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*

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 assess the effect of tumor cell lysate (TCL) with low high-mobility group B1 (HMGB1) content for enhancing immune responses of dendritic cells (DCs) against lung cancer. METHODS: TCLs with low HMGB1 content (LH-TCL) and normal HMGB1 content (NH-TCL) were prepared using Lewis lung cancer (LLC) cells in which HMGB1 was inhibited with 30 nmol/L glycyrrhizic acid (GA) and using LLC cells without GA treatment, respectively. Cultured mouse DCs were exposed to different doses of NH-TCL and LH-TCL, using PBS as the control. Flow cytometry was used to detect the expressions of CD11b, CD11c and CD86 and apoptosis of the stimulated DCs, and IL-12 levels in the cell cultures were detected by ELISA. Mouse spleen cells were co-cultured with the stimulated DCs, and the activation of the spleen cells was assessed by detecting CD69 expression using flow cytometry; TNF-β production in the spleen cells was detected with ELISA. The spleen cells were then co-cultured with LLC cells at the effector: target ratios of 5:1, 10:1 and 20:1 to observe the tumor cell killing. In the animal experiment, C57/BL6 mouse models bearing subcutaneous LLC xenograft received multiple injections with the stimulated DCs, and the tumor growth was observed. RESULTS: The content of HMGB1 in the TCL prepared using GA-treated LLC cells was significantly reduced (P < 0.01). Compared with NH-TCL, LH-TCL showed a stronger ability to reduce apoptosis (P < 0.001) and promote activation and IL- 12 production in the DCs. Compared with those with NH-TCL stimulation, the DCs stimulated with LH-TCL more effectively induced activation of splenic lymphocytes and enhanced their anti-tumor immunity (P < 0.05). In the cell co-cultures, the spleen lymphocytes activated by LH-TCL-stimulated DCs showed significantly enhanced LLC cell killing activity (P < 0.01). In the tumor-bearing mice, injections of LH-TCL-stimulated DCs effectively activated host anti-tumor immunity and inhibited the growth of the tumor xenografts (P < 0.05). CONCLUSION Stimulation of the DCs with LH-TCL enhances the anti-tumor immune activity of the DCs and improve the efficacy of DCbased immunotherapy for LLC in mice.
Animals ; Humans ; Mice ; Apoptosis ; Dendritic Cells/immunology* ; Glycyrrhizic Acid/pharmacology* ; HMGB1 Protein ; Lung Neoplasms/immunology*

OBJECTIVETo investigate the effect of hydrogen dioxide (H(2)O(2)) on the release and translocation of high mobility group box 1 release (HMGB1) from normal human bronchiolar epithelial cells (HBE).

METHODSMTT assay was used to assess the viability of HBE135-E6E7 cells exposed to different concentrations of H(2)O(2). The expression and location of HMGB1 in the cytoplasm, nuclei and culture medium of the exposed cells were determined using Western blotting and immunofluorescence assay.

RESULTSExposure to 125 µmmol/L H(2)O(2) did not obviously affect the cell viability. At the concentration of 250 µmmol/L, H(2)O(2) significantly decreased the cell viability (P<0.05), but significant cell death occurred only after exposure to 400 µmmol/L H(2)O(2) (P=0.000). Compared with the control cells, the cells exposed to 12.5, 125 and 250 µmmol/L H(2)O(2) for 24 h showed significantly increased levels of HMGB1 in the culture medium (P<0.05), and exposure to 125 µmmol/L H(2)O(2) for 12 and 24 h also caused significantly increased HMGB1 level (P<0.05). Exposure to 125 µmmol/L H(2)O(2) for 24 h significantly increased HMGB1 expression in the cytoplasm but decreased its expression in the nucleus. HMGB1 translocation from the nuclei to the cytoplasm and to the plasmalemma occurred after 125 µmmol/L H(2)O(2) exposure for 12 h and 24 h, respectively.

CONCLUSIONH(2)O(2) can induce HMGB1 translocation and release in human bronchial epithelial cells, suggesting the involvement of HMGB1 in airway oxidative stress in chronic inflammatory diseases such as asthma and COPD.

Bronchi ; cytology ; Cell Line ; Epithelial Cells ; drug effects ; metabolism ; HMGB1 Protein ; drug effects ; metabolism ; Humans ; Hydrogen Peroxide ; pharmacology ; Protein Transport

The cell counting kit-8 (CCK-8) proliferation assay and diameter measure of neurospheres were used to investigate the effect of high-mobility group box 1 (HMGB1) on proliferation of primary rat neural stem cells (NSCs) in vitro, and c-Jun N-terminal protein kinase (JNK) potent inhibitor SP600125 was used to study the mechanism. The results demonstrated that HMGB1 significantly increased CCK-8 absorbance values and neurosphere diameters at concentrations of 1 and 10 ng/mL at 48 h and 72 h (P < 0.05), and the other HMGB1 concentration groups (0.01, 0.1, 100 ng/mL) showed no significant difference, compared with control group (P > 0.05). HMGB1 at 10 ng/mL significantly increased the NSCs proliferation accompanied by the rising of phosphorylated JNK levels (P < 0.01), and 10 μmol/L SP600125 prevented these effects in HMGB1-cultured NSCs (P < 0.01). In conclusion, low concentration of HMGB1 (1-10 ng/mL) can increase NSCs proliferation, which may play a role by promoting JNK phosphorylation.
Animals ; Cell Proliferation ; Cells, Cultured ; HMGB1 Protein ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Neural Stem Cells ; cytology ; Phosphorylation ; Rats

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

OBJECTIVETo test the effect of high-mobility group box protein 1 (HMGB1) alone or in synergy with interleukin-1β (IL-1β) on the expression of IL-8 in human airway epithelial cells in vitro.

METHODSHuman airway epithelial 16HBE and A549 cell lines were incubated with HMGB1 (100 ng/ml) in the absence or presence of IL-1β (10 ng/ml) for 24 h, and the changes of IL-8 mRNA and protein expressions were assessed using quantitative PCR and enzyme-linked immunosorbent assay (ELISA).

RESULTSIn the two human airway epithelial cell lines, HMGB1 alone did not produce obvious effect on the expression of IL-8, but in the presence of IL-1β, HMGB1 caused a significant increase of IL-8 expressions at both the mRNA and protein levels.

CONCLUSIONHMGB1 in synergy with IL-1β increases the expression of IL-8 in human airway epithelial cells, which provides new evidence that HMGB1 contributes to neutrophilic airway inflammation by regulating IL-8 expression.

Bronchi ; cytology ; drug effects ; Cell Line ; Epithelial Cells ; drug effects ; metabolism ; HMGB1 Protein ; pharmacology ; Humans ; Inflammation ; Interleukin-1beta ; pharmacology ; Interleukin-8 ; metabolism ; RNA, Messenger

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

OBJECTIVE: To investigate the protective effect against intestinal mucosal injury in rats following traumatic brain injury (TBI) and explore the underlying mechanism. METHODS: SD rat models of TBI were established by fluid percussion injury (FPI), and the specimens were collected at 12, 24, 48, and 72 h after TBI. Another 15 rats were randomly divided into shamoperated group (n=5), TBI with saline treatment (TBI+NS) group (n=5), and TBI with PD treatment (TBI+PD) group (treated with 30 mg/kg PD after TBI; n=5). Body weight gain and fecal water content of the rats were recorded, and after the treatments, the histopathology of the jejunum was observed, and the levels of D-lactic acid (D-LAC), diamine oxidase (DAO), ZO-1, claudin-5, and reactive oxygen species (ROS) were detected. Lipid peroxide (LPO) and superoxide dismutase (SOD) 2 content, jejunal pro-inflammatory factors (IL-6, IL-1β, and TNF- α), Sirt1 activity, SOD2 and HMGB1 acetylation level were also determined after the treatments. RESULTS: The rats showed significantly decreased body weight and fecal water content and progressively increased serum levels of D-LAC and DAO after TBI (P < 0.05) with obvious jejunal injury, significantly decreased expression levels of ZO-1 and claudin-5, lowered SOD2 and Sirt1 activity (P < 0.05), increased expression levels of LPO, ROS, and pro-inflammatory cytokines, and enhanced SOD2 and HMGB1 acetylation levels (P < 0.05). Compared with TBI+NS group, the rats in TBI+PD group showed obvious body weight regain, increased fecal water content, reduced jejunal pathologies, decreased D-LAC and DAO levels (P < 0.05), increased ZO-1, claudin-5, SOD2 expression levels and Sirt1 activity, and significantly decreased ROS, LPO, pro-inflammatory cytokines, and acetylation levels of SOD2 and HMGB1 (P < 0.05). CONCLUSION PD alleviates oxidative stress and inflammatory response by activating Sirt1-mediated deacetylation of SOD2 and HMGB1 to improve intestinal mucosal injury in TBI rats.
Animals ; Brain Injuries, Traumatic ; Glucosides/pharmacology* ; HMGB1 Protein/metabolism* ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Sirtuin 1/metabolism* ; Stilbenes/pharmacology* ; Superoxide Dismutase/metabolism*

OBJECTIVETo examine the synergistic effect of recombinant human high mobility group box 1 (HMGB1) protein and lipopolysaccharides (LPS) on the release of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) in human umbilic vein endothelial cells (HUVECs), and explore the role of mitogen-activated protein kinases (MAPK) signal transduction in cytokine release.

METHODSHUVECs were incubated with recombinant HMGB1 (0-75 ng/ml) for 24 h and the culture medium supernatant was harvested for detection of IL-8 and MCP-1 with LiquiChip system. At 0, 1, 3, 6, 12 and 24 h after stimulation with 15 ng/ml HMGB1 or 15 ng/ml HMGB1 plus 10 ng/ml LPS, the levels of IL-8 and MCP-1 in the HUVECs were examined. To test the effect of MAPK inhibitors, HUVCs were pretreated with the inhibitors SB203580 (20 mol/L), PD98059 (20 mol/L), and JNK inhibitor II (50 nmol/L) 1 h before HMGB1 and LPS stimulation.

RESULTSThe levels of IL-8 and MCP-1 were significantly increased in the HUVECs stimulated with HMGB1 protein at the concentrations of 3, 15 and 75 ng/ml in comparison with the control levels (P<0.01). Since 3-6 h after the stimulation with HMGB1, the levels of IL-8 and MCP-1 began to increase gradually, and steadily increased at 12 and 24 h, all significantly higher than those of the control group (P<0.01). Stimulation of the HUVECs with LPS (10 ngHMGB1 (15 ng/ml) alone resulted in significantly increased levels of IL-8 and MCP-1 (P<0.01), which were further increased after costimulation with LPS and HMGB1, suggesting a synergistic effect between HMGB1 and LPS (P<0.01). This synergistic effect was significantly inhibited by pretreatment with MAPK signaling kinases inhibitors, especially the p38 MAP kinase inhibitor SB203580, and the cocktail of MAP kinase inhibitors almost totally blocked the expression of these chemokines in HUVECs treated with HMGB1 and LPS.

CONCLUSIONHMGB1 protein can activate HUVECs to produce the chemokines IL-8 and MCP-1 in a dose- and time-dependent manner. HMGB1 also acts synergistically with LPS to induce IL-8 and MCP-1 release, which might play an important role in the development of sepsis. MAPK signal transduction plays an important role in HMGB1 and LPS-induced IL-8 and MCP-1 release.

Cell Line ; Chemokine CCL2 ; blood ; metabolism ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; metabolism ; HMGB1 Protein ; pharmacology ; Humans ; Interleukin-8 ; blood ; metabolism ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Time Factors