OBJECTIVETo investigate the effect of heat shock factor 1 (HSF1) on airway hyperresponsiveness and airway inflammation in mice with asthma and possible mechanisms.

METHODSA total of 36 mice were randomly divided into four groups: control, asthma, HSF1 small interfering RNA negative control (siHSF1-NC), and siHSF1 intervention (n=9 each). Ovalbumin (OVA) sensitization and challenge were performed to induce asthma in the latter three groups. The mice in the siHSF1-NC and siHSF1 groups were treated with siHSF1-NC and siHSF1, respectively. A spirometer was used to measure airway responsiveness at 24 hours after the last challenge. The direct count method was used to calculate the number of eosinophils. ELISA was used to measure the serum level of OVA-specific IgE and levels of interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), and interferon-γ (IFN-γ) in lung tissues and bronchoalveolar lavage fluid (BALF). Quantitative real-time PCR was used to measure the mRNA expression of HSF1 in asthmatic mice. Western blot was used to measure the protein expression of HSF1, high-mobility group box 1 (HMGB1), and phosphorylated c-Jun N-terminal kinase (p-JNK).

RESULTSThe asthma group had significant increases in the mRNA and protein expression of HSF1 compared with the control group (P<0.05). The siHSF1 group had significantly reduced mRNA and protein expression of HSF1 compared with the siHSF1-NC group (P<0.05). The knockdown of HSF1 increased airway wall thickness, airway hyperresponsiveness, OVA-specific IgE content, and the number of eosinophils (P<0.05). Compared with the siHSF1-NC group, the siHSF1 group had significantly increased levels of IL-4, IL-5, and IL-13 and significantly reduced expression of IFN-γ in lung tissues and BALF (P<0.05), as well as significantly increased expression of HMGB1 and p-JNK (P<0.05).

CONCLUSIONSKnockdown of HSF1 aggravates airway hyperresponsiveness and airway inflammation in asthmatic mice, and its possible mechanism may involve the negative regulation of HMGB1 and JNK.

Animals ; Asthma ; etiology ; Bronchial Hyperreactivity ; etiology ; immunology ; Cytokines ; biosynthesis ; DNA-Binding Proteins ; analysis ; physiology ; Eosinophils ; physiology ; Female ; HMGB1 Protein ; analysis ; Heat Shock Transcription Factors ; Immunoglobulin E ; blood ; Mice ; Mice, Inbred BALB C ; Transcription Factors ; analysis ; physiology

miR-200c has been shown to regulate the epithelial-mesenchymal transition (EMT) by inhibiting ZEB1 and ZEB2 expression in breast cancer cells. This study further examined the role of miR-200c in the invasion and metastasis of breast cancer that goes beyond the regulation on ZEB1 and ZEB2 expression. In this study, the bioinformatics software (miRanda) was used to predict the target gene of miR-200c and Renilla luciferase assay to verify the result. The metastatic breast cancer cells MDA-MB-231 were cultured and transfected with the miR-200c mimic or inhibitor. The expressions of miR-200c and HMGB1 were detected by RT-PCR and Western blotting, respectively. Transwell assay and wound healing assay were employed to examine the invasive and migrating ability of transfected cells. Target prediction and Renilla luciferase analysis revealed that HMGB1 was a putative target gene of miR-200c. After transfection of MDA-MB-231 cells with the miR-200c mimic or inhibitor, the expression of miR-200c was significantly increased or decreased when compared with cells transfected with the miR-200c mimic NC or inhibitor NC. Moreover, the expression of HMGB1 was reversely correlated with that of miR-200c in transfected cells. Tranwell assay showed that the number of invasive cells was significantly reduced in miR-200c mimic group when compared with miR-200c inhibitor group. It was also found that the migrating ability of cells transfected with miR-200c mimics was much lower than that of cells transfected with miR-200c inhibitors. It was suggested that miR-200c can suppress the invasion and migration of breast cancer cells by regulating the expression of HMGB1. miR-200c and HMGB1 may become useful biomarkers for progression of breast cancer and targets of gene therapy.
Biomarkers, Tumor ; Breast Neoplasms ; genetics ; metabolism ; Cell Movement ; genetics ; Epithelial-Mesenchymal Transition ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; HEK293 Cells ; HMGB1 Protein ; genetics ; Homeodomain Proteins ; biosynthesis ; Humans ; MicroRNAs ; genetics ; Neoplasm Invasiveness ; genetics ; Neoplasm Metastasis ; genetics ; pathology ; Repressor Proteins ; biosynthesis ; Transcription Factors ; biosynthesis ; Zinc Finger E-box Binding Homeobox 2 ; Zinc Finger E-box-Binding Homeobox 1

OBJECTIVETo explore the effect of HMGB1 on the VEGF-C expression and proliferation of esophageal squamous cancer cells as well as its possible mechanism.

METHODSA cassette encoding siRNA targeting HMGB1 mediated by rAAV was constructed, the rAAV-siHMGB1-hrGFP, and a vector encoding siRNA mismatching HMGB1 was constructed, the rAAV-miHMGB1-hrGFP. This experiment in vitro included three groups, namely, the blank control group (group A) of KYSE150 cells transfected by rAAV-hrGFP, negative mismatch control group (group B) of KYSE150 cells transfected with rAAV-miHMGB1-hrGFP, and RNA interference group (group C) of KYSE150 cells transfected with rAAV-siHMGB1-hrGFP. We examined the expression of HMGB1 mRNA and protein in the three group cells by real-time PCR and Western blot after 24 h and 48 h, respectively. Then, VEGF-C expression and cell proliferation in the three group cells with or without sRAGE, as an inhibitor of RAGE signal pathway, were assayed by ELISA and MTT after 24 h.

RESULTSThe expression of HMGB1 mRNA and protein in KYSE150 cells in vitro in the group C transfected with rAAV-siHMGB1-hrGFP at the final concentration of 2×10(6) v.g/cell was significantly lower than that of the group A or B after 24 h and 48 h (P < 0.01). The VEGF-C expression of KYSE150 cells was (502.43 ± 13.10) pg/ml in the group C, significantly reduced in comparison with that of the group A (686.40 ± 10.94) pg/ml or group B (682.31 ± 9.61) pg/ml after 24 h (P < 0.05). At the same time, the proliferation of KYSE150 cells in the group C was significantly inhibited compared with that of groups A and B after 24 h (P < 0.01). Moreover, sRAGE at the final concentration of 0.2 µg/ml inhibited the VEGF-C expression and proliferation of KYSE150 cells compared with the corresponding group without sRAGE after 24 h (P < 0.01 or P < 0.05). However, there was no significant difference of the VEGF-C expression and proliferation of KYSE150 cells with sRAGE in the group C compared with that of cells with sRAGE of the group A or group B after 24 h (P > 0.05).

CONCLUSIONSIn esophageal squamous cell carcinoma, HMGB1 can promote the VEGF-C expression and proliferation of the cancer cells through RAGE signal pathway, and HMGB1-RAGE may become a potential target for cell proliferation and lymph node metastasis of this cancer.

Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Dependovirus ; genetics ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; HMGB1 Protein ; biosynthesis ; genetics ; Humans ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism ; Signal Transduction ; Transfection ; Vascular Endothelial Growth Factor C ; metabolism

OBJECTIVETo investigate the expression and release of high mobility group Box-1 protein (HMGB1) in the lung tissue of mice with respiratory syncytial virus (RSV) infection.

METHODSEighteen mice were randomized into PBS control group, RSV group and RSV/ribavirin group. Seven days after RSV infection in the mice in the latter two groups, the bronchoalveolar lavage fluid (BALF) was collected for cell counting and classification, and the levels of IL-4, IFN-gamma and HMGB1 in the supernatants of the BALF were detected. The left lungs of the mice were harvested for pathological examination with HE staining, and the right lungs were taken for detecting the expression of HMGB1 by Western blotting.

RESULTSRSV induced a TH1 inflammation in the lung tissue as shown by significantly increased IFN-gamma and decreased IL-4 levels in the BALF. The total BALF cells, neutrophils and macrophages in the RSV group were significantly higher than those in the control group (P<0.05), and the cell counts were significantly decreased by ribavirin treatment (P<0.05). HE staining showed neutrophil and lymphocyte infiltration in the lumen and submucous layer of the airway in RSV group. The level of HMGB1 in the BALF significantly increased in the RSV group as compared with that in the control group (P<0.05), but was lowered by ribavirin treatment (P<0.05). The expression of the HMGB1 in the lung tissue significantly increased in the RSV group in comparison with that in the control group (P<0.05), and was not significantly decreased by ribavirin treatment (P>0.05).

CONCLUSIONSThe increased expression and release of HMGB1 in the lung tissue of mice with RSV infection is probably involved in the development of RSV infection-related lung diseases.

Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; Female ; HMGB1 Protein ; biosynthesis ; genetics ; Lung ; metabolism ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Respiratory Syncytial Virus Infections ; metabolism

OBJECTIVETo investigate the time course change in myocardial high mobility group box-1 (HMGB1) after myocardial infarction in rats.

METHODSMyocardial infarction (MI) was induced in SD rats by ligation of the anterior descending coronary artery. At 1, 2, 4, and 8 weeks after MI, the cardiac function of the rats was examined, and the expressions of HMGB1 at mRNA and protein levels in the myocardium were detected using real-time RT-PCR and Western blotting, respectively.

RESULTSCardiac function test confirmed that the MI model was successfully induced. The expression of HMGB1 mRNA was increased in early stage (1 week) after MI, while significantly down-regulated in later stage (4-8 weeks after MI). HMGB1 protein showed a similar biphasic pattern of changes, and was up-regulated early (1-2 weeks) after MI (P<0.05) and decreased markedly (P<0.01) at 8 weeks.

CONCLUSIONSAs an inflammatory regulator, HMGB1 can modulate inflammatory response early time after MI and functions later as a transcriptional modulator, thus contributing to the myocardial repair after MI. Interventions targeting HMGB1 in different stages after MI may prove helpful in reducing the complications, improving the prognosis and promoting long-term survival.

Animals ; Blotting, Western ; HMGB1 Protein ; genetics ; metabolism ; Male ; Myocardial Infarction ; physiopathology ; Myocardium ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

OBJECTIVETo study the effect of progesterone on interleukin-6 (IL-6) release from human umbilic vein endothelial cells (HUVECs) induced by high mobility group box-1 protein (HMGB1).

METHODThe recombinant expression plasmid pET14b-HMGB1 was constructed and transformed into competent E.coli BL21 cells to obtain HMGB1 protein, which was purified with chromatography on Ni-NTA Sepharose column. Cultured HUVECs were treated with purified HMGB1 protein alone at the concentrations 0, 10, 100, 500, and 1000 ng/ml, progesterone alone at the concentrations of 0, 0.1, 1, 10, 100 mmol/L, or with both HMGB1 protein (500 ng/ml) and progesterone at the terminal concentrations of 0, 0.1, 1, 10, and 100 mmol/L. Twenty-four hours later, the supernatant of the cell culture medium was collected to detect the levels of IL-6 using enzyme-linked immunosorbent assay (ELISA).

RESULTSThe IL-6 levels in HUVEC culture medium was slightly decreased after treatment with low-concentration HMGB1 but increased obviously following treatment with high-concentration HMGB1, and these effects could be dose-dependently inhibited by progesterone. Progesterone alone did not result in any noticeable changes of IL-6 levels in the cell culture medium.

CONCLUSIONSProgesterone can dose-dependently inhibit HMGB1-induced IL-6 release from HUVECs, suggesting the protective role of progesterone in endotoxemia.

Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelial Cells ; cytology ; metabolism ; HMG-Box Domains ; HMGB1 Protein ; pharmacology ; Humans ; Interleukin-6 ; biosynthesis ; Progesterone ; pharmacology ; Umbilical Veins ; cytology ; metabolism

OBJECTIVETo investigate the expression of HMGB1 and RAGE mRNA in the lungs of asthmatic mice and the effect of N-acetylcysteine (NAC) on their expression.

METHODSTwenty-one female BALB/c mice were randomly divided into control group, asthma group and NAC group (n=7). The expressions of HMGB1 and RAGE mRNA and their distributions in the lungs were detected by RT-PCR and immunohistochemical method.

RESULTSThe expression levels of HMGB1 and RAGE mRNA were not significantly different between the control group (0.88-/+0.02 and 1.20-/+0.20, respectively) and the asthma model group (0.86-/+0.05 and 1.21-/+0.08, P>0.05). After NAC treatment, both of HMGB1 and RAGE mRNA levels (0.98-/+0.05 and 1.58-/+0.21) were significantly higher than those in the other two groups (P<0.05). HMGB1 was found in the nuclei and membrane of the bronchial and alveolar epithelial cells, and RAGE was located on the membrane of the alveolar epithelial cells.

CONCLUSIONHMGB1 and RAGE may play a role in the oxidative stress during asthma, but the exact mechanism needs further investigation.

Acetylcysteine ; pharmacology ; Animals ; Asthma ; physiopathology ; Female ; Free Radical Scavengers ; pharmacology ; Gene Expression ; drug effects ; HMGB1 Protein ; biosynthesis ; genetics ; Immunohistochemistry ; Lung ; drug effects ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Mitogen-Activated Protein Kinases ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the change in renal high mobility group box-1 protein (HMGB1) levels, and the effect of Chinese traditional medicine-Xuebijing injection on HMGB1 expression as well as acute kidney injury in rats after scald injury.

METHODSWistar rats were subjected to 30% full-thickness scald injury followed with delayed resuscitation. Totally 78 animals were divided into sham scald group (n=18), scald injury group (n=30), and Xuebijing injection treatment group (n=30). All animals were sacrificed at 8, 24, and 72 hours postburn. Renal tissue and blood samples were harvested to determine HMGB1 mRNA as well as protein expression and organ functional parameters. HMGB1 mRNA level was semi-quantitatively measured by the reverse transcription polymerase chain reaction taking GAPDH as an internal standard, and protein expressions of HMGB1 were detected by both Western blot and immunohistochemistry. Serum creatinine (Cr) contents were measured by automatic biochemistry analyzer. In addition, pathological lesions in kidney were observed under light microscope using HE staining.

RESULTSCompared with sham scald group, both mRNA and protein expressions of HMGB1 were significantly enhanced in the kidney at 8, 24, and 72 hours after scald injury (P<0.05, P<0.01), meanwhile serum Cr contents were markedly increased following acute insults (P<0.05, P<0.01). Treatment with Xuebijing injection could markedly down-regulated renal HMGB1 mRNA expression and protein release at 24 hours and 72 hours (P<0.05, P<0.01), and significantly reduced serum Cr content following scald injury (P<0.05). Many inflammatory cells in renal tissues were observed using light microscope following scald. The histological morphology of kidney lesions was a-HMGB1, a late mediator, appears to be inmeliorated after treatment with Xuebijing injection.

CONCLUSIONSvolved in the pathogenesis of excessive inflammatory response and acute kidney damage. Treatment with Xuebijing injection can inhibit HMGB1 synthesis and release in renal tissues, and may prevent the development of acute kidney injury induced by serious scald injury.

Acute Kidney Injury ; etiology ; metabolism ; prevention & control ; Animals ; Burns ; complications ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; HMGB1 Protein ; biosynthesis ; Injections ; Kidney ; drug effects ; metabolism ; Rats ; Rats, Wistar

High mobility group box-1 protein (HMGB1) has recently been shown as a crucial late mediator of inflammation and sepsis, and is involved in mediating multi-organ functional lesions, including acute lung, liver, and intestine injuries. As a delayed inflammatory cytokine, HMGB1 provides a wider therapeutic time window for clinical intervention. HMGB1 has been proven to be a promising therapeutic target to prevent the development of multiple organ dysfunction syndrome in experimental models of severe sepsis. The pharmacological strategies include neutralization of antibodies or specific HMGB1 antagonists, suppression of HMGB1 secretion (ethyl pyruvate, agonists for alpha7-nicotinic acetylcholine receptors), and down-regulation of HMGB1 expression (sodium butyrate, signaling inhibitors for Janus kinase/signal transducer and activator of transcription).
HMGB1 Protein ; antagonists & inhibitors ; biosynthesis ; physiology ; Humans ; Multiple Organ Failure ; immunology ; metabolism ; prevention & control ; Sepsis ; immunology ; metabolism ; therapy

Animals ; Endotoxins ; Female ; HMGB1 Protein ; biosynthesis ; Liver Failure, Acute ; chemically induced ; metabolism ; Male ; Rats ; Rats, Wistar