YAP, in adipocytes. Based on ChIP and Dual-Glo luciferase experiments, STAT3 promoted ADPN transcription by binding to its promoter in adipocytes. In vivo, ADPN promoted lymphangiogenesis and decreased myocardial injury after MI. These phenotypes were rescued by macrophage depletion or HMGB1 knockdown in macrophages. Supplying adipocytes overexpressing STAT3 decreased collagen disposition, increased lymphangiogenesis, and impaired myocardial injury. However, these effects were rescued after HMGB1 knockdown in macrophages. Overall, the IL6/ADPN/HMGB1 axis was validated using human subepicardial tissue and blood samples. This axis could serve as an independent factor in overweight MI patients who need coronary artery bypass grafting (CABG) treatment. Conclusion: The IL6/ADPN/HMGB1 loop between adipocytes and macrophages in the border zone contributes to different clinical outcomes post-infarction. Thus, targeting the IL6/ADPN/HMGB1 loop may be a novel therapeutic approach for cardiac lymphatic regulation and reduction of cell senescence post-infarction.

Icariin attenuates vascular endothelial dysfunction by inhibiting inflammation through GPER/Sirt1/HMGB1 signaling pathway in type 1 diabetic rats.

Icariin, a flavonoid glycoside, is extracted from Epimedium. This study aimed to investigate the vascular protective effects of icariin in type 1 diabetic rats by inhibiting high-mobility group box 1 (HMGB1)-related inflammation and exploring its potential mechanisms. The impact of icariin on vascular dysfunction was assessed in streptozotocin (STZ)-induced diabetic rats through vascular reactivity studies. Western blotting and immunofluorescence assays were performed to measure the expressions of target proteins. The release of HMGB1 and pro-inflammation cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The results revealed that icariin administration enhanced acetylcholine-induced vasodilation in the aortas of diabetic rats. It also notably reduced the release of pro-inflammatory cytokines, including interleukin-8 (IL-8), IL-6, IL-1ß, and tumor necrosis factor-alpha (TNF-α) in diabetic rats and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs). The results also unveiled that the pro-inflammatory cytokines in the culture medium of HUVECs could be increased by rHMGB1. The increased release of HMGB1 and upregulated expressions of HMGB1-related inflammatory factors, including advanced glycation end products (RAGE), Toll-like receptor 4 (TLR4), and phosphorylated p65 (p-p65) in diabetic rats and HG-induced HUVECs, were remarkably suppressed by icariin. Notably, HMGB1 translocation from the nucleus to the cytoplasm in HUVECs under HG was inhibited by icariin. Meanwhile, icariin could activate G protein-coupled estrogen receptor (GPER) and sirt1. To explore the role of GPER and Sirt1 in the inhibitory effect of icariin on HMGB1 release and HMGB-induced inflammation, GPER inhibitor and Sirt1 inhibitor were used in this study. These inhibitors diminished the effects of icariin on HMGB1 release and HMGB1-induced inflammation. Specifically, the GPER inhibitor also negated the activation of Sirt1 by icariin. These findings suggest that icariin activates GPER and increases the expression of Sirt1, which in turn reduces HMGB1 translocation and release, thereby improving vascular endothelial function in type 1 diabetic rats by inhibiting inflammation.

CAMSAP3-mediated regulation of HMGB1 acetylation and subcellular localization in lung cancer cells: Implications for cell death modulation.

BACKGROUND: Deregulation of cell death is a common characteristic of cancer, and resistance to this process often occurs in lung cancer. Understanding the molecular mechanisms underlying an aberrant cell death is important. Recent studies have emphasized the involvement of calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) in lung cancer aggressiveness, its influence on cell death regulation remains largely unexplored. METHODS: CAMSAP3 was knockout in lung cancer cells using CRISPR-Cas9 system. Cell death and autophagy were evaluated using MTT and autophagic detection assays. Protein interactions were performed by proteomic analysis and immunoprecipitation. Protein expressions and their cytoplasmic localization were analyzed through immunoblotting and immunofluorescence techniques. RESULTS: This study reveals a significant correlation between low CAMSAP3 expression and poor overall survival rates in lung cancer patients. Proteomic analysis identified high mobility group box 1 (HMGB1) as a candidate interacting protein involved in the regulation of cell death. Treatment with trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs) resulted in increased HMGB1 acetylation and its translocation to the cytoplasm and secretion, thereby inducing autophagic cell death. However, this process was diminished in CAMSAP3 knockout lung cancer cells. Mechanistically, immunoprecipitation indicated an interaction between CAMSAP3 and HMGB1, particularly with its acetylated form, in which this complex was elevated in the presence of TSA. CONCLUSIONS: CAMSAP3 is prerequisite for TSA-mediated autophagic cell death by interacting with cytoplasmic acetylated HMGB1 and enhancing its release. SIGNIFICANT: This finding provides molecular insights into the role of CAMSAP3 in regulating cell death, highlighting its potential as a therapeutic target for lung cancer treatment.

High mobility group box 1 mediates inflammatory responses in malignant peritoneal mesothelioma.

BACKGROUND: Serum high mobility group box 1 (HMGB1) serves as a diagnostic biomarker for malignant peritoneal mesothelioma (MPM) patients, yet its diagnostic significance within MPM tumor tissues remains uncertain. This study aims to elucidate the roles of HMGB1 in MPM. METHODS: HMGB1 expression analysis was conducted in both tumor and adjacent non-cancerous tissues collected from MPM patients. The two-year follow-up of MPM patients commenced from the diagnosis date. Inflammatory cytokine analysis was performed on these tissues, and Pearson correlation coefficient analysis was applied to examine variable relationships. In vitro assays included constructing an HMGB1 knockdown cell line, assessing cell viability, apoptosis, and inflammatory cytokine levels to delineate HMGB1's roles in MPM. RESULTS: HMGB1 overexpression was observed in MPM tumor tissues, particularly in stages III-IV. Diagnostic implications of HMGB1 for MPM were evident, augmenting its diagnostic value. HMGB1 overexpression correlated with diminished survival rates. Positive correlations existed between inflammatory cytokines and HMGB1 in MPM tumor tissues and cell lines. Suppression of HMGB1 regulated cell growth and apoptosis in MPM cell lines. CONCLUSION: HMGB1 exhibits diagnostic potential for MPM and modulates inflammatory responses within the disease context.

Targeting the MCP-GPX4/HMGB1 Axis for Effectively Triggering Immunogenic Ferroptosis in Pancreatic Ductal Adenocarcinoma.

Induction of ferroptosis can inhibit cancer cells in vitro, however, the role of ferroptosis in treatment in vivo is controversial. The immunosuppressive cells activated by the ferroptotic tumor cells can promote the growth of residual tumor cells, hindering the application of ferroptosis stimulation in tumor treatment. In this study, a new strategy is aimed to be identified for effectively triggering immunogenic ferroptosis in pancreatic ductal adenocarcinoma (PDAC) and simultaneously stimulating antitumor immune responses. Toward this, several molecular and biochemical experiments are performed using patient-derived organoid models and a KPC mouse model (LSL-KrasG12D /+, LSL-Trp53R172H/+, Pdx-1-Cre). It is observed that the inhibition of macrophage-capping protein (MCP) suppressed the ubiquitin fold modifier (UFM)ylation of pirin (PIR), a newly identified substrate of UFM1, thereby decreasing the transcription of GPX4, a marker of ferroptosis, and promoting the cytoplasmic transportation of HMGB1, a damage-associated molecular pattern. GPX4 deficiency triggered ferroptosis, and the pre-accumulated cytosolic HMGB1 is released rapidly. This altered release pattern of HMGB1 facilitated the pro-inflammatory M1-like polarization of macrophages. Thus, therapeutic inhibition of MCP yielded dual antitumor effects by stimulating ferroptosis and activating antitumor pro-inflammatory M1-like macrophages. The nanosystem developed for specifically silencing MCP is a promising tool for treating PDAC.

Transcriptome and single-cell analysis reveal disulfidptosis-related modification patterns of tumor microenvironment and prognosis in osteosarcoma.

Osteosarcoma (OS) is the most common malignant bone tumor with high pathological heterogeneity. Our study aimed to investigate disulfidptosis-related modification patterns in OS and their relationship with survival outcomes in patients with OS. We analyzed the single-cell-level expression profiles of disulfidptosis-related genes (DSRGs) in both OS microenvironment and OS subclusters, and HMGB1 was found to be crucial for intercellular regulation of OS disulfidptosis. Next, we explored the molecular clusters of OS based on DSRGs and related immune cell infiltration using transcriptome data. Subsequently, the hub genes of disulfidptosis in OS were screened by applying multiple machine models. In vitro and patient experiments validated our results. Three main disulfidptosis-related molecular clusters were defined in OS, and immune infiltration analysis suggested high immune heterogeneity between distinct clusters. The in vitro experiment confirmed decreased cell viability of OS after ACTB silencing and higher expression of ACTB in patients with lower immune scores. Our study systematically revealed the underlying relationship between disulfidptosis and OS at the single-cell level, identified disulfidptosis-related subtypes, and revealed the potential role of ACTB expression in OS disulfidptosis.

The Effect of HMGB1 and HMGB2 on Transcriptional Regulation Differs in Neuroendocrine and Adenocarcinoma Models of Prostate Cancer.

Human high-mobility group-B (HMGB) proteins regulate gene expression in prostate cancer (PCa), a leading cause of oncological death in men. Their role in aggressive PCa cancers, which do not respond to hormonal treatment, was analyzed. The effects of HMGB1 and HMGB2 silencing upon the expression of genes previously related to PCa were studied in the PCa cell line PC-3 (selected as a small cell neuroendocrine carcinoma, SCNC, PCa model not responding to hormonal treatment). A total of 72% of genes analyzed, using pre-designed primer panels, were affected. HMGB1 behaved mostly as a repressor, but HMGB2 as an activator. Changes in SERPINE1, CDK1, ZWINT, and FN1 expression were validated using qRT-PCR after HMGB1 silencing or overexpression in PC-3 and LNCaP (selected as an adenocarcinoma model of PCa responding to hormonal treatment) cell lines. Similarly, the regulatory role of HMGB2 upon SERPINE1, ZWINT, FN1, IGFPB3, and TYMS expression was validated, finding differences between cell lines. The correlation between the expression of HMGB1, HMGB2, and their targets was analyzed in PCa patient samples and also in PCa subgroups, classified as neuroendocrine positive or negative, in public databases. These results allow a better understanding of the role of HMGB proteins in PCa and contribute to find specific biomarkers for aggressive PCa.

LncRNA AA465934 Improves Podocyte Injury by Promoting Tristetraprolin-Mediated HMGB1 DownRegulation in Diabetic Nephropathy.

Although LncRNA AA465934 expression is reduced in high glucose (HG)-treated podocytes, its role in HG-mediated podocyte injury and diabetic nephropathy (DN) remains unknown. Herein, we investigated the role of AA465934 in HG-mediated podocyte injury and DN using a spontaneous type II diabetic nephropathy (T2DN) model. The model was created by injecting AA465934 overexpressed adeno-associated virus (AAV) or control into mice. The levels of renal function, proteinuria, renal structural lesions, and podocyte apoptosis were then examined. Furthermore, AA465934 and autophagy levels, as well as tristetraprolin (TTP) and high mobility group box 1 (HMGB1) expression changes were detected. We also observed podocyte injury and the binding ability of TTP to E3 ligase proviral insertion in murine lymphomas 2 (PIM2), AA465934, or HMGB1. According to the results, AA465934 improved DN progression and podocyte damage in T2DN mice. In addition, AA465934 bound to TTP and inhibited its degradation by blocking TTP-PIM2 binding. Notably, TTP knock-down blocked the ameliorating effects of AA465934 and TTP bound HMGB1 mRNA, reducing its expression. Overexpression of HMGB1 inhibited the ability of AA465934 and TTP to improve podocyte injury. Furthermore, AA465934 bound TTP, inhibiting TTP-PIM2 binding, thereby suppressing TTP degradation, downregulating HMGB1, and reversing autophagy downregulation, ultimately alleviating HG-mediated podocyte injury and DN. Based on these findings, we deduced that the AA465934/TTP/HMGB1/autophagy axis could be a therapeutic avenue for managing podocyte injury and DN.

Downregulation of HMGB1 carried by macrophage-derived extracellular vesicles delays atherosclerotic plaque formation through Caspase-11-dependent macrophage pyroptosis.

BACKGROUND: Macrophage-derived extracellular vesicle (macrophage-EV) is highly studied for its regulatory role in atherosclerosis (AS). Our current study tried to elucidate the possible role of macrophage-EV loaded with small interfering RNA against high-mobility group box 1 (siHMGB1) affecting atherosclerotic plaque formation. METHODS: In silico analysis was performed to find critical factors in mouse atherosclerotic plaque formation. EVs secreted by RAW 264.7 cells were collected by ultracentrifugation and characterized, followed by the preparation of macrophage-EV-loaded siHMGB1 (macrophage-EV/siHMGB1). ApoE-/- mice were used to construct an AS mouse model by a high-fat diet, followed by injection of macrophage-EV/siHMGB1 to assess the in vivo effect of macrophage-EV/siHMGB1 on AS mice. RAW264.7 cells were subjected to ox-LDL, LPS or macrophage-EV/siHMGB1 for analyzing the in vitro effect of macrophage-EV/siHMGB1 on macrophage pyrophosis and inflammation. RESULTS: In silico analysis found that HMGB1 was closely related to the development of AS. Macrophage-EV/siHMGB could inhibit the release of HMGB1 from macrophages to outside cells, and the reduced HMGB1 release could inhibit foam cell formation. Besides, macrophage-EV/siHMGB also inhibited the LPS-induced Caspase-11 activation, thus inhibiting macrophage pyroptosis and preventing atherosclerotic plaque formation. CONCLUSION: Our results proved that macrophage-EV/siHMGB could inhibit foam cell formation and suppress macrophage pyroptosis, finally preventing atherosclerotic plaque formation in AS mice.

IGF2BP3 prevent HMGB1 mRNA decay in bladder cancer and development.

BACKGROUND: IGF2BP3 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. METHODS: The 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. RESULTS: We 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. CONCLUSIONS: Our 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.

Effects of HMGB1/TLR4 on secretion IL-10 and VEGF in human jaw bone-marrow mesenchymal stem cells.

OBJECTIVE: We aimed to investigate the regulatory effects of HMGB1/TLR4 signaling pathway on the expression of IL-10 and VEGF in human bone marrow mesenchymal stem cells. METHODOLOGY: Human JBMSCs were isolated and cultured. Then, HMGB1 was added into the JBMSCs culture medium, and the protein and mRNA expression levels of IL-10 and VEGF were assessed. Moreover, cells were pretreated with a specific TLR4 inhibitor (TAK-242), and the expression changes of IL-10 and VEGF were compared. RESULTS: Compared with the control group, exposure to HMGB1 in human JBMSCs up-regulated TLR4, IL-10, and VEGF secretion at both protein and mRNA levels (P<0. 05). In addition, the increased expression of IL-10 and VEGF could be restrained in TAK-242 group compared with the HMGB1 group (P<0.05). CONCLUSIONS: The results indicated that HMGB1 activate TLR4 signaling pathway in Human JBMSCs, which plays a regulatory role in cytokines expression.

Imp7 siRNA nanoparticles protect against mechanical ventilation-associated liver injury by inhibiting HMGB1 production and NETs formation.

Mechanical ventilation (MV) has the potential to induce extra-pulmonary organ damage by adversely affecting the lungs and promoting the secretion of inflammatory cytokines. High-mobility group box 1 protein (HMGB1) is a pro-inflammatory mediator in ventilator-induced lung injury (VILI), but its effect on MV-associated liver injury and the mechanisms are poorly understood. In the present study, mice were subjected to high-volume MV (20 ml/kg) to induce VILI. MV-induced HMGB1 prompted neutrophil extracellular traps (NETs) formation and PANoptosis within the liver. Inhibiting NETs formation by DNase I or PAD4 inhibitor, or by HMGB1 neutralizing ameliorated the liver injury. HMGB1 activated neutrophils to form NETs through TLR4/MyD88/TRAF6 pathway. Importantly, Importin7 siRNA nanoparticles inhibited HMGB1 release and protected against MV-associated liver injury. These data provide evidence of MV-induced HMGB1 prompted NETs formation and PANoptosis in the liver via the TLR4/MyD88/TRAF6 pathway. HMGB1 is a potential therapeutic target for MV-associated liver injury.