High-mobility Group Box 1 mRNA Levels Research Articles

Sirtuin 1 alleviates alcoholic liver disease by inhibiting HMGB1 acetylation and translocation.

Alcoholic liver disease (ALD) encompasses a spectrum of liver disorders resulting from prolonged alcohol consumption and is influenced by factors such as oxidative stress, inflammation, and apoptosis. High Mobility Group Box 1 (HMGB1) plays a pivotal role in ALD due to its involvement in inflammation and immune responses. Another key factor, Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, is known for its roles in cellular stress responses and metabolic regulation. Despite individual studies on HMGB1 and SIRT1 in ALD, their specific molecular interactions and combined effects on disease advancement remain incompletely understood. Alcohol-induced liver injury (ALI) models were established using HepG2 cells and male C57BL/6 mice. HMGB1 and SIRT1 expressions were assessed at the mRNA and protein levels usingreverse transcription-quantitative polymerase chain reaction, western blot, and immunofluorescence staining. The physical interaction between HMGB1 and SIRT1 was investigated using co-immunoprecipitation and immunofluorescence co-expression analyses. Cellular viability was evaluated using the CCK-8 assay. In patients with clinical ALI, HMGB1 mRNA levels were elevated, while SIRT1 expression was reduced, indicating a negative correlation between the two. ALI models were successfully established in cells and mice, as evidenced by increased markers of cellular and liver damage. HMGB1 acetylation and translocation were observed in both ALI cells and mouse models. Treatment with the SIRT1 agonist, SRT1720, reversed the upregulation of HMGB1 acetylation, nuclear translocation, and release in the ethyl alcohol (EtOH) group. Furthermore, SIRT1 significantly attenuated ALI. Importantly, in vivo binding was confirmed between SIRT1 and HMGB1. SIRT1 alleviates HMGB1 acetylation and translocation, thereby ameliorating ALI.

Emodin improves glucose metabolism and ovarian function in PCOS mice via the HMGB1/TLR4/NF-ĸB molecular pathway.

Polycystic ovary syndrome (PCOS) is a reproductive disorder with an unclear etiology. It affects 5-10% of women worldwide and is largely associated with impaired glucose metabolism and obesity. High mobility group box 1 (HMGB1) is a nuclear protein associated with impaired glucose metabolism and PCOS. We sought to investigate the potential therapeutic effects of emodin on glucose metabolism and ovarian functions in PCOS mice via the HMGB1 molecular pathway. A high-fat diet (HFD) and dehydroepiandrosterone (DHEA) induced PCOS mouse model comprising four experimental groups was established: control, PCOS, PCOS plus emodin, and PCOS plus vehicle groups. Emodin administration attenuated obesity, elevated fasting glucose levels, impaired glucose tolerance, and insulin resistance, and improved the polycystic ovarian morphology of PCOS mice. Additionally, it lowered elevated serum HMGB1, LH, and testosterone levels in PCOS mice. Elevated ovarian protein and mRNA levels of HMGB1 and TLR4 in PCOS mice were also lowered following emodin treatment. Furthermore, emodin lowered high NF-ĸB/65 protein levels in the ovaries of PCOS mice. Immunohistochemical staining of the ovaries revealed strong HMGB1, TLR4, and AR expressions in PCOS mice, which were lowered by emodin treatment. Moreover, emodin significantly increased GLUT4, Irs2, and Insr levels that were PCOS. Overall, our study showed that emodin alleviated the impaired glucose metabolism and improved ovarian function in PCOS mice, possibly via the HMGB1/TLR4/NF-ĸB signaling pathway. Thus, emodin could be considered a potential therapeutic agent in the management of PCOS.

Ethyl Pyruvate Alleviating Inflammatory Response after Diabetic Cerebral Hemorrhage.

This study's purpose is to investigate the neuroprotective role of ethyl pyruvate (EP) in the pathogenesis of diabetic intracerebral hemorrhage. The present study used a mouse model of collagenase-induced intracerebral hemorrhage (ICH) and streptozotocin-induced diabetes. The C57BL/6 mice were randomly divided into 3 groups: sham operation, diabetic cerebral hemorrhage, and diabetic cerebral hemorrhage with EP. The EP (80 mg/kg) and EP (50 mg/kg) were injected intraperitoneally one day and one hour before modeling. The protein expression levels of high mobility group box 1 (HMGB1) and NOD-like receptors 3 (NLRP3) were detected with western blot. The mRNA levels of HMGB1 and toll-like receptor 4 (TLR4) were measured by quantitative real-time polymerase chain reaction (PCR). Immunofluorescence and ELISA were performed to confirm some inflammatory factors. Compared to the normal diabetic intracerebral hemorrhage group, the mRNA and protein expression levels of HMGB1 and TLR4 were downregulated in the EP-affected group with diabetic cerebral hemorrhage, together with the downregulation of the expression of inflammasomes, including NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), and caspase 1. EP can reduce the inflammatory response after diabetic intracerebral hemorrhage and may inhibit the activation of inflammasomes by the HMGB1/TLR4 pathway.

HMGB1 is increased in patients with immune thrombocytopenia and negatively associates with Tregs

IntroductionRefractory or recurrent immune thrombocytopenia (ITP) patients suffer from the dual threat of high mortality and drug toxicity in addition to a very poor quality of life. Previous studies have shown that high mobility group box 1 (HMGB1) can promote the development of rheumatoid arthritis, systemic lupus erythematosus, and other autoimmune diseases. However, there is still a lack of research on the role of HMGB1 in the pathogenesis of ITP and whether it can be used as a predictor of efficacy and prognosis. Methods20 patients of adult ITP with splenectomy were chosen as the experimental group, while 19 adults underwent splenectomy for traumatic splenic rupture without other diseases as the control group. We measured the expression of HMGB1, RORγt and Foxp3 in spleen tissues by immunohistochemistry. Another 50 patients, of which 20 were newly diagnosed without treatment and 30 were refractory ITP, and 25 healthy controls were enrolled to analyse the expression levels and mRNA levels of HMGB1, RORγt and Foxp3 in peripheral blood by Western blot and RT-qPCR. The expression of HMGB1, IL-17 and IL-10 in serum was assayed by ELISA. PBMCs from newly diagnosed ITP patients were cultured in vitro which stimulated with recombinant humanHMGB1 (rHMGB1) and its inhibitors, in which the expressions of RORγt and Foxp3 were measured. ResultsThe expression of HMGB1 in the spleen with refractory ITP was significantly higher, while Foxp3 was decreased. A significant negative correlation was found between HMGB1 and Foxp3, and the overexpression of HMGB1 was significantly correlated with poor efficacy after splenectomy. The expression of HMGB1 and IL-17 increased and showed a positive correlation in serum, while IL-10 decreased and was negatively correlated with HMGB1. In PBMCs, the expression of HMGB1 and RORγt increased, while Foxp3 decreased, and the differences were more obvious in the refractory chronic ITP group. In a coculture system with PBMCs of untreated ITP patients, rHMGB1 increased RORγt expression and decreased Foxp3 expression, while an antiHMGB1 antibody partially corrected the above changes. ConclusionOur results suggest that HMGB1 is associated with the imbalance of Treg/Th17 cells and is involved in the pathogenesis of ITP.

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

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

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

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

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.

Effects of hypoxia on the expression of HMGB1 and related inflammatory factors in human pulmonary artery smooth muscle and pulmonary artery endothelial cells

To investigate the effects of hypoxia on the expressions of high mobility group box-1(HMGB1), HMGB1 receptors and inflammatory factors in human pulmonary artery smooth muscle cell( HPASMC) and human pulmonary artery endothelial cells (HPAEC).The effects of HMGB1 on the proliferation and migration activity of the two kinds of cells were detected. HPASMC and HPAEC were cultured under hypoxic conditions (Hypoxia at 1% oxygen, Hypoxia group) and normoxic conditions(Control group) . The mRNA levels of HMGB1, Toll-like receptors 2,4,9 (TLR2 , TLR4, TLR9), the receptor of advanced glycation end products(RAGE) , CD24 and IL-6 ,TNF-α,CXCL8 were detected by real-time PCR. Cell proliferation was measured by MTS. Cell migration was analysed by wound healing test. Compared with control group, the expressions of HMGB1 mRNA and RAGE mRNA in both HPASMC and HPAEC were increased significantly (P＜0.05 and 0.01). Meanwhile, the expressions of CD24 mRNA in HPAEC and IL-6 mRNA in HPASMC of hypoxia group were increased significantly (P＜0.05). MTS results showed that HMGB1 inhibited the proliferation of HPAEC at 345 pmol/L significantly (P＜0.01). HMGB1 had no effect on the proliferation of HPASMC. Wound healing test showed that HMGB1 had no significant effect on HPASMC and HPAEC. HMGB1 was produced by HPAEC and HPASMC induced by hypoxia. HMGB1 induces endothelial barrier dysfunction by inhibiting HPAEC proliferation. Hypoxia stimulates HPASMC to produce inflammatory cytokines.

Overexpression of high mobility group box 1 (HMGB1)has no correlation with the prognosis in glioma.

Aim: We aimed to characterize the role of HMGB1overexpression in glioma and to evaluate its use as a biomarker. Materials & methods: We used the gene expression datasets and tissue microarray to assess the expression levels of HMGB1 among gliomas of all grades; We then assessed its correlation with the malignancy and outcome of glioma. Results: The increase in HMGB1 mRNA and protein levels was found in glioma, but there was no correlation between HMGB1 expression and glioma malignancy, and overall survival and vital status of glioma patients. Conclusion: Overexpression of HMGB1 is not associated with the malignancy and outcome in glioma. And it is not the valuable biomarker for the early diagnosis of glioma.

Hyperbaric oxygen inhibits the HMGB1/RAGE signaling pathway by upregulating Mir-107 expression in human osteoarthritic chondrocytes

MicroRNA (miRNA)107 expression is downregulated but high mobility group box 1 (HMGB-1), Toll-like receptors (TLRs), and receptor for advanced glycation end products (RAGE) are upregulated in osteoarthritic (OA) cartilage. We investigated mir-107/HMGB-1 signaling in OA after hyperbaric oxygen (HBO) treatment. MiR-107 mimic was transfected and the HMGB-1 was analyzed in OA chondrocytes. MiRNA targets were identified using bioinformatics and a luciferase reporter assay. After HBO treatment, the mRNA or protein levels of HMGB-1, RAGE, TLR2, TLR4, and inducible nitric oxide (NO) synthase (iNOS) and phosphorylation of mitogen-activated protein kinase (MAPK) were evaluated. The secreted HMGB-1 and matrix metalloproteases (MMPs) levels were quantified. Finally, we detected the HMGB-1 and iNOS expression in rabbit cartilage defects. Overexpression of miR-107 suppressed HMGB-1 expression in OA chondrocytes. The 3'UTR of HMGB-1 mRNA contained a 'seed-matched-sequence' for miR-107. MiR-107 was induced by HBO and a marked suppression of HMGB-1 was observed simultaneously in OA chondrocytes. Knockdown of miR-107 upregulated HMGB-1 expression in hyperoxic cells. HBO downregulated the mRNA and protein expression of HMGB-1, RAGE, TLR2, TLR4, and iNOS, and the secretion of HMGB-1. HBO decreased the nuclear translocation of nuclear factor (NF)-κB, downregulated the phosphorylation of MAPK, and significantly decreased the secretion of MMPs. Morphological and immunohistochemical observation demonstrated that HBO markedly enhanced cartilage repair and the area stained positive for HMGB-1 and iNOS tended to be lower in the HBO group. HBO inhibits HMGB-1/RAGE signaling related pathways by upregulating miR-107 expression in human OA chondrocytes.

Resveratrol protects against asthma-induced airway inflammation and remodeling by inhibiting the HMGB1/TLR4/NF-κB pathway.

The aim of the present study was to explore the protective role of resveratrol (RES) in asthma-induced airway inflammation and remodeling, as well as its underlying mechanism. An asthma rat model was induced by ovalbumin (OVA) treatment. Rats were randomly assigned into sham, asthma, 10 µmol/l RES and 50 µmol/l RES groups. The amount of inflammatory cells in rat bronchoalveolar lavage fluid (BALF) was detected. Pathological lesions in lung tissues were accessed by hematoxylin and eosin (H&E), and Masson's trichrome staining. Levels of inflammatory factors in lung homogenate were detected via ELISA. The blood serum of asthmatic and healthy children was also collected for analysis. Reverse transcription-quantitative polymerase chain reaction was performed to detect high mobility group box 1 (HMGB1), Τoll-like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (MyD88) and NF-κB expression in asthmatic and healthy children, as well as rats of the different groups. H&E staining demonstrated that multiple inflammatory cell infiltration into the rat airway epithelium of the asthma group occurred whilst the 50 µmol/l RES group displayed alleviated pathological lesions. Masson staining indicated that there was an increased airway collagen deposition area in the asthma and 10 µmol/l RES groups compared with the 50 µmol/l RES group. The number of inflammatory cells in BALF extracted from rats of the asthma and 10 µmol/l RES groups was higher compared with the 50 µmol/l RES group. Treatment with 50 µmol/l RES significantly decreased the thicknesses of the airway wall and smooth muscle. ELISA results illustrated that interleukin (IL)-1, IL-10 and tumor necrosis factor-α (TNF-α) levels were elevated, whereas IL-12 level was reduced in lung tissues of the asthma and 10 µmol/l RES groups whilst the 50 µmol/l RES group demonstrated the opposite trend. HMGB1, TLR4, MyD88 and NF-κB mRNA levels were remarkably elevated in rats of the asthma and 10 µmol/l RES groups compared with the 50 µmol/l RES group. Serum levels of IL-1, IL-10 and TNF-α were elevated, whereas IL-12 was reduced in asthmatic children compared with healthy children. The present results demonstrated that a large dose of RES alleviated asthma-induced airway inflammation and airway remodeling by inhibiting the release of inflammatory cytokines via the HMGB1/TLR4/NF-κB pathway.

High-Mobility Group Box 1 Is Associated with the Inflammatory Pathogenesis of Graves' Orbitopathy.

Background: High-mobility group box 1 (HMGB1) has been implicated in the pathogenesis of inflammatory autoimmune diseases. This study investigated the influence and mechanisms of HMGB1 in Graves' orbitopathy (GO). Methods: HMGB1 and its receptors (receptor for advanced glycation end products [RAGE], Toll-like receptor [TLR] 2, and TLR4) mRNA levels were evaluated by real-time polymerase chain reaction (RT-PCR) in GO and non-GO orbital tissues. The mRNA expressions of HMGB1 and its receptors were evaluated in primary cultured orbital fibroblasts from six GO patients and five healthy control subjects under interleukin (IL)-1β or tumor necrosis factor (TNF)-α stimulation using RT-PCR. HMGB1 secretions under IL-1β or TNF-α stimulation were evaluated by enzyme-linked immunosorbent assay (ELISA). The effects of an anti-HMGB1 antibody, RAGE antagonist (FPS-ZM1), and anti-TLR2 antibody on the expressions of IL-1β or TNF-α induced pro-inflammatory cytokines and phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells were evaluated using ELISA and Western blot analysis, respectively. The plasma levels of HMGB1 were compared among patients with active GO (n = 51), inactive GO (n = 48), Graves' disease without GO (n = 30), and healthy control subjects (n = 46) by ELISA. Results: The genes encoding HMGB1 and its receptors, as well as HMGB1 protein expression, were increased in GO orbital tissues compared to non-GO tissues. IL-1β and TNF-α stimulation increased the mRNA levels of HMGB1, RAGE, and TLR2 and the secretion of HMGB1 protein further in GO cells. Anti-HMGB1 antibody, FPS-ZM1, and anti-TLR2 antibody reduced IL-1β- or TNF-α-induced production of pro-inflammatory cytokines and phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells. The plasma levels of HMGB1 were highly increased in patients with active GO, and were significantly correlated with the clinical activity score (r = 0.566, p = 0.002) and levels of thyrotropin binding inhibitory immunoglobulin (r = 0.506, p < 0.001). Conclusions: This study demonstrates an association of HMGB1 and its receptors in the inflammatory mechanisms of GO. HMGB1, RAGE, and TLR2 blockers reduced the production of pro-inflammatory molecules, providing a rationale for blocking the HMGB1 pathway to treat patients with GO. HMGB1 proteins were secreted further in the plasma of patients with active GO, suggesting that HMGB1 can be used as a biomarker of GO activity.

Upregulation of miR-107 expression following hyperbaric oxygen treatment suppresses HMGB1/RAGE signaling in degenerated human nucleus pulposus cells

BackgroundThe expression of both high-mobility group box 1 (HMGB1) and receptor for advanced glycation end-products (RAGE) is upregulated in degenerated discs. HMGB1 is known to function as a coupling factor between hypoxia and inflammation in arthritis, and this inflammatory response is modulated by microRNAs (miRNAs), with miR-107 expression downregulated during hypoxia. In this study, we investigated the regulation of the miR-107/HMGB1/RAGE pathway in degenerated nucleus pulposus cells (NPCs) after hyperbaric oxygen (HBO) treatment.MethodsNPCs were separated from human degenerated intervertebral disc tissues. The control cells were maintained in 5% CO2/95% air, and the hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The cellular protein and mRNA levels of HMGB1, RAGE, and inducible nitric oxide synthase (iNOS) were assessed, and the phosphorylation of MAPK (p38MAPK, ERK, and JNK) was evaluated. Additionally, cytosolic and nuclear fractions of the IκBα and NF-κB p65 proteins were analyzed, and secreted HMGB1 and metalloprotease (MMP) levels in the conditioned media were quantified.ResultsUsing microarray analyses, 96 miRNAs were identified as upregulated and 66 downregulated following HBO treatment. Based on these results, miR-107 was selected for further investigation. Bioinformatics analyses indicated that the 3′ untranslated region of the HMGB1 mRNA contained the “seed-matched-sequence” for hsa-miR-107, which was validated via dual-luciferase reporter assays. MiR-107 was markedly induced by HBO, and simultaneous suppression of HMGB1 was observed in NPCs. Knockdown of miR-107 resulted in upregulation of HMGB1 expression in HBO-treated cells, and HBO treatment downregulated the mRNA and protein levels of HMGB1, RAGE, and iNOS and the secretion of HMGB1. In addition, HBO treatment upregulated the protein levels of cytosolic IκBα and decreased the nuclear translocation of NF-κB in NPCs. Moreover, HBO treatment downregulated the phosphorylation of p38MAPK, ERK, and JNK and significantly decreased the secretion of MMP-3, MMP-9, and MMP-13.ConclusionsHBO inhibits pathways related to HMGB1/RAGE signaling via upregulation of miR-107 expression in degenerated human NPCs.

Increased HMGB1 expression correlates with higher expression of c-IAP2 and pERK in colorectal cancer.

The aim of this study was to investigate the relationship between high-mobility group box 1 (HMGB1) and colorectal cancer (CRC).In this prospective study, patients with CRC undergoing primary surgery and healthy subjects (control group) were enrolled from July 2013 to December 2014. The serum HMGB1 concentration and HMGB1 mRNA expression were determined using enzyme-linked immunosorbent assay reverse transcription-polymerase chain reaction, respectively. Immunohistochemical analysis was performed to determine HMGB1, pERK, and c-inhibitor of apoptosis protein 2 (c-IAP2) protein expression levels in the cancer tissues.A total 144 patients with CRC and 50 healthy subjects underwent serum HMGB1 testing. Resected specimens of 50 patients were used for HMGB1 mRNA and protein expression analyses. Mean serum HMGB1 level in the patients with CRC was higher than that of the control group (8.42 μg/L vs 1.79 μg/L, P < .05). Mean serum HMGB1 level in the patients with CRC with distant metastasis was significantly higher than that of the controls (13.32 μg/L vs 7.37 μg/L, P < .05). The HMGB1 mRNA and protein expression levels in the CRC tissues were significantly higher than those in the adjacent normal mucosa. HMGB1 protein expression positively correlated with the lymph node metastasis. There were positive correlations between HMGB1 and c-IAP2 (r = 0.457, P < .05), HMGB1 and pERK (r = 0.461, P < .05), as well as pERK and c-IAP2 (r = 0.399, P < .05).HMGB1 expression in CRC correlates with distant and lymph node metastasis. It may inhibit apoptosis by inducing activation of pERK and c-IAP2.

Curcumin inhibits the lymphangiogenesis of gastric cancer cells by inhibiton of HMGB1/VEGF-D signaling.

Accumulating evidence shows that curcumin exerts antitumor activities in a variety of malignancies. High mobility group box 1 (HMGB1) is associated with vascular endothelial growth factor D (VEGF-D)–induced lymphangiogenesis and tumor metastasis in gastric cancer. However, the molecular mechanisms by which curcumin regulates HMGB1-mediated lymphangiogenesis in gastric cancer remain unclear. In this study, the cytotoxic effects of curcumin were investigated in gastric cancer AGS and SGC-7901 cell lines by MTT assay, and curcumin-induced morphological changes and cell apoptosis were assessed by using flow cytometry analysis and caspase-3 activity. The effects of curcumin on HMGB1 and VEGF-D expression were examined by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis. As a result, we found that curcumin decreased cell viability and caused a dose-dependent cell apoptosis through the activation of caspase-3. The mRNA and protein expression levels of HMGB1 and VEGF-D were significantly eliminated by curcumin administration. Pre-treatment with the recombinant HMGB1 (rHMGB1) markedly abolished curcumin-reduced VEGF-D expression. Our findings suggested that curcumin might exert anti-lymphangiogenesis in gastric cancer by inhibition of HMGB1/VEGF-D signaling.

Rosuvastatin reduces the pro-inflammatory effects of adriamycin on the expression of HMGB1 and RAGE in rats.

Rosuvastatin has cardiac protective effects through its anti-inflammatory effects. The nuclear protein high-mobility group box 1 (HMGB1) can activate inflammatory pathways when released from dying cells. The present study aimed to investigate the effects of rosuvastatin in adriamycin (ADR)-treated rats. Adult male rats were randomized to three groups: i) Control group, ii) ADR group, and iii) ADR+rosuvastatin group. Serum biochemical indices were measured using an enzyme-linked immunosorbent assay. Cardiac function was assessed by echocardiography. The expression of HMGB1 and receptors for advanced glycation end products (RAGE) were assessed by reverse transcription-quantitative polymerase chain reaction analysis, western blot analysis, and immunohistochemistry. Cytokines were measured using flow cytometry. Rosuvastatin improved the biochemical indices and cardiac morphology and alleviated the pathological lesions. In the ADR+rosuvastatin group, the mRNA and protein levels of HMGB1 and RAGE in the myocardium were significantly lower compared with those in the ADR group (both P<0.05). The results showed that rosuvastatin significantly reduced the levels of HMGB1 and RAGE in the myocardium of the ADR-treated rats. These results suggest that the protective effects of rosuvastatin may be associated with attenuation of the HMGB1/RAGE-mediated inflammatory response in ADR-treated rats. Despite this protective effect of rosuvastatin in the present study, it did not improve cardiac function in terms of the diastolic left ventricular internal dimension, systolic left ventricular internal dimension, left ventricular ejection fraction and left ventricular fractional shortening; this may be due the observation duration being insufficient.

TLR4/MyD88 signaling determines the metastatic potential of breast cancer cells

The influence of Toll-like receptor (TLR)4/myeloid differentiation factor (MyD)88 signaling on the invasion and metastasis of cancer cells has been previously reported. The purpose of the present study was to determine the role of TLR4/MyD88 in breast cancer cell migration and invasion, and to discover novel therapeutic targets for breast cancer treatment. TLR4, MyD88 and high mobility group box 1 (HMGB1) mRNA expression levels were assessed in highly invasive human MDA-MB-231 breast cancer cells, breast cancer cells with a low rate of invasion (MCF-7) and normal human MDA-Kb2 mammary gland cells by reverse transcription-quantitative polymerase chain reaction. The protein expression levels of these markers were detected by western blotting and immunofluorescence. Randomly selected breast cancer and paracarcinoma tissues were used to measure TLR4 and MyD88 protein expression levels by immunohistochemistry. The mRNA and protein expression levels of TLR4 and MyD88 were significantly higher in MDA-MB-231 cells compared with either MCF-7 cells or MDA-Kb2 cells. The mRNA and protein expression levels of HMGB1 were comparable in the two breast cancer cell lines, with no statistical difference (P>0.05). TLR4 and MyD88 protein expression levels were also significantly higher in breast cancer tissues compared with paracarcinoma tissues (P<0.05). TLR4 and MyD88 protein expression levels were positively correlated with axillary lymph node metastasis and histological grade (P<0.05). TLR4/MyD88 expression levels were positively correlated with the metastasis of breast cancer cells. TLR4/MyD88 may be useful as a novel biomarker to evaluate the prognosis and treatment of patients with breast cancer.

MicroRNA-520a-3p suppresses non-small-cell lung carcinoma by inhibition of High Mobility Group Box 1 (HMGB1).

Currently, pathogenesis of non-small cell lung carcinoma (NSCLC) is still unknown and the treatment is far from ideal. Therefore, we investigated the effect of inhibiting microRNA-520a-3p in NSCLC cells. NCI-H157 cells were treated with microRNA-520a-3p analogs for 48 h, or microRNA-520a-3p analogs and its inhibitor, for a total of 48 h. Many tests were performed, including MTT, flow cytometry, wound healing assay and transwell assay. The tumor model was established, and HMGB1 mRNA was detected by RT-PCR. Protein levels of HMGB1, MMP-2, MMP-9, Bcl-2, Bax, and Caspase-3 were assessed by Western Blot. microRNA-520a-3p could significantly inhibit the proliferation, migration and invasion of NCI-H157 cells, inducing their apoptosis. microRNA-520a-3p inhibited tumor growth and decreased the mRNA and protein levels of HMGB1. Additionally, it decreased the Bcl-2/Bax ratio, MMP-2 and MMP-9 expression, and increased caspase-3 expression. microRNA-520a-3p exhibited an effective inhibition on NCI-H157 tumor growth likely by inhibiting HMGB1 expression.

Systemic lidocaine inhibits high-mobility group box 1 messenger ribonucleic acid expression and protein in BALB/c mice after closed fracture musculoskeletal injury.

Background:Severe musculoskeletal trauma can trigger an inflammatory response, and an excessive inflammatory response can lead to systemic inflammatory response syndrome and multiorgan failure. High-mobility group box 1 (HMGB1) is an early mediator pro-inflammatory cytokine in sterile injuries and a late cytokine mediator in infection and sepsis. Previous research has shown that administration of systemic lidocaine can inhibit HMGB1 expression in macrophages of septic rats. The aim of this study was to demonstrate the efficacy of systemic lidocaine to inhibit HMGB1 mRNA and protein in a BALB/c mouse model of sterile inflammation due to closed fracture musculoskeletal injury.Materials and Methods:Twenty adult male BALB/c mice were divided into lidocaine and control groups. The closed fracture musculoskeletal injury was performed by breaking the left thigh bone of the mice. Four hours after undergoing the closed fracture, the lidocaine group was treated with lidocaine intravenous (2 mg/kg). The same volume of distilled water was injected into the control group instead of lidocaine. HMGB1 mRNA expression was examined with real-time polymerase chain reaction, and HMGB1 protein level was determined with enzyme-linked immunosorbent assay.Results:The expression of HMGB1 mRNA and protein levels in mice that sustained inflammation due to a closed fracture musculoskeletal injury was significantly decreased in the lidocaine group (P < 0.00 and P < 0.00 for mRNA and protein, respectively).Conclusions:Intravenous administration of lidocaine effectively inhibited the inflammatory process in BALB/c mice that underwent closed fracture musculoskeletal injury by suppressing HMGB1 mRNA transcription and HMGB1 protein translation.

Macrophage migration inhibitory factor mediates protease-activated receptor 4-induced bladder pain through urothelial high mobility group box 1.

Macrophage migration inhibitory factor (MIF) mediates pain although the mechanisms are not well understood. Urothelial activation of protease activated receptor 4 (PAR4) results in urothelial MIF release, urothelial high mobility group box 1 (HMGB1) release and bladder pain in mice without bladder inflammation. All three effects are prevented by MIF inhibition while intravesical disulfide HMGB1 alone can induce bladder pain. This study utilizes genetic MIF deletion to determine whether MIF mediates PAR4‐induced bladder pain and is upstream of HMGB1‐induced bladder pain. Wild type (C57/BL6) and MIF knockout (KO) mice were treated with intravesical PAR4 activating peptide or disulfide HMGB1 and tested for abdominal mechanical hypersensitivity at baseline (before treatment) and 24 h after injection. Micturition parameters and bladder histology were examined after behavioral test. Real‐time PCR and western blotting measured HMGB1 mRNA and protein levels in the bladders of naïve wild type and MIF KO mice, while immunofluorescence measured HMGB1 protein levels in the urothelium of both strains. Intravesical PAR4 activation resulted in abdominal mechanical hypersensitivity in wild‐type mice but not MIF KO mice. Intravesical disulfide HMGB1 induced abdominal mechanical hypersensitivity in both strains. Neither treatment resulted in significant changes in micturition or bladder histology in either strain. HMGB1 mRNA and protein levels were higher in MIF KO mouse bladders and the urothelium of MIF KO bladder had greater immunostaining than the wild‐type strain. MIF is a pivotal molecule mediating PAR4‐induced bladder pain and regulating urothelial HMGB1 production and release to elicit bladder pain.