High Mobility Group Box 1 mRNA Expression Research Articles

HMGB1 induces macrophage pyroptosis in chronic endometritis

BackgroundChronic endometritis (CE) reflects the local imbalance in the endometrial immune microenvironment after inflammation. High mobility group box 1 (HMGB1) is highly involved in both immunity and inflammation. In this study, we aimed to explore the roles of HMGB1 in the endometrium of patients with CE. MethodsEndometrium and uterine fluid HMGB1 were tested in a cohort of infertile patients with or without CE. Expression levels of the pyroptosis marker, gasdermin D (GSDMD)-N-terminal (NT), in the human endometrium of patients with CE and controls were determined. Next, the role of HMGB1 as a driver of macrophage pyroptosis was investigated using human THP-1 cells in vitro and a CE mouse model in vivo. ResultsHigh expression levels of HMGB1 in biopsied endometrial tissue and uterine fluid were confirmed in a cohort of patients with CE. Positive correlation between the number of CD138+ cells and HMGB1 mRNA expression level were detected (rs = 0.592, P < 0.001). Meanwhile, we found that GSDMD-NT expression was significantly increased in the CE endometrium at both the transcriptional and translational levels. Moreover, co-localization of GSDMD-NT and macrophages was confirmed via the double immunostaining of GSDMD-NT and CD68. In vitro experiments revealed that macrophage pyroptosis was induced by HMGB1 in human THP-1-derived macrophages. Treatment with glycyrrhizic acid, an inhibitor of HMGB1, significantly suppressed endometrial pyroptosis and inflammation in the CE mouse model. ConclusionsHMGB1 effectively induced macrophage pyroptosis in the human endometrium, suggesting that its inhibition may serve as a novel treatment option for CE.

Effect of Vitamin D on the HMGB1/RAGE Pathway and Adipokines Levels in Obese Asthmatic Mice.

Compared to common asthma, obese asthma is difficult to control. Previous studies have shown that vitamin D (Vit D) has a therapeutic effect on asthma. Nevertheless, the action mechanism of Vit D for obese asthma are not well known. In this study, we, therefore, induced obesity and established an obese asthma mouse model using ovalbumin (OVA) stimulation and applied treatment with Vit D (100 ng/kg). Accordingly, thirty mice were randomly divided into 5 equal groups of normal control, asthma, obese asthma, asthma+Vit D, and obese asthma+ Vit D. The levels of inflammatory factors and adipokines were measured by the ELISA assay; then the quantitative reverse transcription PCR (qRT-PCR) method was used to evaluate the expression of high mobility group box 1(HMGB1) and receptor for advanced glycation end products [RAGE] genes.T he results showed that OVA sensitization significantly increased airway resistance, the levels of inflammatory cytokines, and HMGB and RAGE expression in asthmatic and obese asthmatic mice, as compared to the control group. Also, these changes in the obese asthmatic group were notably higher than those in the asthmatic one. In addition, the treatment of asthmatic and obese asthmatic mice with Vit D significantly reduced the raw, serum and BALF levels of inflammatory cytokines, as well as the expression of HMGB1 and RAGE mRNA. To conclude, the present study showed that vitamin D might attenuate lung injury by up-regulating HMGB1 and RAGE expression. Our findings, thus, may offer new concepts and approaches for the treatment and prevention of obese asthma.

Effect of acupuncture at "Houxi" (SI3) and"Huantiao" (GB30) on high mobility group box 1 protein in spinal nerve trunk of rats with lumbar disc herniation

To observe the effect of acupuncture at "Houxi"(SI3) and "Huantiao"(GB30) on high mobility group box 1(HMGB1) protein and mRNA in spinal nerve trunk(SNT) of rats with lumbar disc herniation(LDH), so as to explore the mechanisms of acupuncture at this paired points on the treatment for LDH. SD rats were randomly divided into sham operation, model, conventional acupuncture(CA) and paired points(PP) groups (with 8 rats in each group). The LDH model was established by injection of autologous suspension made from rats' own nucleus pulsus into the epidural space. Rats in the CA group received acupuncture treatment at bilateral "Weizhong"(BL40), "Dachangshu"(BL25) and "Shenshu"(BL23), while rats in the PP group received acupuncture at bilateral SI3 and GB30, 30 min each time, once daily for 14 consecutive days. The thermal pain threshold of bilateral hind feet of rats was detected by thermal pain stimulator. The contents of serum IL-1β, IL-6 and IL-8 of rats were detected by ELISA. Western blot and immunofluorescence were used to detect the expression of HMGB1 protein in the lumbar(L)5 SNT of rats. The relative expression of HMGB1 mRNA in L5 SNT was determined by qPCR. HE staining was used to observe the morphological changes of L5 SNT. Compared with the sham operation group, the thermal pain threshold of bilateral hind feet in the model group was decreased (P<0.05); compared with the model group, the thermal pain threshold of bilateral hind feet in the CA group and the PP group were increased (P<0.05). The expressions of HMGB1 protein and mRNA in L5 SNT, and the contents of serum IL-1β, IL-6 and IL-8 of rats in the model group were significantly increased(P<0.000 1, P<0.001) in contrast to the sham operation group. The expressions of HMGB1 protein and mRNA in L5 SNT, and the levels of serum IL-1β, IL-6 and IL-8 were significantly decreased (P<0.01, P<0.000 1, P<0.001, P<0.05) in the CA and PP group, in comparison with those of the model group. Compared with the CA group, the above indexes of rats in the PP group recovered more significantly (P<0.05,P<0.001, P<0.01,P<0.000 1). The histomorphological results showed scattered and various-sized nerve fibers, vacuolation, a large number of disintegrating myelin sheath and lysed Schwann cells in the model group. Myelin sheaths regeneration, regularly-arranged nerve fibers were seen in the CA group and the PP group, with more obvious histopathological recovery observed in the PP group than the CA group. Acupuncture intervention inhibites the expressions of HMGB1 protein and mRNA in rats with LDH, and further reduces the production of IL-1β, IL-6 and IL-8, which is beneficial to inflammatory response inhibition and pain alleviation. The therapeutic effect of the PP group is more obvious than that of the CA group.

Compound Tinglizi Decoction intervenes COPD-associated pulmonary hypertension through regulation of HMGB1-mediated pyroptosis and immune imbalance

This paper aimed to investigate the effects of high mobility group box 1(HMGB1)-mediated pulmonary artery smooth muscle cell pyroptosis and immune imbalance on chronic obstructive pulmonary disease-associated pulmonary hypertension(COPD-PH) in rats and the intervening mechanism of Compound Tinglizi Decoction. Ninety rats were randomly divided into a normal group, a model group, low-dose, medium-dose, and high-dose Compound Tinglizi Decoction groups, and a simvastatin group. The rat model of COPD-PH was established by fumigation combined with lipopolysaccharide(LPS) intravascular infusion, which lasted 60 days. Rats in the low, medium, and high-dose Compound Tinglizi Decoction groups were given 4.93, 9.87, and 19.74 g·kg~(-1) Compound Tinglizi Decoction by gavage, respectively. Rats in the simvastatin group were given 1.50 mg·kg~(-1) simvastatin by gavage. After 14 days, the lung function, mean pulmonary artery pressure, and arterial blood gas of rats were analyzed. Lung tissues of rats were collected for hematoxylin-eosin(HE) staining to observe the pathological changes. Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR) was used to determine the expression of related mRNA in lung tissues, Western blot(WB) was used to determine the expression of related proteins in lung tissues, and enzyme linked immunosorbent assay(ELISA) was used to determine the levels of inflammatory factors in the lung tissues of rats. The ultrastructure of lung cells was observed by transmission electron microscope. The forced vital capacity(FVC), forced expiratory volume in 0.3 second(FEV_(0.3)), FEV_(0.3)/FVC, peek expiratory flow(PEF), respiratory dynamic compliance(Cdyn), arterial partial pressure of oxygen(PaO_2), and arterial oxygen saturation(SaO_2) were increased, and resistance of expiration(Re), mean pulmonary arterial pressure(mPAP), right ventricular hypertrophy index(RVHI), and arterial partial pressure of carbon dioxide(PaCO_2) were decreased by Compound Tinglizi Decoction in rats with COPD-PH. Compound Tinglizi Decoction inhibited the protein expression of HMGB1, receptor for advanced glycation end products(RAGE), pro caspase-8, cleaved caspase-8, and gasdermin D(GSDMD) in lung tissues of rats with COPD-PH, as well as the mRNA expression of HMGB1, RAGE, and caspase-8. Pulmonary artery smooth muscle cell pyroptosis was inhibited by Compound Tinglizi Decoction. Interferon-γ(IFN-γ) and interleukin-17(IL-17) were reduced, and interleukin-4(IL-4) and interleukin-10(IL-10) were incresead by Compound Tinglizi Decoction in lung tissues of rats with COPD-PH. In addition, the lesion degree of trachea, alveoli, and pulmonary artery in lung tissues of rats with COPD-PH was improved by Compound Tinglizi Decoction. Compound Tinglizi Decoction had dose-dependent effects. The lung function, pulmonary artery pressure, arterial blood gas, inflammation, trachea, alveoli, and pulmonary artery disease have been improved by Compound Tinglizi Decoction, and its mechanism is related to HMGB1-mediated pulmonary artery smooth muscle cell pyroptosis and helper T cell 1(Th1)/helper T cell 2(Th2), helper T cell 17(Th17)/regulatory T cell(Treg) imbalance.

The effects of etomidate on expression of high mobility group box 1 via the nuclear factor kappa B pathway in rat model of sepsis

ABSTRACT Etomidate is an anesthetic agent used in hemodynamically unstable patients, but its use has been controversial in septic patients. The response of high-mobility group box 1 (HMGB1), a late-phase lethal cytokine in sepsis, to etomidate has not been reported. This study investigated the effects of etomidate on the expression and release of HMGB1 and the underlying mechanism using a cecal ligation and puncture (CLP) model. Thirty-six male Sprague–Dawley rats were divided into sham, CLP, and Etomi groups. Sepsis was induced in the CLP and Etomi groups, and intravenous etomidate (4 mg/kg) was infused for 40 min immediately after operation in the Etomi group. Serum creatinine, alanine aminotransferase (ALT), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and HMGB1 levels were measured 6 and 24 hours after surgery. Activation of nuclear factor (NF)-ĸB and HMGB1 mRNA expression in the liver, lung, kidney, and ileum tissues were measured, and immunohistochemical staining of HMGB1 was implemented. Increases of the TNF-α level 6 h after CLP and ALT and IL-6 levels 24 h after CLP were significantly inhibited by etomidate treatment. Etomidate treatment also significantly attenuated the increase in serum HMGB1 level at 6 and 24 h after CLP and suppressed the NF-ĸB and HMGB1 mRNA in multiple organs 24 h after CLP. Immunohistochemical staining also revealed that etomidate treatment inhibited HMGB1 expression. Etomidate inhibited the systemic release of HMGB1 and its expression in various organs. The mechanism may be associated with the inhibitory effects of etomidate on pro-inflammatory cytokine release and NF-ĸB activity.

CircRTN1 stimulates HMGB1 to regulate the malignant progression of papillary thyroid cancer by sponging miR-101-3p.

The important role played by circular RNA (circRNA) in promoting the progression of papillary thyroid cancer (PTC) is attracting ever more attention among medical researchers. However, what the precise contribution is of circRTN1 in PTC progression remains unclear. The study was designed to analyze the role and mechanism of circRTN1 in regulating PTC progression. Human PTC cell lines (TPC-1 and IHH-4) and human thyroid normal cells (Nthy-ori 3-1) were used for in vitro assays. mRNA or protein expression of circRTN1, miR-101-3p, and high mobility group box 1 (HMGB1) were detected by quantitative real-time polymerase chain reaction or western blot. Cell proliferation was investigated by cell counting kit-8 assay, cell colony formation assay, and 5-ethynyl-2'-deoxyuridine assay. Wound-healing assay and transwell invasion assay were conducted to evaluate cell migration and invasion. Dual-luciferase reporter assay and RNA immunoprecipitation assay were applied to verify the target relations between circRTN1, miR-101-3p, and HMGB1. A xenograft tumor model was established to demonstrate the effect of circRTN1 on tumor formation in vivo. An immunohistochemistry assay was used to detect protein expression of HMGB1, ki-67, E-cadherin, and vimentin. In comparison with healthy thyroid tissues and cells, PTC tissues and cells displayed high circRTN1 RNA expression and high HMGB1 mRNA and protein expression but low miR-101-3p expression. Silencing of circRTN1 suppressed PTC cell proliferation, migration, and invasion in vitro. MiR-101-3p was a target of circRTN1, and the knockdown of miR-101-3p relieved circRTN1 absence-mediated suppressive effects on PTC cell malignancy. HMGB1 was identified as a target gene of miR-101-3p, and overexpressed HMGB1 almost reverted the inhibitory impacts induced by miR-101-3p mimic in PTC cells. Moreover, circRTN1 silencing hampered tumor formation in vivo. CircRTN1 depletion impeded PTC cell malignancy via the miR-101-3p/HMGB1 pathway, which provided a possible circRNA-targeted therapeutic strategy for PTC.

CXCL12/CXCR4 Axis is Involved in the Recruitment of NK Cells by HMGB1 Contributing to Persistent Airway Inflammation and AHR During the Late Stage of RSV Infection.

We previously showed that both high-mobility group box-1 (HMGB1) and natural killer (NK) cells contribute to respiratory syncytial virus (RSV)-induced persistent airway inflammation and airway hyperresponsiveness (AHR). Meanwhile, Chemokine (C-X-C motif) ligand 12 (CXCL12) and its specific receptor (chemokine receptor 4, CXCR4) play important roles in recruitment of immune cells. CXCL12 has been reported to form a complex with HMGB1 that binds to CXCR4 and increases inflammatory cell migration. The relationship between HMGB1, NK cells and chemokines in RSV-infected model remains unclear. An anti-HMGB1 neutralizing antibody and inhibitor of CXCR4 (AMD3100) was administered to observe changes of NK cells and airway disorders in nude mice and BALB/c mice. Results showed that the mRNA expression and protein levels of HMGB1 were elevated in late stage of RSV infection and persistent airway inflammation and AHR were diminished after administration of anti-HMGB1 antibodies, with an associated significant decrease in CXCR4+ NK cells. In addition, CXCL12 and CXCR4 were reduced after HMGB1 blockade. Treatment with AMD3100 significantly suppressed the recruitment of NK cells and alleviated the airway disorders. Thus, CXCL12/CXCR4 axis is involved in the recruitment of NK cells by HMGB1, contributing to persistent airway inflammation and AHR during the late stage of RSV infection.

Glycyrrhizin ameliorates vascular endothelial cell senescence by inhibiting HMGB1 in HFD/STZ-induced diabetic rats and human umbilical vein endothelial cells

The senescence and dysfunction of vascular endothelial cells are important features of diabetic vascular disease. High mobility group box-1(HMGB1) may be involved in vascular injury in response to high glucose. Glycyrrhizin (GL) is an HMGB1 inhibitor that significantly reduces HMGB1. However, the relationship between HMGB1 and vascular ageing in diabetes is not clear, the protective mechanism of GL against vascular injury in type 2 diabetes mellitus (T2DM) is unclear too. This study aims to examine the role of HMGB1 in vascular endothelial cell senescence and the protective effects of GL on vascular aging in high fat diet/streptozotocin (HFD/STZ) induce type 2 diabetic rats.After induction of diabetes, GL (150 mg/kg/d) was treated by gavage for 4 weeks. Results showed that compared with the Control group, the serum level of HMGB1 was increased in rats with type 2 diabetes, while the expression of HMGB1 mRNA and protein in the thoracic aorta was upregulated, with a decrease in endothelium-dependent vasodilation function and an increase in aging degree in the thoracic aorta. However, the above indicators were significantly improved after GL treatment. In HUVECs, we found that treated with HMGB1 (50, 100 and 200 ng/ml) for 48 h induced cells senescence and GL (50, 100 mg/L) significantly inhibited high-glucose-induced endothelial cell senescence, meanwhile GL (50, 100 mg/L) significantly inhibited the high-glucose-induced HMGB1 release and upregulated p53 expression. In conclusion, GL as an HMGB1 inhibitor, attenuates endothelium-dependent relaxation impairment and vascular ageing in an animal model of diabetes and high-glucose-induced endothelial cell senescence.

LncRNA OIP5-AS1 promotes the proliferation and migration of vascular smooth muscle cells via regulating miR-141-3p/HMGB1 pathway

BackgroundLong non-coding RNAs (lncRNAs) have been reported to play critical roles in the pathogenesis of cardiovascular diseases. However, whether lncRNA opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) regulates the pathogenesis of atherosclerosis (AS) is still unknown. MethodsHuman vascular smooth muscle cells (VSMCs) were treated with oxidized low-density lipoprotein (ox-LDL). OIP5-AS1, miR-141-3p and HMGB1 mRNA expressions were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation, migration, and apoptosis of VSMCs were measured using MTT method, Transwell assay and TUNEL assay, respectively. Dual-luciferase reporter gene assay, qRT-PCR, and Western blot were conducted to investigate the interactions among OIP5-AS1, miR-141-3p and high mobility group box 1 (HMGB1). ResultsOIP5-AS1 expression was markedly increased in serum samples of AS patients and VSMCs treated with ox-LDL. OIP5-AS1 over-expression remarkably promoted proliferation, migration and inhibited apoptosis of VSMCs while miR-141-3p exerted the opposite effects. Furthermore, the binding sites between OIP5-AS1 and miR-141-3p were identified. OIP5-AS1 indirectly increased HMGB1 expression in VSMCs by targeting miR-141-3p. ConclusionsOIP5-AS1 promotes the proliferation, migration and suppresses apoptosis of VSMCs through regulating miR-141-3p/HMGB1 axis.

Soluble receptor for advanced glycation end products protects from ischemia- and reperfusion-induced acute kidney injury.

ABSTRACTThe full-length receptor for advanced glycation end products (RAGE) is a multiligand pattern recognition receptor. High-mobility group box 1 (HMGB1) is a RAGE ligand of damage-associated molecular patterns that elicits inflammatory reactions. The shedded isoform of RAGE and endogenous secretory RAGE (esRAGE), a splice variant, are soluble isoforms (sRAGE) that act as organ-protective decoys. However, the pathophysiologic roles of RAGE/sRAGE in acute kidney injury (AKI) remain unclear. We found that AKI was more severe, with enhanced renal tubular damage, macrophage infiltration, and fibrosis, in mice lacking both RAGE and sRAGE than in wild-type (WT) control mice. Using murine tubular epithelial cells (TECs), we demonstrated that hypoxia upregulated messenger RNA (mRNA) expression of HMGB1 and tumor necrosis factor α (TNF-α), whereas RAGE and esRAGE expressions were paradoxically decreased. Moreover, the addition of recombinant sRAGE canceled hypoxia-induced inflammation and promoted cell viability in cultured TECs. sRAGE administration prevented renal tubular damage in models of ischemia/reperfusion-induced AKI and of anti-glomerular basement membrane (anti-GBM) glomerulonephritis. These results suggest that sRAGE is a novel therapeutic option for AKI.

Prunella vulgaris L. Attenuates Experimental Autoimmune Thyroiditis by Inhibiting HMGB1/TLR9 Signaling.

BackgroundPrunella vulgaris L. (PV) has been used to treat autoimmune thyroiditis (AIT), but the underlying mechanism remains unknown. The present study was designed to evaluate the effect of PV on AIT and explore the role of high-mobility group box-1 (HMGB1) signaling in PV-mediated effects in vivo and in vitro.MethodsIn the present study, bioactive components of PV were identified using UPLC-ESI-MS. The protective effects and potential mechanisms critical for the anti-inflammatory and immunomodulatory effects of PV in AIT were investigated in a rat model of thyroglobulin-induced experimental autoimmune thyroiditis (EAT) and in lipopolysaccharide (LPS)-induced thyroid follicular cells (TFCs).ResultsThe main bioactive compound identified in PV was rosmarinic acid. The thyroid volume, thyroiditis inflammation score and serum thyroglobulin antibody levels of EAT rats were attenuated by PV treatment (P<0.01). In addition, PV significantly reduced the elevated levels of the proinflammatory cytokines TNF-α, IL-6, IL-1β and monocyte chemoattractant protein-1 (MCP-1) both in vivo (P<0.01) and in vitro (P<0.05). PV downregulated HMGB1 mRNA and protein expression, reduced HMGB1 secretion, and inhibited TLR9 signaling pathways (TLR9 and MyD88) in PV-treated EAT rats and TFCs. Moreover, PV reversed the increases in the numbers of splenic Th1, Th2, and Th17 cells. Finally, our results acquired following administration of ethyl pyruvate, an HMGB1 inhibitor, to splenocytes cultured in vitro supported the hypothesis that the HMGB1/TLR9 pathway is involved in the PV-mediated reductions in Th1, Th2 and Th17 cells.ConclusionPV decreased the activity of the TLR9/MyD88 pathway and proinflammatory cytokines through HMGB1. In addition, we are the first to show that PV attenuated the HMGB1-induced increases in Th1, Th2 and Th17 cells in AIT models. These findings provide new evidence for the potential therapeutic value of PV as a treatment for AIT and other autoimmune diseases.

Aerobic exercise improves LPS-induced sepsis via regulating the Warburg effect in mice

We investigated the impact of aerobic exercise (AE) on multiple organ dysfunction syndrome (MODS), aortic injury, pathoglycemia, and death during sepsis. ICR mice were randomized into four groups: Control (Con), Lipopolysaccharide (LPS), Exercise (Ex), and Exercise + LPS (Ex + LPS) groups. Mice were trained with low-intensity for 4 weeks. LPS and Ex + LPS mice received 5 mg/kg LPS intraperitoneally for induction of sepsis. Histopathological micrographs showed the organ morphology and damage. This study examined the effects of AE on LPS-induced changes in systemic inflammation, pulmonary inflammation, lung permeability, and bronchoalveolar lavage fluid (BALF) cell count, oxidative stress-related indicators in the lung, blood glucose levels, plasma lactate levels, serum insulin levels, plasma high-mobility group box 1 (HMGB1) levels, glucose transporter 1 (Glut1) and HMGB1, silent information regulator 1 (Sirt-1), and nuclear factor erythroid 2-related factor 2 (Nrf-2) mRNA expression levels in lung tissue. AE improved sepsis-associated multiple organ dysfunction syndrome (MODS), aortic injury, hypoglycemia, and death. AE prominently decreased pulmonary inflammation, pulmonary edema, and modulated redox balance during sepsis. AE prominently decreased neutrophil content in organ. AE prominently downregulated CXCL-1, CXCL-8, IL-6, TNF-α, Glu1, and HMGB1 mRNA expression but activated IL-1RN, IL-10, Sirt-1, and Nrf-2 mRNA expression in the lung during sepsis. AE decreased the serum levels of lactate and HMGB1 but increased blood glucose levels and serum insulin levels during sepsis. A 4-week AE improves sepsis-associated MODS, aortic injury, pathoglycemia, and death. AE impairs LPS-induced lactate and HMGB1 release partly because AE increases serum insulin levels and decreases the levels of Glut1. AE is a novel therapeutic strategy for sepsis targeting aerobic glycolysis.

Gycyrrhizic acid alleviates atherosclerotic lesions in rats with diabetes mellitus.

Gycyrrhizic acid (GA), an inhibitor of high mobility group box 1 (HMGB1), inhibits inflammatory responses and is involved in the occurrence and development of several inflammation-related diseases. However, the role of GA in the atherosclerotic lesions caused by diabetes mellitus (DM) remains unknown. In the present study, Sprague Dawley rats were selected to desi=gn a diabetic atherosclerosis (AS) model. Rats from the DM-AS group were subsequently divided into DM-AS, DM-AS + GA (50 mg/kg) and DM-AS + GA (150 mg/kg) groups. Biochemical analyzers were used to measure levels of blood glucose, fasting insulin, total cholesterol, total triglyceride, low-density lipoprotein and high-density lipoprotein. The number of plaques was recorded after collection of thoracic aortas from the rats. The intimal thickness of arterial tissue was detected by hematoxylin and eosin staining. The expression levels of CD68 and α-smooth muscle actin (α-SMA) were detected by immunohistochemistry. The expression of tumor necrosis factor-α, interleukin (IL)-6 and IL-1β in the serum of the rats was detected by ELISA. The expression of fatty acid synthetase, sterol regulatory element binding protein 1C, HMGB1 and receptor for advanced glycation end products (RAGE) was detected by western blotting. Reverse transcription quantitative PCR was used to detect the mRNA expression of HMGB1 and RAGE. The results demonstrated that GA treatment could decrease the body weight, blood glucose level and biochemical parameters of AS DM rats in a dose-dependent manner. In addition, GA decreased the intimal thickness of carotid artery and the formation of plaque in rats with diabetic AS. Furthermore, GA inhibited macrophage activation and decreased α-SMA expression in vascular smooth muscle cells, and decreased the expression of proteins (FAS and SREBP-1c) and inflammatory factors. Taken together, the findings from the present study demonstrated that GA may have a therapeutic effect on DM-associated AS. This study provides a theoretical basis for the treatment of diabetic AS.

Naturally Occurring HMGB1 Inhibitor, Glycyrrhizin, Modulates Chronic Seizures-Induced Memory Dysfunction in Zebrafish Model.

Glycyrrhizin (GL) is a well-known pharmacological inhibitor of high mobility group box 1 (HMGB1) and is abundantly present in the licorice root (Glycyrrhiza radix). HMGB1 protein, a key mediator of neuroinflammation, has been implicated in several neurological disorders, including epilepsy. Epilepsy is a devastating neurological disorder with no effective disease-modifying treatment strategies yet, suggesting a pressing need for exploring novel therapeutic options. In the current investigation, using a second hit pentylenetetrazol (PTZ) induced chronic seizure model in adult zebrafish, regulated mRNA expression of HMGB1 was inhibited by pretreatment with GL (25, 50, and 100 mg/kg, ip). A molecular docking study suggests that GL establishes different binding interactions with the various amino acid chains of HMGB1 and Toll-like receptor-4 (TLR4). Our finding suggests that GL pretreatment reduces/suppresses second hit PTZ induced seizure, as shown by the reduction in the seizure score. GL also regulates the second hit PTZ induced behavioral impairment and rescued second hit PTZ related memory impairment as demonstrated by an increase in the inflection ratio (IR) at the 3 h and 24 h T-maze trial. GL inhibited seizure-induced neuronal activity as demonstrated by reduced C-fos mRNA expression. GL also modulated mRNA expression of BDNF, CREB-1, and NPY. The possible mechanism underlying the anticonvulsive effect of GL could be attributed to its anti-inflammatory activity, as demonstrated by the downregulated mRNA expression level of HMGB1, TLR4, NF-kB, and TNF-α. Overall, our finding suggests that GL exerts an anticonvulsive effect and ameliorates seizure-related memory disruption plausibly through regulating of the HMGB1-TLR4-NF-kB axis.

High Mobility Group Box 1 in Pig Amniotic Membrane Experimentally Infected with E. coli O55.

Intra-amniotic infections (IAI) are one of the reasons for preterm birth. High mobility group box 1 (HMGB1) is a nuclear protein with various physiological functions, including tissue healing. Its excessive extracellular release potentiates inflammatory reaction and can revert its action from beneficial to detrimental. We infected the amniotic fluid of a pig on the 80th day of gestation with 1 × 104 colony forming units (CFUs) of E. coli O55 for 10 h, and evaluated the appearance of HMGB1, receptor for glycation endproducts (RAGE), and Toll-like receptor (TLR) 4 in the amniotic membrane and fluid. Sham-infected amniotic fluid served as a control. The expression and release of HMGB1 were evaluated by Real-Time PCR, immunofluorescence, immunohistochemistry, and ELISA. The infection downregulated HMGB1 mRNA expression in the amniotic membrane, changed the distribution of HMGB1 protein in the amniotic membrane, and increased its level in amniotic fluid. All RAGE mRNA, protein expression in the amniotic membrane, and soluble RAGE level in the amniotic fluid were downregulated. TLR4 mRNA and protein expression and soluble TLR4 were all upregulated. HMGB1 is a potential target for therapy to suppress the exaggerated inflammatory response. This controlled expression and release can, in some cases, prevent the preterm birth of vulnerable infants. Studies on suitable animal models can contribute to the development of appropriate therapy.

β-catenin has potential effects on the expression, subcellular localization, and release of high mobility group box 1 during bovine herpesvirus 1 productive infection in MDBK cell culture

ABSTRACT High mobility group box 1 (HMGB1), a ubiquitous DNA-binding protein, can be released into extracellular space and function as a strong proinflammatory cytokine, which plays critical roles in the pathogenesis of various inflammatory diseases. Here, we showed that BoHV-1 productive infection in MDBK cells at later stage significantly increases HMGB1 mRNA expression and the protein release, but decreases the steady-state protein levels. Virus infection increases accumulation of HMGB1 protein in both nucleus and mitochondria, and relocalizes nuclear HMGB1 to assemble in highlighted foci via a confocal microscope assay. Interestingly, β-catenin-specific inhibitor iCRT14 is able to increase HMGB1 transcription and the protein release, and subcellular translocation in virus-infected cells. HMGB1-specific inhibitor, glycyrrhizin, could differentially affect virus gene transcription such as, the viral regulatory protein bICP0, bICP4 and bICP22, as well as glycoprotein gD. In summary, our data provides a novel mechanism that β-catenin signaling may regulate inflammatory response via affecting HMGB1 signaling.

Association of High-Mobility Group Box 1 (HMGB1) Gene Polymorphisms with Susceptibility and Better Survival Prognosis in Chinese Han Neonatal Necrotizing Enterocolitis.

BackgroundHigh-mobility group box 1 (HMGB1) plays a crucial role in a variety of diseases, including neonatal necrotizing enterocolitis (NEC). The purpose of this study was to investigate the association of HMGB1 gene single-nucleotide polymorphisms (SNPs) with susceptibility and survival prognosis in Chinese Han neonates with NEC.Material/MethodsThe HMGB1 gene rs1360485, rs1045411, and rs2249825 site SNPs were genotyped in all participants. The mRNA expression of serum HMGB1 was examined using quantitative reverse transcription-polymerase chain reaction. The correlation of the HMGB1 rs1360485 SNP with NEC neonatal survival prognosis was evaluated by univariate analysis and logistic multivariate regression analysis.ResultsThe TC and CC genotype and C allele distribution frequencies of the rs1360485 SNP were lower in the NEC group, and the differences were statistically significant (all P<0.05). Individuals carrying the TC and CC genotype or C allele had a low risk of being affected by NEC. However, the genotype and allele distributions of rs1045411 and rs2249825 were not significantly different between the patient and control groups (P>0.05). NEC neonates with HMGB1 gene rs1360485 site mutations had lower mRNA levels of serum HMGB1 than those with rs1360485 site wild-type, and the rs1360485 genotypes TC and CC could independently predict better survival outcomes in NEC neonates.ConclusionsThis study demonstrated that the rs1360485 SNP of the HMGB1 gene is associated with susceptibility of NEC in neonates, and the rs1360485 genotypes TC and CC may affect HMGB1 expression and are associated with the survival prognosis of neonates with NEC.

WITHDRAWN: Tanshinone-IIA inhibits the inflammatory response and proliferation of PAECs under hypoxic conditions by repressing HMGB1 via the TLR4/NF-κB signalling pathway

To explore the potential molecular mechanism of Tanshinone-IIA (Tan-IIA) in pulmonary artery endothelial cells (PAECs) under hypoxic conditions. Rat PAECs were observed under an inverted microscope, and the biomarkers CD31 and factor VIII related antigen (FVIII-Rag) were identified by immunofluorescence (IF). The PAECs were divided into a 20% normoxia group and a 4% hypoxia group, and the cells were subjected to Tan-IIA treatment, high mobility group box 1 (HMGB1) overexpression, or the relevant controls. ELISA and MTT were performed to evaluate proinflammatory factor production and cell proliferation, respectively. RT-qPCR was used to measure HMGB1 mRNA expression, and western blotting was performed to evaluate the protein levels of HMGB1, Toll-like receptor 4 (TLR4) and nuclear factor kappa-B (NF-κB)-p65. Immunocytochemistry (ICC) and western blotting indicated the subcellular distribution of NF-κB-p65. Rat PAECs were round or short spindle-shaped and abundantly expressed CD31 and FVIII-Rag. Compared with the normoxia group, the hypoxia group produced significantly increased levels of proinflammatory factors, which decreased with increasing concentrations of Tan-IIA (p<0.05). The expression of HMGB1 and TLR4 and the phosphorylation of NF-κB-p65 presented gradually decreasing trends in the 4% hypoxia group with increasing concentrations of Tan-IIA. Moreover, ICC indicated that in the 4% hypoxia group, NF-κB-p65 translocated to the nucleus (p<0.05), and western blotting analysis showed that the higher the Tan-IIA concentration was, the lower the NF-κBp65 level in the nucleus was. Compared with the Hypoxia + Vector group, the Hypoxia+ pHMGB1 group exhibited significantly enhanced effects of the hypoxia-induced inflammatory response (p<0.05), and the inhibitory effects of Tan-IIA on the hypoxia-induced inflammatory response were reversed in the Hypoxia+Tan-IIA+pHMGB1 group due to the overexpression of HMGB1 (p<0.05); HMGB1 exerted similar effects on cell proliferation, as illustrated by MTT assay. Tan-IIA inhibits the proliferation and inflammatory response of PAECs by downregulating the expression of HMGB1 via the TLR4/NF-κB signalling pathway under hypoxic conditions.

Long Non-Coding RNA PITPNA-AS1 Accelerates the Progression of Colorectal Cancer Through miR-129-5p/HMGB1 Axis.

BackgroundLong non-coding RNAs (lncRNAs) are essential regulators in colorectal cancer (CRC) progression. This work aimed to delve into the characteristics of lncRNA PITPNA-AS1 in CRC.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was adopted to examine PITPNA-AS1, miR-129-5p, high-mobility group box-1 (HMGB1) mRNA expressions in CRC tissues and cell lines. Functionally, cell counting kit-8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU) incorporation assays were employed to examine cell proliferation; wound healing assay was utilized to detect cell migration; and flow cytometry was used to detect the cell apoptosis. Luciferase reporter assay, RNA immunoprecipitation assay and Western blot were conducted to detect the regulatory relationships among PITPNA-AS1, miR-129-5p and HMGB1.ResultsPITPNA-AS1 and HMGB1 mRNA expressions were observably elevated in CRC tissues and cell lines. Knocking down PITPNA-AS1 could significantly inhibit cell proliferation and migration, and promote apoptosis of CRC cells. PITPNA-AS1 could serve as a competitive endogenous RNA, and up-regulate HMGB1 expression by adsorbing miR-129-5p.ConclusionPITPNA-AS1 can expedite CRC cell proliferation and migration, and inhibit apoptosis through miR-129-5p/HMGB1 axis.

MiR-708 affords protective efficacy in anoxia/reoxygenation-stimulated cardiomyocytes by blocking the TLR4 signaling via targeting HMGB1

Ischemic heart disease is a proverbial and common cardiovascular disease, and constitutes a leading cause of disability and mortality globally. Myocardial ischemic/reperfusion (MI/R) injury is a highly orchestrated phenomenon that involves the excessive activation of high mobility group box 1 (HMGB1) signaling. In the present study, we sought to investigate the function of miR-708 in MI/R injury due to the predicted binding to HMGB1. Intriguingly, down-regulation of miR-708 and up-regulation of HMGB1 were observed in MI/R rat model and H9c2 cardiomyocytes exposed to hypoxia/reoxygenation (H/R) conditions. Dual luciferase reporter assays substantiated that HMGB1 was a direct target of miR-708. Moreover, miR-708 overexpression suppressed the mRNA and protein expression of HMGB1. Noticeably, elevation of miR-708 antagonized H/R-induced inhibition in cell viability; whilst, increased cell apoptosis evoked by H/R was restrained after miR-708 up-regulation. Simultaneously, miR-708 elevation suppressed H/R exposure-increased lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) generation, but elevated the activity of anti-oxidative stress superoxide dismutase (SOD). Additionally, H/R-increased production of pro-inflammatory cytokine TNF-α and IL-6 was offset following miR-708 overexpression. Moreover, enhancement of miR-708 inhibited H/R-evoked activation of the HMGB1-TLR4-NF-κB pathway by inhibiting the protein levels of HMGB1, TLR4 and p-p65 NF-κB. Specially, restoring this pathway offset the protective effects of miR-708 on H/R-induced cardiomyocyte injury. Together, these data indicate that miR-708 may protect against H/R-induced cardiomyocyte damage by directing targeting HMGB1 signaling, implying a promising therapeutic agent against ischemic heart disease including myocardial infarction.