Lentivirus-mediated RNA interference targeting HMGB1 modulates AQP1 to reduce pain induced by chronic compression of the dorsal root ganglia.

Backgrounds: Neuropathic pain (NP) is a kind of chronic pain that has attracted much attention in clinical practice, characterized by high morbidity, complex mechanisms, and difficulties in clinical treatment, with which the activation of High mobility group box 1 (HMGB1) is closely related. The aim of this study was to investigate the effects of lentivirus-mediated RNA interference gene therapy targeting HMGB1 on neuropathic pain in rats with chronic dorsal root ganglion compression (CCD) and its specific mechanisms, so as to explore new pharmacological targets. Methods: Adult male Wistar rats were surgically subjected to chronic compression of the dorsal root ganglia (CCD). Behavioral tests were performed by calculating the paw withdrawal mechanical threshold (PWMT) and the thermal paw withdrawal latency (TPWL). Co-immunoprecipitation (CO-IP) was used to clarify protein interactions. Gene silencing was induced by injecting lentivirus expressing HMGB1 short hairpin RNA (shRNA) into rats. An LPS-inflammation-stimulated rat astrocyte model was established to validate the animal experiment results further. Western blot analysis and real-time quantitative PCR were used to detect pathway protein expression. Results: After first establishing the rat CCD model, both PWMT and PTWL were significantly reduced in rats, indicating that the model construction was successful. After lentiviral silencing of HMGB1 expression, NP was significantly alleviated in CCD rats. CO-IP experiments showed a link between HMGB1 and AQP1; After silencing HMGB1 expression, the expression of AQP1 was significantly reduced, and HMGB1 was able to modulate the effect of AQP1 on NP. Further use of an inhibitor of the HMGB1 receptor showed that after inhibition of RAGE, AQP1 was significantly reduced; HMGB1 may regulate AQP1 through its receptor RAGE to affect NP. Silencing of HMGB1 resulted in a significant decrease in NF-κB, and HMGB1 affects the inflammatory pathways it mediates. After silencing AQP1, NF-κB also decreased significantly, indicating that AQP1 is an upstream regulator of NF-κB. Conclusion: Lentivirus-mediated RNA interference (RNAi) silencing targeting HMGB1 may play a key role in the development of neuropathic pain in rats by regulating AQP1 expression via RAGE and ultimately activating NF-κB.

HMGB1 promotes M1 polarization of macrophages and induces COPD inflammation.

Chronic obstructive pulmonary disease (COPD) is a pervasive and incapacitating respiratory condition, distinguished by airway inflammation and the remodeling of the lower respiratory tract. Central to its pathogenesis is an intricate inflammatory process, wherein macrophages exert significant regulatory functions, and High mobility group box 1 (HMGB1) emerges as a pivotal inflammatory mediator potentially driving COPD progression. This study explores the hypothesis that HMGB1, within macrophages, modulates COPD through inflammatory mechanisms, focusing on its influence on macrophage polarization. Our investigation uncovered that HMGB1 is upregulated in the context of COPD, associated with an enhanced proinflammatory M1 macrophage polarization induced by cigarette smoke. This polarization is linked to suppressed cell proliferation and induced apoptosis, indicative of HMGB1's role in the disease's inflammatory trajectory. The study further implicates HMGB1 in the activation of the Nuclear factor kappa-B (NF-κB) signaling pathway and chemokine signaling within macrophages, which are likely to amplify the inflammatory response characteristic of COPD. The findings underscore HMGB1's critical involvement in COPD pathogenesis, presenting it as a significant target for therapeutic intervention aimed at modulating macrophage polarization and inflammation.

Alarmin-loaded extracellular lipid droplets induce airway neutrophil infiltration during type 2 inflammation.

Group 2 innate lymphoid cells (ILC2s) play a crucial role in allergic diseases by coordinating a complex network of various effector cell lineages involved in type 2 inflammation. However, their function in regulating airway neutrophil infiltration, a deleterious symptom of severe asthma, remains unknown. Here, we observed ILC2-dependent neutrophil accumulation in the bronchoalveolar lavage fluid (BALF) of allergic mouse models. Chromatography followed by proteomics analysis identified the alarmin high mobility group box-1 (HMGB1) in the supernatant of lung ILC2s initiated neutrophil chemotaxis. Genetic perturbation of Hmgb1 in ILC2s reduced BALF neutrophil numbers and alleviated airway inflammation. HMGB1 was loaded onto the membrane of lipid droplets (LDs) released from activated lung ILC2s. Genetic inhibition of LD accumulation in ILC2s significantly decreased extracellular HMGB1 abundance and BALF neutrophil infiltration. These findings unveil a previously uncharacterized extracellular LD-mediated immune signaling delivery pathway by which ILC2s regulate airway neutrophil infiltration during allergic inflammation.

Tumour cell-released autophagosomes promote lung metastasis by upregulating PD-L1 expression in pulmonary vascular endothelial cells in breast cancer.

PURPOSE: Establishing an immunosuppressive premetastatic niche (PMN) in distant organs is crucial for breast cancer metastasis. Vascular endothelial cells (VECs) act as barriers to transendothelial cell migration. However, the immune functions of PMNs remain unclear. Tumour cell-released autophagosomes (TRAPs) are critical modulators of antitumour immune responses. Herein, we investigated the mechanism through which TRAPs modulate the immune function of pulmonary VECs in lung PMN in breast cancer. METHODS: Immortalised mouse pulmonary microvascular endothelial cells were incubated with TRAPs in vitro. RNA sequencing, flow cytometry, and western blotting were employed to assess immunosuppressive function and mechanism. In vivo, TRAP-trained and autophagy-deficient tumour mice were used to detect immunosuppression, and high-mobility group box 1 (HMGB1)-deficient TRAP-trained and TLR4 knockout mice were utilised to investigate the underlying mechanisms of pulmonary VECs. Additionally, the efficacy of anti-programmed cell death ligand-1 (PD-L1) immunotherapy was evaluated in early tumour-bearing mice. RESULTS: HMGB1 on TRAPs surfaces stimulated VECs to upregulate PD-L1 via a TLR4-MyD88-p38/STAT3 signalling cascade that depended on the cytoskeletal movement of VECs. Importantly, PD-L1 on TRAP-induced VECs can inhibit T cell function, promote lung PMN immunosuppression, and result in more pronounced lung metastasis. Treatment with anti-PD-L1 reduces lung metastasis in early stage tumour-bearing mice. CONCLUSIONS: These findings revealed a novel role and mechanism of TRAP-induced immunosuppression of pulmonary VECs in lung PMN. TRAPs and their surface HMGB1 are important therapeutic targets for reversing immunosuppression, providing a new theoretical basis for the treatment of early stage breast cancer using an anti-PD-L1 antibody.

Endogenous Ligands of TLR4 in Microglia: Potential Targets for Related Neurological Diseases.

Chronic inflammation mediated by microglia is a cause of some neuroinflammatory diseases. TLR4, a natural immune receptor on microglia, plays an important role in the occurrence of inflammation and the process of diseases. TLR4 can be activated by a variety of ligands to trigger inflammatory responses, including endogenous ligands HMGB1, S100A8/9, Heme, and Fetuin-A. As ligands derived from the body itself, they have the ability to bind directly to TLR4 and can be used as inducers of aseptic inflammation. In the past 20 years, targeting ligands rather than receptors has become an emerging therapeutic strategy for the treatment of diseases, so understanding the relationship between microglia, TLR4, TLR4 ligands, and corresponding diseases may have new implications for the treatment of diseases. In the article, we will discuss the TLR4 and the endogenous substances that can activate the TLR4 signaling pathway and present literature support for their role in neuroinflammatory diseases.

Exosomal miR-142-3p from M1-polarized macrophages suppresses cell growth and immune escape in glioblastoma through regulating HMGB1-mediated PD-1/PD-L1 checkpoint.

Glioblastoma (GBM) is one of the most prevalent cancerous brain tumors. Former studies have reported that exosomes derived from M1-polarized macrophages (M1 exosomes) inhibit tumor occurrence and development through delivery of tumor suppressor genes. Also, microRNA-142-3p (miR-142-3p) has been verified to function as a tumor suppressor. GBM cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8), colony formation assay and 5-ethynyl-2'-deoxyuridine (EdU) assay; cell apoptosis was determined by flow cytometry analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Mechanism investigations were conducted for analyzing the molecular mechanism by which miR-142-3p and M1 exosomes affect GBM progression. Upregulation of miR-142-3p expression was detected in M1-polarized macrophages and M1 exosomes. M1 exosomes inhibit GBM cell proliferation and trigger cell apoptosis. Functionally, miR-142-3p silencing promotes the proliferation and inhibits the apoptosis of GBM cells treated with M1 exosomes. As for molecular mechanism, miR-142-3p inhibits GBM cell growth via targeting high-mobility group box 1 (HMGB1). In addition, miR-142-3p/HMGB1 axis affects GBM cell immune escape through modulation of programmed death-1/programmed death ligand-1 (PD-1/PD-L1) checkpoint. Our study demonstrated that exosomal miR-142-3p from M1-polarized macrophages suppresses cell growth and immune escape in GBM through regulating HMGB1-mediated PD-1/PD-L1 checkpoint.

Cytosolic Hmgb1 accumulation in mesangial cells aggravates diabetic kidney disease progression via NFκB signaling pathway.

Diabetic kidney disease (DKD) is the predominant type of end-stage renal disease. Increasing evidence suggests thatglomerular mesangial cell (MC) inflammation is pivotal for cell proliferation and DKD progression. However, the exactmechanism of MC inflammation remains largely unknown. This study aims to elucidate the role of inflammatoryfactor high-mobility group box 1 (Hmgb1) in DKD. Inflammatory factors related to DKD progression are screened viaRNA sequencing (RNA-seq). In vivo and in vitro experiments, including db/db diabetic mice model, CCK-8 assay, EdUassay, flow cytometric analysis, Co-IP, FISH, qRT-PCR, western blot, single cell nuclear RNA sequencing (snRNA-seq),are performed to investigate the effects of Hmgb1 on the inflammatory behavior of MCs in DKD. Here, wedemonstrate that Hmgb1 is significantly upregulated in renal tissues of DKD mice and mesangial cells cultured withhigh glucose, and Hmgb1 cytopasmic accumulation promotes MC inflammation and proliferation. Mechanistically,Hmgb1 cytopasmic accumulation is two-way regulated by MC-specific cyto-lncRNA E130307A14Rik interaction andlactate-mediated acetylated and lactylated Hmgb1 nucleocytoplasmic translocation, and accelerates NFκB signalingpathway activation via directly binding to IκBα. Together, this work reveals the promoting role of Hmgb1 on MCinflammation and proliferation in DKD and helps expound the regulation of Hmgb1 cytopasmic accumulation in twoways. In particular, Hmgb1 may be a promising therapeutic target for DKD.

Direct reinfusion of pericardial blood complications: A case of acute respiratory distress syndrome and disseminated intravascular coagulation.

Background: Direct reinfusion of pericardial blood during cardiac surgery triggers a systemic inflammatory response. Although various inflammatory mediators have been identified as triggers, the role of damage-associated molecular patterns (DAMPs) remains poorly understood. Despite guidelines recommending against this practice owing to its harmful effects, it is sometimes used in emergencies. Case Presentation: A 72-year-old man with atrial fibrillation and cerebral infarction developed cardiac tamponade during catheter ablation. He underwent pericardial drainage and direct blood reinfusion. He was transferred to our ICU, where he developed acute respiratory distress syndrome (ARDS) and disseminated intravascular coagulation (DIC). Despite aggressive management, the patient died 41 days after admission. Conclusion: This case highlights severe adverse events following direct reinfusion of pericardial blood. These findings suggest a significant role for DAMPs in mediating these inflammatory responses. Direct reinfusion of pericardial drainage blood should be avoided during emergencies to prevent life-threatening complications.

IFN-γ induces acute graft-versus-host disease by promoting HMGB1-mediated nuclear-to-cytoplasm translocation and autophagic degradation of p53.

Acute graft-versus-host disease (aGVHD) poses a significant impediment to achieving a more favourable therapeutic outcome in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our prior investigations disclosed a correlation between p53 downregulation in CD4+ T cells and the occurrence of aGVHD. Notably, the insufficiency of the CCCTC-binding factor (CTCF) emerged as a pivotal factor in repressing p53 expression. However, the existence of additional mechanisms contributing to the reduction in p53 expression remains unclear. Interferon (IFN)-γ, a pivotal proinflammatory cytokine, assumes a crucial role in regulating alloreactive T cell responses and plays a complex part in aGVHD development. IFN-γ has the capacity to induce autophagy, a vital catabolic process facilitating protein degradation, in various cell types. Presently, whether IFN-γ participates in the development of aGVHD by instigating the autophagic degradation of p53 in CD4+ T cells remains an unresolved question. In this study, we demonstrated that heightened levels of IFN-γ in the plasma during aGVHD promoted the activation, proliferation, and autophagic activity of CD4+ T cells. Furthermore, IFN-γ induced the nuclear-to-cytoplasm translocation and autophagy-dependent degradation of p53 in CD4+ T cells. The translocation and autophagic degradation of p53 were contingent upon HMGB1, which underwent upregulation and translocation from the nucleus to the cytoplasm following IFN-γ stimulation. In conclusion, our data unveil a novel mechanism underlying p53 deficiency in CD4+ T cells among aGVHD patients. This deficiency is induced by IFN-γ and relies on autophagy, establishing a link between IFN-γ, HMGB1-mediated translocation, and the autophagic degradation of p53.

High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury.

Astrocytes act as immune cells that can produce a series of chemokines to attract large numbers of leucocytes to the lesion site, where they contribute to excessive inflammation following spinal cord injury (SCI). However, the relevant regulatory mechanism involved in chemokine production by astrocytes has not been fully elucidated. In the present study, we examined the correlation between C-C motif chemokine ligand 5 (CCL5) and high mobility group box-1 protein (HMGB1) in a T8-T10 spinal cord contusion model. Our results revealed that SCI-induced CCL5 protein levels increased synchronously with the increase in HMGB1. Administration of an HMGB1-neutralizing antibody significantly reduced the protein expression of CCL5 in the context of SCI. An in vitro study revealed that HMGB1 binding with TLR2/4 receptors potently facilitates the production of CCL5 by astrocytes by activating the intracellular ERK/JNK-mediated NF-κB pathway. Furthermore, the HMGB1-induced release of CCL5 from astrocytes is involved in promoting microglia/macrophage accumulation and M1 polarization. The inhibition of HMGB1 activity reduces microglia/macrophage infiltration by decreasing the expression of CCL5 and improves motor functional recovery following SCI. Our results provide insights into the new functions of HMGB1-mediated astrocytic CCL5 production, which elicits inflammatory cell recruitment to the site of injury; this recruitment is associated with excessive inflammation activation. These data may provide a new therapeutic strategy for central nervous system (CNS) inflammation.

Oleuropein Relieves Pancreatic Ischemia Reperfusion Injury in Rats by Suppressing Inflammation and Oxidative Stress through HMGB1/NF-κB Pathway.

Oleuropein (OLP) is a naturally occurring phenolic compound in olive plant with antioxidant and anti-inflammatory potential and can possibly be used in treating pancreatic injuries. This investigation aimed to follow the molecular mechanism behind the potential therapeutic effect of OLP against pancreatic injury persuaded by ischemia-reperfusion (I/R). Pancreatic I/R injury was induced by splenic artery occlusion for 60 min followed by reperfusion. Oral administration of OLP (10 and 20 mg/kg) for 2 days significantly alleviated I/R-persuaded oxidative damage and inflammatory responses in pancreatic tissue as indicated by the decreased malondialdehyde (MDA) content and increased glutathione peroxidase (GPx) activity, accompanied by the suppression of myeloperoxidase (MPO) activity and reduced levels of interleukin-1beta (IL-1ß), nuclear factor kappa B (NF-κB), and tumor necrosis factor alpha (TNF-α) in pancreatic tissues. Furthermore, OLP treatment markedly restored the serum levels of amylase, trypsinogen-activated peptide (TAP), and lipase, with concurrent improvement in pancreatic histopathological alterations. Moreover, treatment with OLP regulated the pancreatic expression of inducible nitric oxide synthase (iNOS) and high-mobility group box 1 (HMGB1) relative to rats of the pancreatic IR group. Thus, OLP treatment significantly alleviates the I/R-induced pancreatic injury by inhibiting oxidative stress and inflammation in rats through downregulation of HMGB1 and its downstream NF-κB signaling pathway.

PARP1 inactivation increases regulatory T / Th17 cell proportion in intestinal inflammation. Role of HMGB1.

Inflammatory bowel diseases (IBD) are chronic relapsing disorders with increasing prevalence. Knowledge gaps still limit the possibility to develop more specific and effective therapies. Using a dextran sodium sulfate colitis mouse model, we found that inflammation increased the total number and altered the frequencies of leukocytes within colon mesenteric lymph nodes (cMLNs). Although the inflammation reduced the frequency of regulatory T (Treg) cells, their absolute numbers were increased. Increased frequency of colitogenic Th17 cells was also observed. Noteworthy, untreated mice lacking Poly(ADP-ribose)-Polimerase-1 functional gene (PARP-1KO) displayed higher frequency of Treg cells and lower percentage of Th17 cells in cMLNs. In colitic PARP-1KO mice the inflammation driven expansion of the Foxp3 Treg population was more pronounced than in WT mice. Conversely, colitis increased Th17 cells to a lower extent in PARP-1KO mice compared with WT mice, resulting in a more protective Treg/Th17 cell ratio. Consequently PARP-1KO mice developed less severe colitis with reduced expression of inflammatory cytokines. In ex vivo experiments PARP-1KO and WT CD11c dendritic cells (DCs) promoted naïve CD4 T cell differentiation differently, the former sustaining more efficiently the generation of Treg cells, the latter that of Th17 cells. Addition of HMGB1 B box or of dipotassium glycyrrhizate, which sequesters extracellular HMGB1, revealed a role for this alarmin in the regulation exerted by PARP-1 on the stimulating vs. tolerogenic function of DCs during colitis. Moreover, a higher percentage of CD11c DC from PARP-1KO mice expressed CD103, a marker associated with the ability of DC to induce Treg cells, compared with WT DC. Conversely, PARP-1KO DC were including a reduced percentage of CX3CR1+ DC, described to induce Th17 cells. These findings were observed in both splenic and colon lamina propria DC.

Sphk1 regulates HMGB1 via HDAC4 and mediates epithelial pyroptosis in allergic rhinitis.

Background: Allergic rhinitis (AR) is a global health issue affecting millions of individuals worldwide. Pyroptosis has emerged as a major player in the development of AR, and targeting its inhibition with specific drugs holds promise for AR treatment. However, a comprehensive understanding of the precise mechanisms underlying pyroptosis in AR remains to be explored, warranting further investigation. Objective: This study aims to elucidate the roles of HMGB1, Sphk1, and HDAC4 in regulating human nasal epithelial cell (hNEC) pyroptosis and AR. Methods: An in vitro AR cell culture model and an in vivo AR mouse model were established. Western blot, ELISA, histological staining, and flow cytometry were utilized to confirm the gene and protein expression. The interactions among Sphk1, HDAC4, and HMGB1 were validated through ChIP, Co-IP, and Dual-luciferase assay. Results and conclusion: We identified that the expression levels of Sphk1, HMGB1, and inflammasome components, including IL-18, and IL-1ß were elevated in AR patients and mouse models. Knockdown of Sphk1 inhibited hNEC pyroptosis induced by dust mite allergen. Overexpression of HDAC4 suppressed HMGB1-mediated pyroptosis in hNECs. In addition, HDAC4 was found to mediate the transcriptional regulation of HMGB1 via MEF2C, a transcription factor. Additionally, Sphk1 was shown to interact with CaMKII-δ, promoting the phosphorylation of HDAC4 and inhibiting its cytoplasmic translocation. Knockdown of HDAC4 reversed the effect of Sphk1 knockdown on pyroptosis. These discoveries offer a glimpse into the molecular mechanisms underlying AR and suggest potential therapeutic targets for the treatment of this condition.

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.

Remimazolam ameliorates postoperative cognitive dysfunction after deep hypothermic circulatory arrest through HMGB1-TLR4-NF-κB pathway.

BACKGROUND: Postoperative cognitive dysfunction (POCD) is a complication of deep hypothermic circulatory arrest (DHCA). Various amounts of neurologic dysfunctions have been shown after DHCA, which has often been attributed to systemic inflammatory response syndrome and cerebral ischemia/reperfusion injury. Remimazolam is one of the commonly used anesthetic drugs with protective actions against inflammatory diseases, such as sepsis and cerebral ischemia/reperfusion injury. Here, we determined the protective effect and potential mechanism of action of remimazolam against neuronal damage after DHCA. METHODS: A rat model of DHCA was established, and a gradient dosage of remimazolam was administered during cardiopulmonary bypass (CPB). The cognitive function of rats was evaluated by Morris water maze. Hematoxylin and eosin and TUNEL staining were performed to assess hippocampus tissue injury and neuronal apoptosis. Inflammatory cytokines concentration were analyzed by enzyme-linked immunosorbent assay. The protein expression was analyzed using automated electrophoresis western analysis and immunohistochemical analysis. RESULTS: The appropriate dosage of remimazolam reduced histologic injury, neuronal apoptosis, microglia activation, and secondary inflammatory cascades, as well as the downregulation of the expression of the HMGB1-TLR4-NF-κB pathway after DHCA, improved the memory and learning abilities in DHCA rats. Further, administration of a TLR4 antagonist TAK-242 had a similar effect to remimazolam, while the TLR4 agonist LPS attenuated the effect of remimazolam. CONCLUSIONS: Remimazolam could ameliorate POCD after DHCA through the HMGB1-TLR4-NF-κB signaling pathway.

The role of high mobility group box-1 on the development of diabetes complications: A plausible pharmacological target.

BACKGROUND: Diabetes mellitus has emerged as a pressing global concern, with a notable increase in recent years. Despite advancements in treatment, existing medications struggle to halt the progression of diabetes and its associated complications. Increasing evidence underscores inflammation as a significant driver in the onset of diabetes mellitus. Therefore, perspectives on new therapies must consider shifting focus from metabolic stress to inflammation. High mobility group box (HMGB-1), a nuclear protein regulating gene expression, gained attention as an endogenous danger signal capable of sparking inflammatory responses upon release into the extracellular environment in the late 1990s. PURPOSE: Given the parallels between inflammatory responses and type 2 diabetes (T2D) development, this review paper explores HMGB-1's potential involvement in onset and progression of diabetes complications. Specifically, we will review and update the understanding of HMGB-1 and its inflammatory pathways in insulin resistance, diabetic nephropathy, diabetic neuropathy, and diabetic retinopathy. CONCLUSIONS: HMGB-1 and its receptors i.e. receptor for advanced glycation end-products (RAGE) and toll-like receptors (TLRs) present promising targets for antidiabetic interventions. Ongoing and future projects in this realm hold promise for innovative approaches targeting HMGB-1-mediated inflammation to ameliorate diabetes and its complications.

Electroacupuncture inhibits TLR4/NF-κB signaling in the dorsal root ganglion of rats with spared nerve injury.

OBJECTIVE: Neuropathic pain can be provoked by high mobility group box 1 (HMGB1) activation of toll-like receptor (TLR)4/nuclear factor (NF)-κB signaling in the dorsal root ganglion (DRG). Electroacupuncture (EA) has been reported to effectively alleviate neuropathic pain with few side effects, but its precise mechanism of action remains unknown. The aim of this study was to explore whether 2 Hz EA stimulation suppresses TLR4/NF-κB signaling in the DRG following spared nerve injury (SNI) in a rat model. METHODS: In this experiment, SNI rats were given 2 Hz EA once every other day for a total of 21 days. Paw withdrawal threshold (PWT) was measured to assess SNI-induced mechanical hypersensitivity, and western blotting and immunofluorescence staining were used to determine the levels of pain-related signaling molecules and pro-inflammatory mediators in the DRG. RESULTS: SNI up-regulated HMGB1, TLR4, myeloid differentiation factor-88 adaptor protein (MyD88) and NF-κB p65 protein expression in the DRG. In addition, immunofluorescence staining demonstrated that SNI induced higher levels of TLR4 and MyD88 in the DRG. We also demonstrated co-localization of TLR4 and MyD88 with both calcitonin gene-related peptide (CGRP) and isolectin GS-IB4 in the DRG of SNI rats, respectively. Meanwhile, 2 Hz EA stimulation effectively reversed the elevations of HMGB1, TLR4, MyD88 and NF-κB p65 induced by SNI in the DRG, which was coupled with amelioration of SNI-induced mechanical hypersensitivity. CONCLUSIONS: The results of this study suggested that inhibition of the TLR4/NF-κB signaling pathway in the DRG by 2 Hz EA might be exploited as a therapeutic option for neuropathic pain.

Discovery and synthesis of novel glyrrhizin-analogs containing furanoylpiperazine and the activity against myocardial injury in sepsis.

The signaling pathway mediated by high mobility group protein B1 (HMGB1) plays a key role in myocardial injury during sepsis. Glyrrhizin (GL) is a natural product that inhibits HMGB1 biological activities through forming GL-HMGB1 complex; the research shows its aglycone (GA) is the main pharmacophore binding to HMGB1, while the glycosyl mainly altering its pharmacokinetic properties and enhances the stability of the complex. GL is often metabolized to GA in the gastrointestinal tract, which has a lower efficacy in the treatment of HMGB1-mediated diseases. To obtain the GL analogs with higher activity and better pharmacokinetic properties, 24 GL analogs were synthesized by simplification the glycosyl of GL. Among all the compounds, compound 11 with furanoylpiperazine was screened. The pharmacokinetics experiments showed that compound 11 is converted to 11a in vivo, and 11 serves as its prodrug. Compound 11a displayed a lower cytotoxicity to RAW264.7 cells and three types of cardiomyocyte lines, with IC50 > 800 µM. In the anti-inflammatory assay, 11a not only strongly inhibited NO production (IC50 5.73 µM), but also down-regulated the levels of HMGB1, IL-1ß and TNF-α in a dose-dependent manner; in the anti-oxidative stress assay, compound 11a reduced the level of ROS and increased the MMP in H9c2 cells. More importantly, in the myocardial injury model of septic mice, compound 11a not only alleviated the symptom of myocardial injury by reducing inflammatory infiltration and oxidative stress, but also improved the myocardial blood supply by shrinking the inner diameter of the left ventricle and increasing the ejection fraction (EF) more dramatically (155.8 %); meanwhile, compound 11a adjusted myocardial enzymes in serum of septic mice. In addition, in molecular docking experiments, compound 11a showed stronger HMGB1 binding ability than GL. In summary, compound 11 is a prodrug, which can be converted to 11a in vivo. And compound 11a has a good activity against septic myocardial injury, as well as improving the myocardial blood supply function. This suggests compound 11 is a potential drug candidate for the treatment of septic myocardial injury and deserves further investigate.

Plasmodium berghei HMGB1 controls the host immune responses and splenic clearance by regulating the expression of pir genes.

High mobility group box (HMGB) proteins belong to high mobility group (HMG) superfamily of non-histone nuclear proteins that are involved in chromatin remodeling, regulation of gene expression and DNA repair. When extracellular, HMGBs serve as alarmins inducing inflammation and this is attributed to the proinflammatory activity of box B. Here, we show that Plasmodium HMGB1 has key amino acid changes in box B resulting in the loss of TNF-α stimulatory activity. Site-directed mutagenesis of the critical amino acids in box B with respect to mouse HMGB1 renders recombinant Plasmodium berghei (Pb) HMGB1 capable of inducing TNF-α release. Targeted deletion of PbHMGB1 and a detailed in vivo phenotyping show that PbHMGB1 knockout (KO) parasites can undergo asexual stage development. Interestingly, Balb/c mice-infected with PbHMGB1KO parasites display a protective phenotype with subsequent clearance of blood parasitemia, and develop long-lasting protective immunity against the challenges performed with Pb wildtype parasites. The characterization of splenic responses show prominent germinal centres leading to effective humoral responses and enhanced T follicular helper cells. There is also a complete protection from experimental cerebral malaria in CBA/CaJ mice susceptible for cerebral pathogenesis with subsequent parasite clearance. Transcriptomic studies suggest the involvement of PbHMGB1 in pir expression. Our findings highlight the gene regulatory function of parasite HMGB1 and its in vivo significance in modulating the host immune responses. Further, clearance of asexual stages in PbHMGB1KO-infected mice underscores the important role of parasite HMGB1 in host immune evasion. These findings have implications in developing attenuated blood-stage vaccine for malaria.

Glucocorticoids induce HMGB1 release in primary cultured rat cortical microglia.

Stress, a risk factor for major depressive disorder and Alzheimer disease, leads to the release of high-mobility group box-1 (HMGB1) protein, which in turn causes neuroinflammation. The mechanism underlying stress-induced HMGB1 release is unknown, but stress-associated glucocorticoids could be involved. Primary cultured rat cortical microglia and neurons were treated with corticosterone, a stress-associated glucocorticoid, and HMGB1 release was measured by ELISA and western blotting to test this hypothesis. With corticosterone treatment, significant HMGB1 was released in microglia but not in neuronal cell cultures. HMGB1 mRNA expression and HMGB1 protein expression in microglia were not affected by corticosterone treatment. Thus, the source of extracellular HMGB1 released into the medium is likely to be existing nuclear HMGB1 rather than newly synthesized HMGB1. Corticosterone-induced HMGB1 release in microglia culture was significantly attenuated by blocking glucocorticoid receptors but not mineralocorticoid receptors. Dexamethasone, a selective glucocorticoid receptor agonist, and dexamethasone-bovine serum albumin (BSA), a membrane-impermeable glucocorticoid receptor agonist used to confirm the membrane receptor-mediated effects of glucocorticoids, increased the release of HMGB1. Immunocytochemistry showed that HMGB1 translocated from the nucleus to the cytoplasm following dexamethasone or dexamethasone-BSA treatment through glucocorticoid receptors. The present findings suggest that glucocorticoids stimulate microglial membrane glucocorticoid receptors and trigger cytoplasmic translocation and extracellular release of nuclear HMGB1. Thus, under stress conditions, glucocorticoids induce microglial HMGB1 release, leading to a neuroinflammatory state that could mediate neurological disorders.