HMGB1 mRNA Expression Research Articles

Effect of HMGB1 on human hepatoma cell line-HepG2 proliferation

To investigate the effect of high mobility group box-1 protein (HMGB1) on the proliferative activity of human hepatoma cell line HepG2 and its potential regulating mechanism. The cultured HepG2 cells were treated with recombinant HMGB1 (0, 10, 50, and 100 ng/mL, respectively) for 24 h. Cell proliferation was observed by MTT analysis. Western blot and reverse transcriptase-polymerase chain reaction were used to detect the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 protein and mRNA, respectively. Compared with the control group, HMGB1 at 10, 50, and 100 ng/mL obviously increased HepG2 cells proliferation, cyclin D1 and PCNA protein and mRNA expression after the treatment for 24 h, respectively (P<0.05). Anti-HMGB1 significantly inhibited the proliferation and cyclin D1 and PCNA mRNA and protein expression of HMGB1 on HepG2 cells (P<0.05). Proliferation of HMGB1 on HepG2 cells may be associated with increasing cyclin D1 and PCNA expression. Anti-HMGB1 may have a therapeutic effect on hepatocellular carcinoma.

Role of high-mobility group box 1 protein in post-infarction healing process and left ventricular remodelling

High-mobility group box 1 protein (HMGB1) is one of the recently defined damage-associated molecular pattern molecules derived from necrotic cells and activated macrophages. We investigated clinical implications of serum HMGB1 elevation in patients with acute myocardial infarction (MI). Then, we evaluated the effect of HMGB1 blockade on post-MI left ventricular (LV) remodelling in a rat MI model. Serum HMGB1 levels were examined in patients with ST-elevation MI (n = 35). A higher peak serum HMGB1 level was associated with pump failure, cardiac rupture, and in-hospital cardiac death. Then, an experimental MI model was induced in male Wistar rats. The mRNA and protein expression of HMGB1 were increased in the infarcted area compared with those values observed in sham-operated rats. We administered neutralizing anti-HMGB1 antibody (MI/anti-H) or control antibody (MI/C) to MI rats subcutaneously for 7 days. The mRNA levels of tumour necrosis factor-alpha and interleukin-1beta and the number of macrophages in the infarcted area were reduced on day 3 in MI/anti-H rats compared with MI/C rats. Interestingly, HMGB1 blockade resulted in thinning and expansion of the infarct scar and marked hypertrophy of the non-infarcted area on day 14. Elevated serum HMGB1 levels were associated with adverse clinical outcomes in patients with MI. However, HMGB1 blockade in a rat MI model aggravated LV remodelling, possibly through impairment of the infarct-healing process. HMGB1, a novel predictor of adverse clinical outcomes after MI, may have an essential role in the appropriate healing process after MI.

Effects of HMGB1 on PMN apoptosis during LPS-induced acute lung injury

Objective To observe the effects of intravenous injection of HMGB1 inhibitor sodium butyrate on changes in apoptosis of PMN during LPS-induced acute lung injury in rats and HMGB1 in vitro on human circulating PMN apoptosis, in order to clarify the role of HMGB1 in the pathogenesis of acute lung injury. Methods (1) LPS-induced acute lung injury rat model was developed by LPS infusion. At different time-points after LPS challenge in the presence or absence of sodium butyrate (SB), the rat tissue sample, peripheral blood PMNs and BALF were collected. RT-PCR was applied to examining rat lung tissue HMGB1 mRNA expression level, and Western blotting analysis was adopted to determine expression of rat lung tissue HMGB1 protein. PMN apoptotic changes were determined by flow cytometric (FCM) analysis, Giemsa staining and TdT-mediated dUTP nick end labeling (TUNEL) method. (2) Separated and purified human circulating PMN were coincubated for 24 h with different doses of HMGB1 (0, 10, 100, 1000 ng/ml, respectively) at 37 °C in 5% CO 2. PMN apoptosis rate was determined by flow cytometric (FCM) analysis and by TdT-mediated dUTP nick end labeling (TUNEL) method. Results (1) The percentage of apoptosis of PMN in rat model of LPS-induced ALI was gradually decreased as compared with that of normal control. The PMN apoptosis-initiation time and non-survival time in rat BALF prolonged significantly as compared with that of normal control. The injured rat lung tissue HMGB1 mRNA and protein expression was upregulated 6–24 h after LPS exposure; SB intervention significantly ameliorated the upregulation. In addition, the morphologic examination indicated that the edema severity and pathological changes of lung tissues were excessively aggravated in rats after LPS administration. By comparison, SB treatment diminished the severity of lung damage. Combined with lung HMGB1 expression level, the above changes indicate that the pathological changes of lung tissue were related to the injured lung HMGB1 expression, as well as apoptotic changes in PMN. (2) After coincubation of HMGB1 with human circulating PMNs, TUNEL and flow cytometry were performed. The study revealed that PMN apoptosis ratios was (40.53 ± 4.12) % in control group (PMNs + RPMI 1640 medium), (19.05 ± 2.44) % in LPS group (PMNs + RPMI 1640 medium + 10 μg/ml LPS), (40.52 ± 2.73) % in HMGB1-1 group (PMNs + RPMI 1640 medium + 10 ng/ml HMGB1), (34.89 ± 1.15) % in HMGB1-2 group (PMNs + RPMI 1640 medium + 100 ng/ml HMGB1), and (18.77 ± 3.02) % in HMGB1-3 group (PMNs + RPMI 1640 medium + 1 000 ng/ml HMGB1). There was statistical significance. Meanwhile, PMN TUNEL positive rate was (31.42 ± 4.40) %, (31.39 ± 3.80) %, (25.62 ± 2.46) %, and (17.98 ± 3.20) % in control group, HMGB1-1 group, HMGB1-2 group and HMGB1-3 group, respectively. The inhibitory effect was HMGB1 dose-depended as compared with that of control group. Conclusion After LPS challenge, high expression of rats' lung HMGB1 mRNA occurs at a later phase, but keeps for a long time. Sodium butyrate (SB) treatment attenuated LPS-induced PMN apoptosis delay and inhibition, and down-regulated HMGB1 mRNA expression of injured lung. HMGB1 in vitro inhibited human circulating PMN apoptosis markedly, and the inhibitory effect was HMGB1 dose-depended. The results demonstrated that HMGB1 may play an important role as a modulator in apoptotic changes in PMN during LPS-induced ALI. It concludes that HMGB1 may contribute to the development of PMN apoptotic changes during LPS-induced acute lung injury.

SODIUM BUTYRATE PREVENTS LETHALITY OF SEVERE SEPSIS IN RATS

This study was performed to investigate a novel strategy to pharmacologically inhibit high-mobility group box 1 protein (HMGB1) expression with sodium butyrate, a short-chain fatty acid. Using a sepsis model induced by cecal ligation and puncture (CLP), 100 male Wistar rats were randomly divided into 4 groups as follows: control group (10 rats), sham operation group (10 rats), CLP group (further randomized into 2, 6, 12, 24, 48, and 72 h post-CLP subgroups, each 10 rats), and sodium butyrate treatment group (further randomized into 12 and 24 h post-CLP subgroups, each 10 rats). Animals of all groups were killed at designated time points, and blood and tissue samples from livers, lungs, kidneys, and small intestines were harvested to determine organ damage-related variables, and HMGB1 mRNA expression was assessed by the reverse-transcription-polymerase chain reaction. In addition, we observed the effect of treatment with sodium butyrate on survival rate in septic rats. The results showed that early treatment with sodium butyrate can markedly reduce serum alanine aminotransferase, creatinine levels at 12 h, and pulmonary myeloperoxidase activity at 24 h post-CLP, and significantly improve the 1- to 6-day survival rates in animals subjected to CLP (P < 0.05-0.01). These findings suggest that HMGB1 is excessively expressed and produced in sepsis. Sodium butyrate can markedly inhibit HMGB1 mRNA expression and may have protective effect on multiple organ damage in sepsis.

The potential mechanism on signal transduction pathway in regulation of mRNA expression of high mobility group box-1 protein in septic rats

To investigate the potential role of Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway in regulation of gene expression of high mobility group box-1 protein (HMGB1) in various tissues in rats with sepsis. A sepsis model reproduced by cecal ligation and puncture (CLP), and 128 male Wistar rats were randomly divided into normal control group (n = 10), sham operation group (n = 10), CLP group (n = 60), AG490 treatment group (n = 24), and rapamycin (RPM) treatment group (n = 24). At serial time points animals in each group were sacrificed after CLP, then tissue samples were harvested to determine HMGB1 mRNA expression and STAT1/3 DNA binding activity. STAT1 activities increased rapidly in the liver, lungs and small intestine after CLP, peaking at 6 - 12 h, while it increased slowly, and still kept at mild level from 2 to 48 h in the kidneys. Compared with STAT1, lower STAT3 activities were detected only in the liver and lungs, with negative detection in the small intestine and kidneys. HMGB1 mRNA levels significantly increased in liver, lungs and small intestine at various time points after CLP respectively (P < 0.05 or P < 0.01), while they didn't change in the kidneys. Treatment with AG490 could markedly inhibit HMGB1 mRNA expression in the liver and small intestine at 24 and 48 h (P < 0.05 or P < 0.01), and in lungs at 2 h following CLP (P < 0.01). Similarly, treatment with RPM significantly decreased HMGB1 mRNA expression in the lungs at 2, 6, 24 and 48 h, in the liver at 6 and 24 h, and in the small intestine at 24 and 48 h (P < 0.05 or P < 0.01). In addition, STAT1 and STAT3 activities in the liver and lungs were significantly correlated with corresponding tissue HMGB1 mRNA expression. Peritoneal infection could extensively activate STAT1 and limitedly activate STAT3 in vital organs. Activation of JAK/STAT pathway might be involved in up-regulating the gene expression of HMGB1 and systemic inflammation secondary to severe septic challenge.

HEAT SHOCK RESPONSE INHIBITS RELEASE OF HIGH MOBILITY GROUP BOX 1 PROTEIN INDUCED BY ENDOTOXIN IN MURINE MACROPHAGES

The purpose of this study was to evaluate the kinetic changes and the localization of high-mobility group box 1 protein (HMGB1) and to observe the effect of heat shock response (HSR) on the expression and release of HMGB1 in lipopolysaccharide (LPS)-activated murine macrophage-like RAW 264.7 cells. Reverse transcriptase (RT)-PCR and Western blot were used to examine HMGB1 expression after LPS treatment. The intracellular localization of HMGB1 in normal or LPS-activated cells was investigated by immunocytochemical analysis and HMGB1 released from cultured macrophages by Western blot. HSR was performed by incubating RAW 264.7 cells at 42.5 degrees C for 1 h then recovery at 37 degrees C for 12 h. The effect of HSR on expression and release of HMGB1 was observed. The results showed that a decrease of HMGB1 mRNA expression was observed at 18 h after LPS (500 ng/mL) treatment, although the total intracellular HMGB1 protein levels were not affected. A visible translocation of HMGB1 from the nuclear to the cytoplasm was observed at 20 h after stimulation with LPS (500 ng/mL). Furthermore, HMGB1 was released into the medium by LPS-activated RAW 264.7 cells in a time- and dose-dependent manner. Heat shock pretreatment significantly inhibited LPS-induced release of HMGB1 and the translocation of HMGB1 from the nucleus to the cytoplasm in RAW 264.7 cells. These findings suggest that the release of HMGB1 by LPS-activated macrophages is a late event in the pathogenesis of sepsis and that HSR could inhibit the release and translocation of HMGB1 induced by LPS.

Effects of escharectomy during shock stage on tissue high mobility group box-1 expression and balance of pro-/anti-inflammatory response in rats after severe thermal injury

To investigate the effects of escharectomy during shock stage on tissue high mobility group box-1 protein (HMGB1) expression and balance of pro-/anti-inflammatory cytokines, and to elucidate the potential mechanism underlying beneficial effect of early escharectomy after severe burns. Wistar rats inflicted by 30% full-thickness thermal injury were randomly divided into thermal injury group, 24 h escharectomy group and 72 h escharectomy group, in which escharectomy were performed at 24 and 72 h postburn, respectively. Gene expression of HMGB1, interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-alpha) in liver and lungs was detected with reverse-transcription PCR, and protein levels of IL-10 and TNF-alpha in liver and lung tissues were measured by ELISA. The plasma AST and ALT contents, and pulmonary myeloperoxidase (MPO) activity were also assayed. The mRNA expression of HMGB1 and TNF-alpha in liver and lungs was up-regulated on postburn day 2, with IL-10 over-expression on postburn day 8. In the 24 h escharectomy group, HMGB1 and TNF-alpha mRNA expression in liver and lungs was down-regulated on postburn day 4, and IL-10 expression returned to normal range on postburn day 8, while the down-regulation of HMGB1, TNF-alpha and IL-10 were not noted in the 72 h escharectomy group. There were two peaks in liver TNF-alpha protein levels appearing on postburn days 2 and 8, respectively, with an unexpected marked decrease on day 4 in thermal injury controls, yet liver TNF-alpha levels maintained in normal range in animals of 24 h and 72 h escharectomy groups. The ratios of TNF-alpha to IL-10 protein levels in liver tissue were significantly increased on postburn days 2 and 4 (P = 0.0001 and 0.002, respectively), while escharectomy during shock stage markedly reduced hepatic TNF-alpha to IL-10 ratios (P = 0.0008 and 0.040, respectively). No significant changes in TNF-alpha protein levels in lung tissue were observed. Additionally, plasma AST as well as ALT contents, and pulmonary MPO activity were markedly decreased on postburn days 4 and 8 in the 24 h escharectomy group compared to the 72 h escharectomy group or thermal injury controls (P < 0.05). Escharectomy during burn shock stage could inhibit the over-expression of both early and late inflammatory mediators, and maintain the balance of pro-/anti-inflammatory response, thereby improving multiple organ functions in rats following severe burns.