The aim of this study was to investigate the effects of dexmedetomidine (Dex) on hepatic ischemia/reperfusion injury (HIRI) and the underlying mechanism. The in vitro HIRI was induced by culturing HL-7702 cells, a human hepatocyte cell line, under 24 h of hypoxia and 12 h of reoxygenation. Quantitative real time PCR (qRT-PCR) and Western blot were performed to detect the expression levels of long non-coding RNA MALAT1, microRNA-126-5p (miR-126-5p) and high mobility group box-1 (HMGB1). Bioinformatics prediction and double luciferase assay were used to verify the targeting relationship between miR-126-5p and MALAT1, HMGB1. Reactive oxygen species (ROS), malondialdehyde (MDA) and ATP levels in culture medium were detected by corresponding kits. The results showed that Dex significantly reduced the levels of ROS and MDA, but increased the level of ATP in HL-7702 cells with HIRI. HIRI up-regulated the expression levels of MALAT1 and HMGB1, and down-regulated the level of miR-126-5p. Dex reversed these effects of HIRI. Furthermore, Dex inhibited HIRI-induced cellular apoptosis, whereas MALAT1 reversed the effect of Dex. This inhibitory effect of Dex could be restored by up-regulation of miR-126-5p. The results suggest that Dex protects hepatocytes from HIRI via regulating MALAT1/miR-126-5p/HMGB1 axis.
Dexmedetomidine/pharmacology* ; HMGB1 Protein/genetics* ; Humans ; MicroRNAs/genetics* ; RNA, Long Noncoding/genetics* ; Reperfusion Injury/genetics*

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.
Animals ; Rats ; Caspase 8 ; Pyroptosis ; HMGB1 Protein/genetics* ; Hypertension, Pulmonary/etiology* ; Pulmonary Disease, Chronic Obstructive/genetics*

Objective: To investigate the effect of silencing the high-mobility group box-1 protein (HMGB1) combined with docetaxel (DTX) on the proliferation and apoptosis of PCa cells and its possible action mechanism. METHODS: The expression of HMGB1 mRNA in different PCa cell lines and normal prostatic epithelial cells was detected by RT-qPCR. The PC-3 cells were transfected with different HMGB1 small interfering RNAs (si-HMGB1, si-HMGB1-2 and si-HMGB1-3), and the silencing effect was detected. The effects of different concentrations of DTX on the proliferation of the PC-3 cells was determined by MTT. Then the PC-3 cells were randomly divided into five groups: control (conventional culture), si-HMGB1-NC (si-HMGB1-NC transfection), si-HMGB1 (si-HMGB1-3 transfection), DTX (20 nmol/L DTX), and si-HMGB1+DTX (si-HMGB1-3+20 nmol/L DTX transfection), followed by measurement of the survival rate of the cells by MTT, their apoptosis rate by flow cytometry, and the expressions of HMGB1, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X (Bax) proteins in different groups by Western blot. RESULTS: The expression of HMGB1 mRNA in the PC-3 cells was the highest and the lowest after transfection with si-HMGB1-3. DTX inhibited the proliferation of the PC-3 cells at various concentrations. Compared with the control group, the si-HMGB1 and DTX groups showed significantly decreased A values, cell survival rates and HMGB1 and Bcl-2 expressions, but increased cell apoptosis rates and Bax expressions (P < 0.05). In comparison with the si-HMGB1 and DTX groups, the si-HMGB1+DTX group exhibited a remarkably decreased A value, cell survival rate and Bcl-2 expression, but increased cell apoptosis and Bax expression. The expression of the HMGB1 protein was markedly lower in the si-HMGB1+DTX than in the DTX group (P < 0.05). CONCLUSIONS Silencing HMGB1 combined with DTX chemotherapy can inhibit the proliferation and promote the apoptosis of PCa cells, which may be attributed to its regulatory effect on the expressions of the Bcl-2 family-related proteins.、.
Apoptosis ; Cell Proliferation ; Docetaxel/pharmacology* ; HMGB1 Protein/genetics* ; Humans ; Male ; Prostatic Neoplasms/genetics*

OBJECTIVE: To study the expression of high-mobility group box 1 (HMGB1) in neonates with sepsis and its role in the pathogenesis of neonatal sepsis. METHODS: A total of 62 neonates with sepsis were enrolled as the sepsis group, 66 neonates with local infection were enrolled as the local infection group, and 70 healthy neonates were enrolled as the healthy control group. Serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-17 (IL-17), interleukin-23 (IL-23), C-reactive protein (CRP) and procalcitonin (PCT) were measured. The mRNA expression of HMGB1, Toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) and the protein expression of TLR4 and NF-κB in peripheral blood mononuclear cells (PBMCs) were also measured. PBMCs from healthy neonates were divided into 4 groups: control, HMGB1 treatment, HMGB1+TAK-242 (a TLR4 inhibitor) treatment and HMGB1+PDTC (an NF-κB inhibitor) treatment, and the mRNA expression of TLR4, NF-κB and IL-8 and the protein expression of TLR4 and NF-κB were measured. PBMCs from healthy neonates were divided into another 3 groups: control, LPS treatment and LPS+glycyrrhizin (an HMGB1 inhibitor) treatment, and the mRNA expression of HMGB1, TLR4, NF-κB and IL-8 and the protein expression of TLR4 and NF-κB were measured. RESULTS: Compared with the local infection and healthy control groups, the sepsis group had significantly higher serum levels of IL-6, IL-8, IL-17, IL-23, CRP and PCT (P<0.05), as well as significantly higher mRNA expression of HMGB1, TLR4 and NF-κB and protein expression of TLR4 and NF-κB in PBMCs (P<0.05). HMGB1 significantly induced the mRNA and protein expression of TLR4 and NF-κB in PBMCs (P<0.05). TAK-242 inhibited the mRNA and protein expression of TLR4 and NF-κB and mRNA expression of IL-8 (P<0.05). PDTC inhibited the mRNA and protein expression of NF-κB and the mRNA expression of IL-8 (P<0.05). LPS significantly induced the mRNA expression of HMGB1 and the mRNA and protein expression of TLR4 and NF-κB and then stimulated the mRNA expression of IL-8 (P<0.05). Glycyrrhizin inhibited the mRNA expression of HMGB1 and the mRNA and protein expression of TLR4 and NF-κB and then reduced the mRNA expression of IL-8 (P<0.05). CONCLUSIONS HMGB1 plays an important role in the pathogenesis of neonatal sepsis by activating the TLR4/NF-κB signaling pathway and inducing the secretion of inflammatory factors including IL-8. The HMGB1 blocker glycyrrhizin can inhibit activation of the TLR4/NF-κB signaling pathway and the secretion of inflammatory factors.
HMGB1 Protein ; genetics ; Humans ; Infant, Newborn ; Leukocytes, Mononuclear ; NF-kappa B ; Sepsis ; genetics ; Signal Transduction

OBJECTIVETo investigate the effect of decreased expression of high mobility group Box-1 on the proliferation and apoptosis of HepG2 cells.

METHODSThree specific siRNAs of HMGB1 were designed and synthesized, and were transiently transfected into HepG2 cells by Lipofectamine 2000. The HMGB1 expression in HepG2 cells was detected by RT-PCR and Western blotting respectively. The proliferation activity in vitro was assessed by MTT assay. In situ apoptosis was evaluated by terminal deoxynucleotidyl transferase-deoxyuridine triphosphate nick end labeling (TUNEL) assay.

RESULTSAll of these specific HMGB1-siRNAs (1, 2, 3) efficiently and specifically inhibited the expression of the HMGB1 gene, and the levels of HMGB1 mRNA were 1.147+/-0.024, 1.014+/-0.042, 0.435+/-0.055, respectively, in HMGB1-siRNAs transfection group, which were significantly lower than that in Lipofectamine 2000 alone group (1.411+/-0.065, P < 0.01). Correspondingly, all of these specific HMGB1-siRNAs (1, 2, 3) could efficiently and specifically inhibit the expression of the HMGB1 protein, and the levels of HMGB1 protein were 0.369+/-0.035, 0.340+/-0.028, 0.097+/-0.020, respectively, in HMGB1-siRNAs transfection group, which were significantly lower than that in Lipofectamine 2000 alone group (0.553+/-0.051, P < 0.01). Of the 3 specific HMGB1-siRNAs, HMGB1-siRNA-3 (siRNAH3) had the highest inhibition rate (80%). The proliferation of HepG2 cells was markedly inhibited by siRNAH3 transfection. Compared to mock-transfection, siRNAH3 transfection dramatically suppressed the proliferation of HepG2 cells (P < 0.01). Moreover, siRNAH3 can induce apoptosis (P < 0.01).

CONCLUSIONsiRNA targeting HMGB1 mRNA can specifically reduce HMGB1 gene and protein expression. siRNAH3 can effectively suppress the proliferation and induce apoptosis of HepG2 cells.

Apoptosis ; Cell Proliferation ; HMGB1 Protein ; genetics ; Hep G2 Cells ; Humans ; RNA, Small Interfering

This study was aimed to establish a stable subline of K562 cells (K562-HMGB1) overexpressing HMGB1 protein and K562-HMGB1 sublines served as control, so as to provide a basis for exploring the role of hmgb1 gene in occurrence and development of leukemia and their mechanism. Protein-coding gene of hmgb1 was amplified by PCR with cDNA as template, which was synthesized by reverse transcription from total RNA extracted from U937 cells. The PCR-amplified hmgb1 gene was ligated into PMD18-T vector (PMD18-T-HMGB1 vector), and then transformed into E. coli strain DH5α. DH5α containing PMD18-T-HMGB1 vector were grown on LB agar plate supplemented with 100 µg/ml ampicillin overnight. The single ampicillin-selected DH5α clone was picked for culturing overnight and then harvested for plasmid extraction. The extracted plasmid was characterized to contain hmgb1 gene digested with the desired restriction enzymes of KpnI/XhoI. The correctness of hmgb1 sequence was confirmed with DNA sequencing. The insert of hmgb1 gene contained in PMD18-T-HMGB1 vector was cut out with restriction enzymes of KpnI/XhoI and then ligated into eukaryotic expression vector pcDNA3.1 to form pcDNA3.1-HMGB1 vector. 10µg of pcDNA3.1-HMGB1 or pcDNA3.1 plasmid was separately electroporated into K562 cells. At 48 hours after electroporation the cells were cultured with G418 at a final concentration of 800 µg/ml for over 2 weeks. Finally stably transfected sublines of K562 cells containing hmgb1 gene (K562-HMGB1), and of K562 containing pcDNA3.1 vector (K562-pcDNA3.1) served as a control, were obtained. The transcriptional or translational expression of hmgb1 gene was detected with RT-PCR or Western blot, respectively, to testify transfected efficiency and validity of stable subline of K562-HMGB1. The results indicated that the eukaryotic expression vector pcDNA3.1-HMGB1 plasmid was successfully constructed and was electroporated into K562 cells. The transcriptional or translational expression of hmgb1 gene in the stable subline of K562 cells containing hmgb1 gene was overexpressed. It indicated that stable subline of K562-HMGB1 cells was successfully established. It is concluded that the stable sublines of K562-HMGB1 cells or K562-pcDNA3.1 cells are successfully established, which provides a basis for exploring the roles and mechanisms of hmgb1 gene in leukemogenesis and development of leukemia.
Gene Expression ; Genes, Regulator ; Genetic Vectors ; HMGB1 Protein ; genetics ; Humans ; K562 Cells ; metabolism ; Plasmids ; Transformation, Genetic

OBJECTIVETo investigate the effect of high mobility group boxl (HMGBI) gene silence on adriamycin (ADM)-induced apoptosis in K562/A02 drug resistance leukemia cells.

METHODSK562/ A02 cells were transient transfected with HMGB1- small interference RNA(siRNA) vector, and the levels of HMGB1 gene differential expression pre-and post-transfection were measured by RT-PCR and Western blotting. 50% inhibition concentration (IC50) of ADM on K562/A02 was determined by WST-8 assay. Cell apoptosis was assessed by flow cytometry. The release of Smac/DIABLO from the mitochondria to the cytoplasm was assayed by Western blotting. Activity of Caspase-3 was assayed with a Caspase Colorimetric Assay Kit.

RESULTS(1) The HMGB1 expression at mRNA and protein levels in HMGB1 siRNA transfected K562/A02 cells were decreased by 86% and 71% respectively compared with control. (2) Suppression of HMGB1 by siRNA in K562/A02 cells resulted in a reversal of the resistance to ADM, and decreased IC50 from (4.83 +/- 0.08) microg/ml to (1.33 +/- 0.10) microg/ml. 1 microg/ml and 5 microg/ml of ADM treatment increased cell apoptotic rate by 27% and 32% respectively. (3) HMGB1 suppression in K562/A02 cells significantly promoted ADM- induced Smac/DIABLO release from the mitochondria to the cytoplasm, and increased the activities of Caspase-3.

CONCLUSIONHMGB1 gene silence can enhance sensitivity of K562/A02 cells to ADM and reverse cell resistant to ADM.

Apoptosis ; drug effects ; genetics ; Doxorubicin ; pharmacology ; Gene Silencing ; HMGB1 Protein ; genetics ; Humans ; K562 Cells ; RNA, Small Interfering ; genetics

Objective: To explore the pathogenic mechanism of the miR-340/high mobility group box 1 (HMGB1) axis in the formation of liver fibrosis. Methods: A rat liver fibrosis model was established by injecting CCl(4) intraperitoneally. miRNAs targeting and validating HMGB1 were selected with gene microarrays after screening the differentially expressed miRNAs in rats with normal and hepatic fibrosis. The effect of miRNA expressional changes on HMGB1 levels was detected by qPCR. Dual luciferase gene reporter assays (LUC) was used to verify the targeting relationship between miR-340 and HMGB1. The proliferative activity of the hepatic stellate cell line HSC-T6 was detected by thiazolyl blue tetrazolium bromide (MTT) assay after co-transfection of miRNA mimics and HMGB1 overexpression vector, and the expression of extracellular matrix (ECM) proteins type I collagen and α-smooth muscle actin (SMA) was detected by western blot. Statistical analysis was performed by analysis of variance and the LSD-t test. Results: Hematoxylin-eosin and Masson staining results showed that the rat model of liver fibrosis was successfully established. Gene microarray analysis and bioinformatics prediction had detected eight miRNAs possibly targeting HMGB1, and animal model validation had detected miR-340. qPCR detection results showed that miR-340 had inhibited the expression of HMGB1, and a luciferase complementation assay suggested that miR-340 had targeted HMGB1. Functional experiments results showed that HMGB1 overexpression had enhanced cell proliferation activity and the expression of type I collagen and α-SMA, while miR-340 mimics had not only inhibited cell proliferation activity and the expression of HMGB1, type I collagen, and α-SMA, but also partially reversed the promoting effect of HMGB1 on cell proliferation and ECM synthesis. Conclusion: miR-340 targets HMGB1 to inhibit the proliferation and ECM deposition in hepatic stellate cells and plays a protective role during the process of liver fibrosis.
Animals ; Rats ; Cell Proliferation ; Collagen Type I/metabolism* ; Fibrosis ; Hepatic Stellate Cells ; HMGB1 Protein/genetics* ; Liver Cirrhosis/pathology* ; MicroRNAs/metabolism*

High-mobility group box 1 (HMGB1) protein has been demonstrated to play an important role in chronic inflammatory diseases including rheumatoid arthritis, and systemic lupus erythematosus. This study investigated the association between extracellular HMGB1 expression and disease activity, and clinical features of Behcet's disease (BD). Extracellular HMGB1 expression in the sera of 42 BD patients was measured and was compared to that of 22 age- and sex-matched healthy controls. HMGB1 expression was significantly increased in BD patients compared to healthy controls (78.70 +/- 20.22 vs 10.79 +/- 1.90 ng/mL, P = 0.002). In addition, HMGB1 expression was significantly elevated in BD patients with intestinal involvement compared to those without (179.61 +/- 67.95 vs 61.89 +/- 19.81 ng/mL, P = 0.04). No significant association was observed between HMGB1 concentration and other clinical manifestations, or disease activity. It is suggested that extracellular HMGB1 may play an important role in the pathogenesis of BD.
Adult ; Aged ; Behcet Syndrome/genetics/*metabolism/pathology ; Extracellular Space/metabolism ; Female ; HMGB1 Protein/genetics/*metabolism ; Humans ; Inflammation ; Intestinal Diseases/blood/genetics ; Male ; Middle Aged ; Young Adult

OBJECTIVETo determine the role of the novel proinflammatory cytokine high mobility group box chromosomal protein 1 (HMGB-1) in the pathogenesis of lupus nephritis.

METHODSSerum levels of anti-dsDNA antibodies were determined by enzyme linked immunosorbent assay (ELISA). Renal morphologic features were examined by light microscopy, electron microscopy, and immunohistologic analyses. The mRNA expression of HMGB-1 and monocyte chemoattractant protein-1 (MCP-1) was detected by RT-PCR.

RESULTMRL/lpr mice demonstrated characteristic alterations of serum immune parameters, with progressively increased anti-dsDNA antibodies with age, compared with age-matched C57BL/6J mice. MRL/lpr mice showed progressive development of renal damage, starting at 12 weeks of age and reached the peak at 20 weeks. The observed lesions included the presence of enlarged hypercellular glomeruli, with increased numbers of both resident cells and infiltrating leukocytes. Higher expression of HMGB-1 mRNA was found in MRL/lpr mice than what in C57BL/6J mice. Expression of HMGB-1 was positively correlated with that of MCP-1 mRNA.

CONCLUSIONThe results demonstrate that the higher expression of HMGB-1 may contribute to the pathogenesis of lupus nephritis.

Animals ; Chemokine CCL2 ; metabolism ; Disease Models, Animal ; HMGB1 Protein ; genetics ; metabolism ; Kidney ; metabolism ; pathology ; Lupus Nephritis ; metabolism ; pathology ; Mice ; Mice, Inbred MRL lpr ; RNA, Messenger ; genetics