ALKBH5-Mediated m6A Demethylation Stabilizes HMGB1 to Drive Hepatocyte Pyroptosis and Liver Injury in Heat Stroke: Targeting via Biomimetic Nanodelivery.

Heatstroke induces liver injury via IL-1β and HMGB1-induced pyroptosis

The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1.

Rhinacanthin-C, A Fat-Soluble Extract from Rhinacanthus nasutus, Modulates High-Mobility Group Box 1-Related Neuro-Inflammation and Subarachnoid Hemorrhage-Induced Brain Apoptosis in a Rat Model

Objective To research the role of high mobility group box 1 (HMGB1) in systemic inflammatory response in rats with acute pancreatitis (AP).Methods The 136 Spragye-Dawley rats were randomly divided into three groups:group A (n =8),group B (n =64) and group C (n =64).AP models were induced by retrograde injection of sodium taucrocholate into the biliopancreatic duct:with 2％ sodium taucrocholate to induce mild AP,and 5％ to induce severe AP in group B and group C respectively.Group C received ethyl pyruvate (EP) solution (28 mmol/L,40 mg/kg·6 h) by tail vein beginning at 2nd h after induction of AP,while group B received the same amount of ringer lactate solution balance as a placebo.The samples of the blood and pancreas were collected directly in group A,and after 6,12,24 and 48 h,the blood and pancreas were collected in groups B and C.Reverse transcription-polymerase chain reaction (RT-PCR) Western blotting were ued to detect mRNA and protein expression of HMGB1 respectively.A point scoring system histologic features were respectively used to evaluate the severity of pancreatitis,and correlation between HMGB1 mRNA expression and pancreatic histopathological score was analyzed.Results The expression levels of HMGB1 mRNA and the pancreas pathological scores in group A were 1.01 ±0.18 and 0.70 ±0.23 separately.The HMGB1 mRNA expression in the pancreatic tissue of group B at 6th,12th,24th and 48th h was separately 1.18 ±0.31,18.23 ± 1.36,201.14 ± 12.01 and 190.66 ± 9.77,with histopathological scores of the pancreas being 9.01 ± 1.94,9.97 ± 1.81,11.45 ±1.43 and 13.17 ± 1.37.The HMGB1 mRNA expression in the pancreatic tissue of group C at at 6th,12th,24th and 48th h was separately 1.11 ±0.27,12.60 ± 1.20,45.44 ±4.45,and 41.41 ±4.11,and histopathological scores of the pancreas were 8.97 ± 1.87,9.92 ± 1.88,9.34 ± 1.63 and 10.26 ± 1.22 separately.The correlation coefficient of HMGB1 mRNA in pancreatic tissues and pancreatic histopathological scores in groups A and B was r =0.847 (P ＜ 0.01).Conclusion The expression of HMGB1 was increased at 12th h,reached the peak at 24th h and stayed at a high level at 48th h.The increased HMGB1 was positively correlated wtih the severity of AP,suggesting its importanthy role in the systemic inflammatory response of AP.Treatment with EP has a therapeutic effect on AP by inhibitting HMGB1 expression,and reducing pancreatic injury. Key words: Acute pancreatitis ; High mobility group box 1 ; Ethyl pyruvate

Effects of Propofol on the Outcomes of Rats with Sepsis

ABSTRACTThe mortality of sepsis is increasing and conventional therapies for it have no better therapeutic effects. We investigated the effects of adiponectin (APN) on mortality and high mobility group box 1 (HMGB1) in polymicrobial sepsis mouse models. Sepsis models were established by cecal ligation and puncture (CLP) in BALB/c mice. Animals were randomly divided into four groups including control group (C group), model group (CLP group), early APN treatment group (APN + CLP group), and late APN treatment group (CLP + APN group). Mice in each group were killed at 6, 12, 24, and 48 hr after CLP, respectively, to collect samples for determining the levels of serum interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), high mobility group protein-1 (HMGB1), and the expression of lung tissue HMGB1 mRNA. The survival curves in the four groups were drawn. The mortality rates were significantly lower in APN + CLP (30%) and CLP + APN (40%) groups than in CLP group (80%) seven days after CLP. Serum levels of cytokines (IL-6 and TNF-α) and HMGB1 were significantly reduced (p < .05) in APN + CLP and CLP + APN groups compared with those of CLP group. There was a significant correlation between serum HMGB1 and lung HMGB1 mRNA (r = 0.891). The levels of HMGB1 and HMGB1 mRNA were higher in CLP, APN + CLP, and CLP + APN groups than in C group (p < .01), but were lower in APN + CLP and CLP + APN groups than in CLP group (p < .01). APN can reduce the mortality rate and plays an anti-inflammatory role in polymicrobial sepsis mouse models through inhibiting HMGB1.

Dietary supplementation of camel whey protein attenuates heat stress-induced liver injury by inhibiting NLRP3 inflammasome activation through the HMGB1/RAGE signalling pathway

Objective: To investigate the expression changes of high mobility group box-1 (HMGB1) and the 2B receptor of N- methyl -D- aspartate receptor (NR2B) in status epilepticus (SE). Methods: (1) Primary hippocampal neurons from SD rats with 16 to 18 days of fetal age were cultured in vitro for 7 days, and exposed to Mg(2+) free media for 3 hours. Those cultured neurons were randomly divided into control group and intermittent hypoxia group. (2) SD rats with similar weight were selected and randomly divided into control group and SE model group. The rat model with SE was established by an intraperitoneal injection of lithium chloride-piloearpine (LI-PILO). Real-time PCR technique was used to detect the expression of HMGB1 and NR2B mRNA. Results: In Sombati's cell model cultured in normal concentration of oxygen, the HMGB1 mRNA expression levels were 0.005 01±0.000 54, 0.026 76±0.003 75, 0.003 52±0.000 33, and the NR2B mRNA expression levels were 0.008 84±0.000 69, 0.012 23±0.000 90, 0.029 11±0.000 71, respectively, at 2, 4 and 6 h; compared with the expressions of HMGB1 and NR2B mRNA at the same time points of Sombatis cell model groups, the differences were also significant (all P<0.05). After the successful establishment of epilepsy model, the HMGB1 mRNA expression levels were 0.000 11±0.000 09, 0.000 18±0.000 01, 0.000 11±0.000 01, and the NR2B mRNA expression levels were 0.196 12±0.009 41, 0.232 11±0.006 27, 0.272 48±0.005 84, respectively, at 6, 8 and 10 h; compared with the expressions of HMGB1 and NR2B mRNA at the same time points of control groups, the differences were all significant (all P<0.05). Conclusion: HMGB1 mRNA expression levels increase at 2, 4 h, decrease at 6 h in the Sombati's cell model in normal oxygen culture, while increase at 6, 8 h, and decrease at 10 h in LI-PILO induced rat model with SE; the NR2B mRNA relative expression increases with time in both the Sombati's cell model in normal oxygen culture and rat model of SE.

What's known on the subject? and What does the study add? Recent studies have indicated that high mobility group box 1 (HMGB1) is related to the development and progression of human carcinomas. However, further studies were required to confirm the roles played by HMGB1 in clinical prostate cancer treatment. We investigated the relationship between HMGB1 expression and the characteristics of prostate cancer, and also evaluated the significance of HMGB1 as a prognostic factor for biochemical recurrence-free survival after radical prostatectomy. • To investigate high mobility group box 1 (HMGB1) expression in human prostate cancer (PC) cell lines and its prognostic significance after radical prostatectomy (RP). • Quantitative reverse-transcription polymerase chain reaction and western blotting were used to detect HMGB1 mRNA and protein expression in PC cell lines. • Immunohistochemistry coupled with the tissue microarray technique was performed to evaluate HMGB1 protein expression in 168 primary prostatectomy tissue samples. • Clinicopathological features were compared between positive and negative HMGB1 protein expression groups. • Kaplan-Meier and multivariate Cox analyses were applied to determine the prognostic value of HMGB1 protein expression on biochemical recurrence (BCR) for patients with PC who were undergoing RP. • There were three PC cells (DU145, PC-3 and LNCaP) with overexpression of HMGB1 mRNA and protein compared to the non-transformed immortalized prostate cell RWPE-1. • A total of 60.1% (101/168) of the PC samples appeared to have positive protein expression of HMGB1. • HMGB1 protein expression was correlated with some clinicopathological parameters, such as pathological stage (pT) (P= 0.011), Gleason score, preoperative prostate-specific antigen concentration and BCR (P < 0.001, respectively). • Positive HMGB1 immunostaining in patients with PC who were undergoing RP was significantly associated with poor median BCR-free survival (23.1 months vs 15.6 months) (P < 0.001). • Multivariate analysis indicated that HMGB1 protein expression was an independent prognostic factor for BCR-free survival after RP (hazard ratio = 2.348, 95% confidence interval = 1.373-6.361, P= 0.001). • Up-regulation of HMGB1 mRNA and protein concentrations was confirmed in PC cells. • HMGB1 expression may contribute to the malignant progression of PC. • HMGB1 presents as a novel prognostic factor for BCR after RP.

017 INHIBITORY INTERACTIONS BETWEEN HMGB1 AND HEME OXYGENASE-1 PATHWAYS IN OSTEOARTHRITIC SYNOVIOCYTES

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.

Background/Aims: Interferon regulatory factor 1(IRF-1) and high mobility group box 1(HMGB1) have been independently identified as being key players in hepatic ischemia-reperfusion injury (IRI). We attempted to determine whether IRF-1 activates autophagy to aggravate hepatic IRI by increasing HMGB1 release. Methods: The hepatic IRI model was generated in C57BL/6 mice, euthanized at 2, 6, 12 or 24 h after reperfusion. To examine the effects of HMGB1 release inhibition, Glycyrrhiza acid (GA) was administered to the mice and at six hours after injectiont. AML12 cells were immersed in mineral oil for 90 min and then cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 to simulate IRI. AML12 cells were treated with IRF-1 siRNA, Ad-IRF-1 or GA. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as histological changes were examined. Next, autophagic vacuoles were detected by transmission electron microscopy (TEM) or LC3 dots. The expression of IRF-1 and HMGB1 mRNA were measured by real-time polymerase chain reaction. The expression of IRF-1, microtubule-associated protein 1 light chain 3 (LC3), Bcl-2, Beclin 1, HMGB1 were detected by western blotting or immunohistochemistry. Results: The levels of hepatic IRF-1, mRNA and protein were significantly increased in livers after exposure to IRI, together with, IRI-induced increase of HMGB1 mRNA and release of HMGB1 in liver tissue. Knockout of IRF-1 decreased expression and release of HMGB1 in liver, and inhibiting the release of HMGB1 could alleviate hepatic IRI. In addition, knockout of IRF-1 downregulated LC3II and Beclin1, while number of autophagosomes or LC3 dots were increased. Up-regulating IRF-1 expression could increase the levels of LC3Ⅱ expression in AML12 cells after exposure to IRI. The levels of HMGB1 in Ad-IRF-1 transfected AML12 cell supernatants increased, together with number of LC3 dots increasing. However, GA could inhibit both Ad-IRF-1 induced HMGB1 release and the increase in the number of LC3 dots. Conclusions: IRF-1 activates autophagy to aggravate hepatic IRI by increasing HMGB1 release.

Objective To investigate the effect of lidocaine on lung injury and expression of inflammatory factors in septic rats. Methods Sixty male adult Sprague Dawley rats were randomly divided into the sham operation group, cecal ligation and puncture (CLP) group, ulinastatin group and lidocaine group, with 15 rats in each group. The abdominal cavity of rats was opened and sutured in the sham operation group, and sepsis models in the other groups were established by the CLP method. Rats in the lidocaine group were continuously pumped with lidocaine through the tail vein at a dose of 10 mg·kg-1·h-1 for 3 h after injecting a loading dose of 10 mg/kg. Rats in the ulinastatin group were given CLP and continuously pumped with ulinastatin through the tail vein at a dose of 100 000 U·kg-1·h-1 for 3 h. Rats in the sham operation and CLP groups were injected with equal amount of isoosmotic NaCl solution. The levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and high mobility group box 1 (HMGB1) in serum were determined by enzyme-linked immunosorbent assay (ELISA), as all rats were sacrificed at 24 h after CLP. The expression of HMGB1 mRNA in lung tissue was detected by real-time fluorescence quantitative PCR, and the pathological changes of lung tissue in each group were observed by hematoxylin-eosin (HE) staining. Another 40 rats (10 in each group) were used to observe the death condition at 72 h in 4 groups. Results The levels of TNF-α, IL-6, HMGB1, and HMGB1 mRNA in serum of the four groups were significantly different (F=189.886, 237.952, 175.999, 179.491; all P 0.008). Conclusions By reducing the expressions of TNF-α, IL-6 and HMGB1 and inhibiting the HMGB1 mRNA expression in lung tissue, continuous intravenous injection of lidocaine can alleviate pulmonary injury induced by sepsis and effectively improve the survival rate. The effect of lidocaine on inflammatory reaction and lung protection after sepsis is similar to that of ulinastatin. Key words: Lidocaine; Ulinastatin; Sepsis; Acute lung injury; High mobility group box 1

Objective To investigate the effect of Xuebijing injection on release of high mobility group box 1 (HMGB1) in endotoxin-induced cells. Methods HMGB1 concentration and mRNA expression were analyzed by enzyme-linked immunosorbent assay (ELISA) and RT-PCR, respectively. The effects of Xuebijing injection with 2, 10 and 50 mg/ml on HMGB 1 concentration in the culture medium of 200 ng/ml lipopolysaccharide-induced macrophage-like RAW 264.7 cells and BRL-3A hepatocytes, and HMGB1 mRNA expression in lipopolysaccharide-induced macrophages were observed. Results 50 mg/ml Xuebijing injection significantly decreased HMGB1 concentration in the culture medium of lipopolysaccharide-induced macrophages and hepatocytes(P< 0.01), and inhibited HMGB1 mRNA expression in macrophages. Conclusion Xuebijing injection inhibits endotoxin-induced release of HMGB1. Key words: HMGB1 protein; XueBiJing; Macrophages; Hepatocytes; Endotoxins

Objective: We investigated the effect of high mobility group box 1 (HMGB1) on intracranial aneurysms (IAs) by analyzing single-nucleotide polymorphisms (SNPs) based on genome-wide association study (GWAS) data. HMGB1 mRNA and protein expression levels in plasma were also analyzed. Methods: This study was a comprehensive analysis of a GWAS dataset, including 250 patients with IAs and 294 controls. The HMGB1 gene region was targeted within SNP rs3742305 ± 10 kbp. Multivariate logistic regression analysis determined its association with IAs after adjusting for relevant clinical factors. HMGB1 mRNA expression was analyzed in the plasma of 24 patients selected from the GWAS dataset. The HMGB1 protein was analyzed by Western blotting. Results: A total of seven polymorphisms, including rs1360485, rs185382445, rs2039338, rs1045411, rs3742305, rs2249825, and rs189034241, were observed. Two SNPs, including rs1045411 (UTR-3) and rs3742305 (intron), showed strong linkage disequilibrium (r2 = 0.99). However, none of the seven SNPs associated with IAs had an adjusted p-value of < 0.0016 on multiple comparison analysis. HMGB1 mRNA levels (2-ΔCt) did not differ significantly between patients with IAs and the control subjects [1.07 (1.00-1.15) in patients with IAs vs. 1.05 (0.94-1.12) in controls; p = 0.67)]. Also, no significant difference in the degree of plasma HMGB1 protein expression was seen between the two groups (p = 0.82). Conclusions: The number of SNPs associated with HMGB1 and the degree of HMGB1 mRNA and protein expression were not significantly different between patients diagnosed with IAs and the controls.