High Mobility Group Box 1 Protein Expression Research Articles

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.

Target Gene-Based Association Study of High Mobility Group Box Protein 1 in Intracranial Aneurysms in Koreans.

Objective: We investigated the effect of high mobility group box 1 (HMGB1) on intracranial aneurysms (IAs) by analyzing single-nucleotide polymorphisms (SNPs) based on genome-wide association study (GWAS) data. HMGB1 mRNA and protein expression levels in plasma were also analyzed. Methods: This study was a comprehensive analysis of a GWAS dataset, including 250 patients with IAs and 294 controls. The HMGB1 gene region was targeted within SNP rs3742305 ± 10 kbp. Multivariate logistic regression analysis determined its association with IAs after adjusting for relevant clinical factors. HMGB1 mRNA expression was analyzed in the plasma of 24 patients selected from the GWAS dataset. The HMGB1 protein was analyzed by Western blotting. Results: A total of seven polymorphisms, including rs1360485, rs185382445, rs2039338, rs1045411, rs3742305, rs2249825, and rs189034241, were observed. Two SNPs, including rs1045411 (UTR-3) and rs3742305 (intron), showed strong linkage disequilibrium (r2 = 0.99). However, none of the seven SNPs associated with IAs had an adjusted p-value of < 0.0016 on multiple comparison analysis. HMGB1 mRNA levels (2-ΔCt) did not differ significantly between patients with IAs and the control subjects [1.07 (1.00-1.15) in patients with IAs vs. 1.05 (0.94-1.12) in controls; p = 0.67)]. Also, no significant difference in the degree of plasma HMGB1 protein expression was seen between the two groups (p = 0.82). Conclusions: The number of SNPs associated with HMGB1 and the degree of HMGB1 mRNA and protein expression were not significantly different between patients diagnosed with IAs and the controls.

Fingolimod Alleviates Inflammation after Cerebral Ischemia via HMGB1/TLR4/NF-κB Signaling Pathway.

Clinically, ischemic reperfusion injury is the main cause of stroke injury. This study aimed to assess the effectiveness of fingolimod in suppressing inflammation caused by ischemic brain injury and explore its pharmacological mechanisms. In total, 75 male Sprague-Dawley rats were randomly and equally assigned to five distinct groups: sham, middle cerebral artery occlusion/reperfusion (MCAO/R) surgery, fingolimod low-dose (F-L), fingolimod medium-dose (F-M), and fingolimod high-dose (F-H). Neurobehavioral tests, 2,3,5-triphenyltetrazolium chloride staining, and the brain tissue drying-wet method were conducted to evaluate neurological impairment, cerebral infarction size, and brain water content. Enzyme-linked immunosorbent assay was employed to quantify pro-inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) protein levels. Western blotting and immunohistochemical staining were performed to assess high mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), and nuclear factor kappa-B p65 (NF-κBp65) levels. Rats in the F-L, F-M, and F-H groups exhibited lower Longa scores, reduced infarction volumes, and decreased brain edema than those in the MCAO/R group. Additionally, the F-L, F-M, and F-H groups exhibited lower serum levels of IL-1β, IL-6, and TNF-α than those of the MCAO/R group. Additionally, F-L, F-M, and F-H treatments resulted in decreased HMGB1, TLR4, and NF-κBp65 protein expression levels in the hippocampus of MCAO/R rats. Fingolimod was found to reduce ischemic brain injury in a dose-dependent manner. Moreover, it was also found to alleviate inflammation following ischemic brain injury via the HMGB1/TLR4/NF‑κB signaling pathway.

N-acetylserotonin alleviates retinal ischemia-reperfusion injury via HMGB1/RAGE/NF-κB pathway in rats.

To observe the effects of N-acetylserotonin (NAS) administration on retinal ischemia-reperfusion (RIR) injury in rats and explore the underlying mechanisms involving the high mobility group box 1 (HMGB1)/receptor for advanced glycation end-products (RAGE)/nuclear factor-kappa B (NF-κB) signaling pathway. A rat model of RIR was developed by increasing the pressure of the anterior chamber of the eye. Eighty male Sprague Dawley were randomly divided into five groups: sham group (n=8), RIR group (n=28), RIR+NAS group (n=28), RIR+FPS-ZM1 group (n=8) and RIR+NAS+ FPS-ZM1 group (n=8). The therapeutic effects of NAS were examined by hematoxylin-eosin (H&E) staining, and retinal ganglion cells (RGCs) counting. The expression of interleukin 1 beta (IL-1β), HMGB1, RAGE, and nod-like receptor 3 (NLRP3) proteins and the phosphorylation of nuclear factor-kappa B (p-NF-κB) were analyzed by immunohistochemistry staining and Western blot analysis. The expression of HMGB1 protein was also detected by enzyme-linked immunosorbent assay (ELISA). H&E staining results showed that NAS significantly reduced retinal edema and increased the number of RGCs in RIR rats. With NAS therapy, the HMGB1 and RAGE expression decreased significantly, and the activation of the NF-κB/NLRP3 pathway was antagonized along with the inhibition of p-NF-κB and NLRP3 protein expression. Additionally, NAS exhibited an anti-inflammatory effect by reducing IL-1β expression. The inhibitory of RAGE binding to HMGB1 by RAGE inhibitor FPS-ZM1 led to a significant decrease of p-NF-κB and NLRP3 expression, so as to the IL-1β expression and retinal edema, accompanied by an increase of RGCs in RIR rats. NAS may exhibit a neuroprotective effect against RIR via the HMGB1/RAGE/NF-κB signaling pathway, which may be a useful therapeutic target for retinal disease.

Lactate promotes HMGB1 phosphorylation and release via Akt signaling pathway in gastric cancer cells HGC-27

Objective: To investigate the molecular mechanism of how lactate induces high mobility group box 1 (HMGB1) release. Methods: Gastric cancer HGC-27 cells were divided into the control group and the lactate group (The cells were treated with lactate for 6 h). The level of HMGB1 in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA), the localization of HMGB1 was detected using laser confocal microscopy, and the nuclear translocation of HMGB1 was detected using the nucleoplasmic separation assay. The phosphorylation and acetylation levels of HMGB1 were determined by co-immunoprecipitation, and Western blot was used to measure the phosphorylation of Akt and protein kinase C (PKC). HGC-27 cells were first treated with lactate and LY294002, the inhibitor of Akt, and then the phosphorylation of HMGB1 and Akt was analyzed by co-immunoprecipitation and Western blot, respectively. The localization of HMGB1 in cells was detected by laser confocal microscopy. EdU and Transwell assays were used to detect the proliferation and migration abilities of HGC-27 cells, respectively. HGC-27 cells were then injected into the BALB/C null mice for subcutaneous tumor implantation. Mice in the lactate group were intraperitoneally injected with lactate (0.2 g/kg/2 d), while those in the control group were intraperitoneally injected with an equal amount of PBS for 20 consecutive days. ELISA was used to detect the HMGB1 levels in the blood samples taken from the medial canthus vein of the mice, while co-immunoprecipitation and Western blot were used to detect the phosphorylation of HMGB1 and Akt in tumor tissue proteins, respectively. Results: The release levels of HMGB1 in the lactate group were (2 995.00±660.91) pg/ml and (696.33±22.03) pg/ml, after lactate treatment for 6 h and 12 h, respectively, both higher than those in the control group (485.00±105.83) pg/ml (P<0.001 and P=0.028, respectively). After lactate treatment for 6 h, the relative expression of HMGB1 protein in the cytoplasm of HGC-27 cells was 1.13±0.09, higher than that of the control group (0.83±0.07, P=0.001), while the relative expression of HMGB1 in the nucleus was 0.79±0.06, lower than that of the control group (1.07±0.06, P=0.007). The phosphorylation level of HMGB1 reached 1.41±0.09, which was higher than that of the control group (0.97±0.10, P=0.031). The phosphorylation level of Akt was 11.16±0.06, higher than that of the control group (0.91±0.022, P=0.002). The phosphorylation level and nuclear translocation of HMGB1 induced by lactate decreased obviously after Akt inhibition; the proliferation and migration abilities induced by lactate were also obviously inhibited after Akt inhibition. In vivo, the HMGB1 level in the peripheral blood was (1 280.70±389.66) pg/ml in the lactate group, which was obviously higher than that in the control group (595.11±44.75) pg/ml (P=0.008), and the phosphorylation levels of HMGB1 and Akt in tumor tissues in the lactate group were obviously enhanced compared with the control group. Conclusion: Lactate induces HMGB1 release through enhancing HMGB1 phosphorylation via the Akt signaling pathway.

Oxaliplatin regulates the autophagy of skin squamous cell carcinoma cell line through HMGB1 pathway

Purpose: To determine the influence of oxaliplatin on skin squamous cell carcinoma cell line, and the involvement of autophagy- regulating pathway of high mobility group box 1 (HMGB1) in the process.&#x0D; Methods: A431 cells cultured in vitro were used. The cells were divided into groups A (treated with different concentrations of oxaliplatin) and B (treated with different concentrations of oxaliplatin combined with autophagy inhibitor 5 mmol/l3-ma). Changes in expression levels of autophagy marker molecules LC3-Ⅰ, LC3-Ⅱ, p62 and HMGB1 in A431 cells treated with different concentrations of oxaliplatin and different concentrations of HMGB1 were evaluated by Western blotting. Viability of A431 cells in both groups was assessed by CCK-8 assay.&#x0D; Results: With increase in oxaliplatin concentration, LC3-Ⅱ levels in A431 cells were up-regulated, p62 expression decreased, while autophagy level was increased significantly (p &lt; 0.05). With increase in HMGB1 protein concentration, LC3-Ⅱ level in A431 cells was raised, while p62 level was reduced, while the level of autophagy was significantly increased (p &lt; 0.05). Oxaliplatin treatment led to significantly higher expression level of HMGB1 in the experimental group than in the control group without oxaliplatin treatment. The viability of oxaliplatin-treated group was dose-dependently and significantly lower (p &lt; 0.05) than that of the control group. Compared with the control group, the cell viability of the 3-mA + oxaliplatin group also showed a downward trend, and the decrease was greater than that of oxaliplatin-treated group (p &lt; 0.05).&#x0D; Conclusion: Oxaliplatin upregulates autophagy by promoting HMGB1 protein expression, which may be a protective mechanism of tumor cells against oxaliplatin cytotoxicity thereby making HMGB1 protein a potential target in skin cancer therapy.

DIFFERENTIAL CELL SURFACE PROTEOMICS OF FLOTILLIN-1, HMGB1 AND AMINOPEPTIDASE IN H3 MUTATED PAEDIATRIC HIGH-GRADE GLIOMA

Abstract AIMS High-grade gliomas (HGG) are the deadliest central nervous system tumour in children. Despite this, treatment options remain elusive, leaving children with a dismal prognosis. Histone H3.3 mutations, K27M and G34V/R have been identified as the two most common recurrent somatic mutations in paediatric HGG (pHGG). These mutations are present in 50% of pHGG compared to only 1% in adults. Our previous research on cell membrane protein analyses have identified different signature of unique and common cell membrane proteins in H3-G34R/V mutated pHGG, in comparison to H3 wild type tumour. Among these we have identified flotillin-1, HMGB1 and aminopeptidase N. Flotillin-1 promotes numerous malignancies, High mobility group box 1(HMGB1) functions as an oncogene and aminopeptidase N is a cell surface metallo- protease involved in tumour progression. We have now verified the expression and localization of these cell surface proteins in H3 mutated and wildtype tumour, in order to target them for developing novel therapeutics. METHOD Cell membrane proteomics were further verified though mass-spectrometric data analyses. Expression and localization of flotillin-1, HMGB1 and Aminopeptidase in H3.3 mutated pHGG cell lines in comparison to H3 wildtype glioma were analysed, using different protein expression assays including immunofluorescence microscopy and western blot assays. RESULTS Experimental analyses showed differential protein expression and localization of Flotillin-1, HMGB1 and Aminopeptidase and verified the mass-spectrometric proteomic data in H3-G34V pHGG, in comparison to wild- type pHGG. CONCLUSIONS This new knowledge will help us utilise Flotillin-1, HMGB1 and Aminopeptidase N, as potential therapeutic targets for these unique H3 mutated children’s brain tumours.

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.

Spinal alarmin HMGB1 and the activation of TLR4 lead to chronic stress-induced nociceptive hypersensitivity in rodents

Chronic stress affects millions of people around the world, and it can trigger different behavioral disorders like nociceptive hypersensitivity and anxiety, among others. However, the mechanisms underlaying these chronic stress-induced behavioral disorders have not been yet elucidated. This study was designed to understand the role of high-mobility group box-1 (HMGB1) and toll-like receptor 4 (TLR4) in chronic stress-induced nociceptive hypersensitivity. Chronic restraint stress induced bilateral tactile allodynia, anxiety-like behaviors, phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) and activation of spinal microglia. Moreover, chronic stress enhanced HMGB1 and TLR4 protein expression at the dorsal root ganglion, but not at the spinal cord. Intrathecal injection of HMGB1 or TLR4 antagonists reduced tactile allodynia and anxiety-like behaviors induced by chronic stress. Additionally, deletion of TLR4 diminished the establishment of chronic stress-induced tactile allodynia in male and female mice. Lastly, the antiallodynic effect of HMGB1 and TLR4 antagonists were similar in stressed male and female rats and mice. Our results suggest that chronic restraint stress induces nociceptive hypersensitivity, anxiety-like behaviors, and up-regulation of spinal HMGB1 and TLR4 expression. Blockade of HMGB1 and TLR4 reverses chronic restraint stress-induced nociceptive hypersensitivity and anxiety-like behaviors and restores altered HMGB1 and TLR4 expression. The antiallodynic effects of HMGB1 and TLR4 blockers in this model are sex independent. TLR4 could be a potential pharmacological target for the treatment of the nociceptive hypersensitivity associated with widespread chronic pain.

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.

The Comprehensive Role of High Mobility Group Box 1 (HMGB1) Protein in Different Tumors: A Pan-Cancer Analysis.

HMGB1 is a highly conserved nuclear protein widely expressed in mammalian cells. This study aimed to comprehensively investigate the roles and mechanisms of HMGB1 in different tumors. Original data on HMGB1 expression, localization, potential interacting proteins, genetics were obtained from The Cancer Genome Atlas, Genotype-Tissue Expression, Cancer Cell Line Encyclopedia, Human Protein Atlas, Compartmentalized Protein-Protein Interaction and cBioPortal databases. Then, correlation between HMGB1 expression levels and tumor stage, prognosis, potential pathways, tumor microenvironment, ESTIMATE score, immune-related genes, immune cell infiltration, microsatellite instability, tumor mutation burden, or anti-tumor drug resistance was investigated. The above results consistently indicated that high expression of HMGB1 protein may be related to clinical prognosis of HCC patients. Therefore, clinical tissues of HCC patients were selected to verify the differential expression of HMGB1 protein in HCC. The sensitivity of HMGB1-siRNA transfected HepG2 cells to sorafenib was assessed. HMGB1 was found to be differentially expressed in many tumors and normal tissues. HMGB1 was mainly located in the nucleus and might interact with proteins such as TLR2 and TLR4. Furthermore, HMGB1 expression was closely related to tumor stage, prognosis, tumor microenvironment, immune-related genes, immune cell infiltration, microsatellite instability, tumor mutation burden, and anti-tumor drug resistance and might be involved in different pathways of various tumors. Immunohistochemistry results further verified the differential expression of HMGB1 in HCC and paracancerous tissues. HMGB1-siRNA transfected HepG2 cells had a tendency to be more insensitive to sorafenib treatment compared to the control group. HMGB1 was differentially expressed in most tumors and normal tissues, and was closely related to the clinical stage, prognosis, immune infiltration, tumor microenvironment, and drug resistance of tumors. Therefore, HMGB1 may serve as a novel biomarker for predicting tumor prognosis, efficacy of immune checkpoint inhibitors, and a potential target for anti-tumor therapy.

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.

MiR-181a-5p Alleviates the Inflammatory Response of PC12 Cells by Inhibiting High-Mobility Group Box-1 Protein Expression

Neuroinflammation triggers sequelae after spinal cord injury (SCI). Inhibition of inflammation promotes recovery after SCI. MicroRNAs regulate many pathophysiological processes, including inflammation. Any role for miR-181a-5p in the inflammatory response after SCI remains unclear. Thus, we evaluated the effects of miR-181a-5p on inflammation in PC12cells and the underlying mechanism in play. Quantitative reverse transcription-polymerase chain reaction was used to measure the levels of miR-181a-5p and high-mobility group box-1 protein (HMGB1) in SCI tissues. Cell-counting kit-8 assays were used to assess the viability of PC12cells treated with lipopolysaccharide (LPS). Plasmids encoding MiR-181a-5p mimics, an miR-181a-5p inhibitor, or/and the HMGB1 weretransfected into PC12cells. Quantitative reverse transcription-polymerase chain reaction or/and Western blotting were performed to assess the expression of miR-181a-5p, HMGB1, and inflammatory factors invitro. MiR-181a-5p expression decreased and HMGB1 expression increased in SCI tissues and LPS-induced PC12cells. Upregulation of miR-181a-5p (via transfection) inhibited inflammation of, and HMGB1 expression by, LPS-induced PC12cells. HMGB1 overexpression reversed the anti-inflammatory effects of miR-181a-5p. Dual-luciferase assays confirmed that HMGB1 was a direct target of miR-181a-5p. miR-181a-5p attenuated the inflammatory response of LPS-induced PC12cells by directly inhibiting HMGB1; thus, miR-181a-5p may serve as a therapeutic target in SCI.

Withdrawn: The LncRNA H19/microRNA-29b-3p/HMGB1 signaling axis contributes to the regulation of lung cancer cell growth.

The long non-coding RNA (lncRNA) H19 acts as a competitive endogenous RNA (ceRNA) of miR-29b-3p and has been reported to exert pro-tumorigenic roles in several cancer types. However, the role of lncRNA H19 in lung cancer is not fully understood. Here, we investigated the role of the lncRNA H19/microRNA-29b-3p (miR-29b-3p)/high mobility group box 1 (HMGB1) signaling pathway in lung cancer cell growth using 293T, NCI-H1975, Calu-3 and 2BS cell lines. Cell viability was determined using a cell counting kit-8 (CCK-8) assay, while apoptosis and cell cycle distribution were assessed by flow cytometry. Cell migration was detected using a wound healing assay. Cell invasion was evaluated by transwell assay. The expression of lncRNA H19, miR-29b-3p, HMGB1, toll-like receptor 4 (TLR4), and matrix metallopeptidase 9 (MMP-9) was measured by fluorescence quantitative PCR or western blotting. We demonstrated that miR-29b-3p could directly bind to both lncRNA H19 and HMGB1 by dual-luciferase reporter assay. Three shRNAs targeting lncRNA H19 (shlncRNA H19) were designed, and shlncRNA H19-2 was selected to investigate the function of lncRNA H19 in tumor cell biology. Compared with controls, lung cancer cells expressing shlncRNA H19 exhibited decreased proliferation, cell-cycle arrest at the G1 phase, increased levels of cell apoptosis, and reduced migration and invasion. Moreover, shlncRNA H19 upregulated the expression of miR-29b-3p and reduced the protein expression of HMGB1, TLR4, and MMP-9 in lung cancer cells. Together, our data indicate that the lncRNA H19/miRNA-29b-3p/HMGB1 signaling axis is involved in the regulation of lung cancer cell growth.

Evaluation of RUNX2 and HMGB1 expression as markers of tumor aggressiveness in urothelial carcinoma

Aim To investigate the expression and the clinical significance of runt-related transcription factor (RUNX2) and high-mobility group box 1 (HMGB1) in urothelial carcinoma of the urinary bladder. The correlation between RUNX2 and HMGB1 expression and the clinicopathological features was analyzed. Patients and methods RUNX2 and HMGB1 expression was examined by immunohistochemistry in 62 urothelial carcinoma and 20 nonneoplastic bladder tissues as control group. Results High RUNX2 and HMGB1 protein expression was observed in 67.7 and 56.5% of cases of urothelial carcinoma, respectively, and showed predominantly nuclear expression. The expression of RUNX2 and HMGB1 in urothelial carcinoma was significantly higher than that in nonneoplastic urothelial tissues (P&lt;0.001 for each). Moreover, high RUNX2 expression was significantly associated with high tumor grade (P=0.003), advanced tumor stage (P=0.011), lymph-node metastasis (P=0.045), and the presence of concomitant carcinoma in situ (P=0.01). HMGB1 showed higher expression with high-grade tumors (P=0.007), advanced tumor stage (P=0.002), and the presence of necrosis (P&lt;0.001). A significant direct relationship was detected between RUNX2 and HMGB1 expression (P&lt;0.001). Conclusion Our data suggest that RUNX2 and HMGB1 overexpression may contribute to urothelial carcinoma tumorigenesis and was associated with aggressive clinicopathologic features. RUNX2 and HMGB1 could be promising potential biomarkers for urothelial carcinoma.

Vitamin D decreases cell death and inflammation in human umbilical vein endothelial cells and placental explants from pregnant women with preeclampsia cultured with TNF-α

ABSTRACT This study evaluated the impact of vitamin D on Human Umbilical Vein Endothelial Cells (HUVEC) and inflammation in placental explants from women with preeclampsia (PE). HUVEC and explants from 10 late-onset PE (LOPE), 10 early-onset (EOPE), and 10 normotensive (NT) pregnant women were cultured with/without tumor necrosis factor (TNF-α) and VD. Interleukin-1β (IL-1β), 18 (IL-18), TNF-α, and TNF-related apoptosis-inducing ligand (TRAIL) were detected by ELISA. High mobility group box 1 (HMGB1) was determined by qPCR/Western blotting, and cell death by flow cytometry. Statistical significance was accepted at p < .05. Compared to the NT group, the endogenous levels of IL-1β, TNF-α, and IL-18 were higher in the PE group. The stimulus with TNF-α increased cytokines in NT, TNF-α in EOPE/LOPE, IL-18 in LOPE, and all cytokines in HUVEC. TNF-α+VD treatment decreased cytokines in explant and HUVEC supernatants. TRAIL was higher in EOPE versus NT, while TNF-α increased this receptor in NT versus control. In HUVEC, TNF-α increased TRAIL versus control, and TNF-α+VD decreased levels compared to only TNF-α stimulus. Protein expression of HMGB1 was higher in explant cultures treated with TNF-α and decreased after TNF-α+VD treatment in all groups, and gene/protein expression in HUVEC. Gene expression was elevated in EOPE versus NT and LOPE, and TNF-α increased HMGB1 in NT versus control, while TNF-α+VD decreased mRNA levels in EOPE. TNF-α stimulus increased late apoptosis in HUVEC, while VD increased viability. These in vitro observations suggest that VD administration to women with preeclampsia may be beneficial in reducing placental inflammation and cell death.

N-Acetyl Cysteine Restores Sirtuin-6 and Decreases HMGB1 Release Following Lipopolysaccharide-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Mice.

Inflammation and neonatal hypoxia-ischemia (HI) are important etiological factors of perinatal brain injury. However, underlying mechanisms remain unclear. Sirtuins are a family of nicotinamide adenine dinucleotide (NAD)+-dependent histone deacetylases. Sirtuin-6 is thought to regulate inflammatory and oxidative pathways, such as the extracellular release of the alarmin high mobility group box-1 (HMGB1). The expression and role of sirtuin-6 in neonatal brain injury are unknown. In a well-established model of neonatal brain injury, which encompasses inflammation (lipopolysaccharide, LPS) and hypoxia-ischemia (LPS+HI), we investigated the protein expression of sirtuin-6 and HMGB1, as well as thiol oxidation. Furthermore, we assessed the effect of the antioxidant N-acetyl cysteine (NAC) on sirtuin-6 expression, nuclear to cytoplasmic translocation, and release of HMGB1 in the brain and blood thiol oxidation after LPS+HI. We demonstrate reduced expression of sirtuin-6 and increased release of HMGB1 in injured hippocampus after LPS+HI. NAC treatment restored sirtuin-6 protein levels, which was associated with reduced extracellular HMGB1 release and reduced thiol oxidation in the blood. The study suggests that early reduction in sirtuin-6 is associated with HMGB1 release, which may contribute to neonatal brain injury, and that antioxidant treatment is beneficial for the alleviation of these injurious mechanisms.

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.

Modulation of the HMGB1/TLR4/NF-κB signaling pathway in the CNS by matrine in experimental autoimmune encephalomyelitis

The inflammatory mediator high-mobility group box 1 (HMGB1)-induced signaling pathway has been shown to play an important role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Matrine (MAT), a quinolizidine alkaloid component derived from the root of Sophorae flavescens, has the capacity to effectively suppress EAE. However, the impact of MAT treatment on HMGB1-induced signaling is not known. In the present study, we show that MAT treatment alleviated disease severity of ongoing EAE, reduced inflammatory infiltration and demyelination, and reduced the production of inflammatory factors including TNF-α, IL-6, and IL-1β in the CNS. Moreover, MAT administration significantly reduced the protein and RNA expression of HMGB1 and TLR4 in the spinal cord, particularly in astrocytes and microglia/infiltrating macrophages. The expression of MyD88 and TRAF6, and the phosphorylation of NF-κB p65, was also down-regulated after MAT treatment. In contrast, the level of IκB-α, an inhibitory molecule for NF-κB activation, was significantly increased. Furthermore, the direct inhibitory effect of MAT on HMGB1/TLR4/NF-κB signaling in macrophages was further confirmed in vitro. Taken together, these findings demonstrate that MAT treatment alleviated CNS inflammatory demyelination and activation of astrocytes and microglia/macrophages in EAE rats, and that the mechanism underlying these effects may be closely related to modulation of HMGB1/TLR4/NF-κB signaling pathway.