Cytoplasmic Translocation Of High-mobility Group Box 1 Research Articles

SFI Reduces the Nucleocytoplasmic Transportation of HMGB1 by Upregulating HDAC3 in LPS-induced RAW264.7 Cells

Shenfu injection (SFI) is widely used for treating endotoxin shock in China. In the present study, to investigate the anti-inflammatory effects of SFI and further explored the potential mechanism of HMGB1 nuclear translocation, we established a vitro cell model provoked by lipopolysaccharide (LPS), observed nucleocytoplasmic translocation of high mobility group box 1 (HMGB1) and the relationship between histone deacetylase 3 (HDAC3) and HMGB1 under SFI intervention. The results showed that SFI upregulated the transcription and expression of HDAC3 in RAW264.7 cells, inhibited the nuclear to cytoplasmic translocation of HMGB1 and its subsequent extracellular release, and depressed the secretion of HMGB1, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). However, targeted knockdown of HDAC3 induced an increase in HMGB1 translocation to the cytoplasm, and HMGB1 localization was not altered significantly following LPS stimulation. SFI failed to reverse the abnormal localization of HMGB1. These results suggested that SFI may inhibit LPS-induced HMGB1 nuclear translocation in RAW264.7 cells through upregulating HDAC3 expression, thereby inhibiting its downstream pathway and suppressing inflammatory response.

Oxidative Stress-Induced HMGB1 Translocation in Myenteric Neurons Contributes to Neuropathy in Colitis.

High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern released by dying cells to stimulate the immune response. During cell death, HMGB1 is translocated from the nucleus to the cytoplasm and passively released. High levels of secreted HMGB1 are observed in the faeces of inflammatory bowel disease (IBD) patients, indicating its role in IBD pathophysiology and potential as a non-invasive IBD biomarker. HMGB1 is important in regulating neuronal damage in the central nervous system; its pathological activity is intertwined with oxidative stress and inflammation. In this study, HMGB1 expression in the enteric nervous system and its relevance to intestinal neuroinflammation is explored in organotypic cultures of the myenteric plexus exposed to oxidative stimuli and in Winnie mice with spontaneous chronic colitis. Oxidative stimuli induced cytoplasmic translocation of HMGB1 in myenteric neurons in organotypic preparations. HMGB1 translocation correlated with enteric neuronal loss and oxidative stress in the myenteric ganglia of Winnie mice. Inhibition of HMGB1 by glycyrrhizic acid ameliorated HMGB1 translocation and myenteric neuronal loss in Winnie mice. These data highlight modulation of HMGB1 signalling as a therapeutic strategy to reduce the consequences of enteric neuroinflammation in colitis, warranting the exploration of therapeutics acting on the HMGB1 pathway as an adjunct treatment with current anti-inflammatory agents.

Jieduan-Niwan Formula Ameliorates Oxidative Stress and Apoptosis in Acute-on-Chronic Liver Failure by Suppressing HMGB1/TLR-4/NF-κB Signaling Pathway: A Study In Vivo and In Vitro.

Jieduan-Niwan (JDNW) formula is a traditional Chinese medicine compound created by the famous Chinese medicine expert Professor Qian Ying, and has been used clinically for decades to treat acute-on-chronic liver failure (ACLF) and exhibits remarkable efficacy. However, the exact mechanism remains to be discovered. As an important hepatocyte damage-associated molecular patterns (DAMP) factor, high mobility group box 1 (HMGB1) is a potential therapeutic target as an accelerator of ACLF in the pathogenesis. Therefore, the present study investigated whether JDNW inhibits the overexpression and cytoplasmic translocation of HMGB1 in ACLF liver tissue and alleviates its mediated oxidative stress and apoptosis. In vivo, an immune-induced ACLF rat model was established, and then treated with JDNW for 5, 10, and 15 d. The results showed that a large number of cytoplasmic translocations of HMGB1 occurred in the ACLF group. And there was an increase in the expression of HMGB1 in the M-5 d group. After the intervention of JDNW, the overexpression and translocation of HMGB1 were inhibited. In vitro, D-GaLN caused an increase in the expression and translocation of HMGB1 in L02 cells. Similar to the inhibitor of HMGB1, JDNW serum alleviated this kind of increase. Further tests showed that JDNW attenuated ACLF-related oxidative stress and apoptosis, and the inhibition was associated with the regulation of TLR-4/NF-κB signaling pathway. In conclusion, our present findings suggest that the therapeutic effect of JDNW on ACLF was associated with the inhibition of high expression and cytoplasmic translocation of HMGB1 during the acute injury phase, thus, attenuating oxidative stress injury and apoptosis induced by HMGB1/TLR-4/NF-κB pathway.

Camel whey protein alleviates heat stress-induced liver injury by activating the Nrf2/HO-1 signaling pathway and inhibiting HMGB1 release.

This study aimed to investigate the mechanism by which camel whey protein (CWP) inhibits the release of high-mobility group box 1 (HMGB1) in heat stress (HS)-stimulated rat liver. Administration of CWP by gavage prior to HS inhibited the cytoplasmic translocation of HMGB1 and consequently reduced the inflammatory response in the rat liver, and downregulated the levels of the NLR pyrin domain containing 3 (NLRP3) inflammasome, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α. The use of N-acetyl-L-cysteine (NAC), an inhibitor of reactive oxygen species (ROS) production, indicated that this downregulation effect may be attributed to the antioxidant activity of CWP. We observed that CWP enhanced nuclear factor erythroid 2-related factor (Nrf)2 and heme-oxygenase (HO)-1 expression, which inhibited ROS production, nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity, and malondialdehyde (MDA) levels, and increased superoxide dismutase 1 (SOD1) activity and reduced glutathione (GSH) content in the HS-treated liver, ultimately increasing the total antioxidant capacity (TAC) in the liver. Administration of Nrf2 or HO-1 inhibitors before HS abolished the protective effects of CWP against oxidative damage in the liver of HS-treated rats, accompanied by increased levels of HMGB1 in the cytoplasm and IL-1β and TNF-α in the serum. In conclusion, our study demonstrated that CWP enhanced the TAC of the rat liver after HS by activating Nrf2/HO-1 signaling, which in turn reduced HMGB1 release from hepatocytes and the subsequent inflammatory response and damage. Furthermore, the combination of CWP and NAC abolished the adverse effects of HS in the rat liver. Therefore, dietary CWP could be an effective adjuvant treatment for HS-induced liver damage.

Isochlorogenic Acid A Attenuates the Progression of Liver Fibrosis Through Regulating HMGB1/TLR4/NF-κB Signaling Pathway.

Liver fibrosis, a chronic damage process related to further progression of hepatic cirrhosis, has yet no truly effective treatment. Isochlorogenic acid A (ICQA), isolated from a traditional Chinese herbal medicine named Laggera alata (DC.) Sch.Bip. ex Oliv. (Asteraceae), is proved to exhibit anti-inflammatory, hepatoprotective and antiviral properties. However, the actions of ICQA on liver fibrosis are poorly understood. The purpose of this study was to evaluate the actions of ICQA on liver fibrosis and clarify the underlying mechanism. It was found that ICQA had significant protective actions on liver injury, inflammation as we as fibrosis in rats. Meanwhile, ICQA prevented hepatic stellate cells (HSC) activation, indicated by its inhibitory effect on the overexpression of α-smooth muscle actin (α-SMA). In addition, the reduced fibrosis was found to be associated with the decreased protein expression of high-mobility group box 1 (HMGB1) as well as toll like receptor (TLR) 4. Simultaneously, ICQA can suppress the cytoplasmic translocation of HMGB1 in rat liver. Further investigations indicated that ICQA treatment dramatically attenuated the nuclear translocation of the nuclear factor-kB (NF-κB) p65 and suppressed the hepatic expression of p−IκBα in rats with liver fibrosis. Taken together, our study indicated that ICQA could protect against CCl4-induced liver fibrosis probably through suppressing the HMGB1/TLR4/NF-κB signaling pathways.

Lidocaine Alleviates Neuropathic Pain and Neuroinflammation by Inhibiting HMGB1 Expression to Mediate MIP-1α/CCR1 Pathway.

High mobility group box1 (HMGB1) released from sensory nerve tissues can induce neuropathic pain. Whether HMGB1 is implicated in the mechanism underlying the effect of lidocaine in pain management remains to be determined. This study aims to explore the effect of lidocaine in a rat model of spared nerve injury (SNI) and the underlying mechanism. An SNI model was established via nerve ligation. Two weeks after the SNI model was established, rats were intrathecally injected with lidocaine, an HMGB1 antibody (HMG Ab), an MIP-1α antibody (MIP-1α Ab), a CCR1 inhibitor (CCR1-RS) or a CCR5 antagonist (CCR5-Mar). Pain behaviors were assessed before and after model establishment to calculate the number of spontaneous flinches (NSF), paw withdrawal threshold (PWT), paw withdrawal thermal latency (PWL) and sciatic function index (SFI). Cell apoptosis and the inflammatory response in the cerebrospinal fluid (CSF) were detected by TUNEL staining and ELISA. The mRNA and protein expression levels of MIP-1α, CCR1 and CCR5 were determined by RT-PCR and Western blotting. The expression levels of HMGB1, MIP-1α, CCR1 and CCR5 were measured by Western blotting and immunofluorescence. Pain behavior testing in SNI rats showed that SNI rats exhibited an increased NSF and adecreased PWT, PWL and SFI. Cell apoptosis in the spinal dorsal horn and the generation of inflammatory cytokines were enhanced in SNI rats, and the expression levelsof HMGB1, MIP-1α, CCR1 and CCR5 were upregulated. HMGB1 cytoplasmic translocation, the coexpression of MIP-1α with NeuN, and the coexpression of CCR1 and CCR5 with OX42 were also observed in SNI rats. Neuropathic pain and neuroinflammation were suppressed by the intrathecal injection of lidocaine, HMG Ab, MIP-1α Ab, CCR1-RS or CCR5-Mar. Lidocaine inhibited the expression levels of HMGB1, MIP-1α, CCR1 and CCR5, and the HMGB1 antibody suppressed the expression of MIP-1α, CCR1 and CCR5. Lidocaine attenuates neuropathic pain and neuroinflammation by inhibiting HMGB1 to regulate the MIP-1α/CCR1/CCR5 pathway. Graphical Abstract.

High-mobility group box-1 translocation and release after hypoxic ischemic brain injury in neonatal rats

Inflammation contributes to neonatal brain injury. Pro-inflammatory cytokines represent key inflammatory meditators in neonatal hypoxic-ischemic (HI) brain injury. The high mobility group box-1 (HMGB1) protein is a nuclear protein with pro-inflammatory cytokine properties when it is translocated from the nucleus and released extracellularly after stroke in adult rodents. We have previously shown that HMGB1 is translocated from the nucleus to cytosolic compartment after ischemic brain injury in fetal sheep. In the current study, we utilized the Rice-Vannucci model to investigate the time course of HMGB1 translocation and release after HI injury in neonatal rats. HMGB1 was located in cellular nuclei of brains from sham control rats. Nuclear to cytoplasmic translocation of HMGB1 was detected in the ipsilateral-HI hemisphere as early as zero h after HI, and released extracellularly as early as 6 h after HI. Immunohistochemical double staining detected HMGB1 translocation mainly in neurons along with release from apoptotic cells after HI. Serum HMGB1 increased at 3 h and decreased by 24 h after HI. In addition, rat brains exposed to hypoxic injury alone also exhibited time dependent HMGB1 translocation at 3, 12 and 48 h after hypoxia. Consequently, HMGB1 responds similarly after HI injury in the brains of neonatal and adult subjects. We conclude that HMGB1 is sensitive early indicator of neonatal HI and hypoxic brain injury.

N-(2'-Hydroxyphenyl)-2-propylpentanamide (OH-VPA), a histone deacetylase inhibitor, induces the release of nuclear HMGB1 and modifies ROS levels in HeLa cells.

N-(2′-Hydroxyphenyl)-2-propylpentanamide (OH-VPA) is a valproic acid (VPA) derivative with improved antiproliferative activity toward breast cancer (MCF-7, MDA-MB-231, and SKBr3) and human cervical cancer cell lines (HeLa) compared to that of VPA. However, the pharmacological mechanism of OH-VPA activity remains unknown. High-mobility group box 1 (HMGB1) is an important enzyme that is highly expressed in tumor cells and has a subcellular localization that is dependent on its acetylation or oxidative state. Therefore, in this study, we analyzed changes in HMGB1 sub-cellular localization and reactive oxygen species (ROS) as well as changes in HeLa cell viability in response to treatment with various concentrations of OH-VPA. This compound is formed by the covalent bond coupling VPA to a phenol group, which is capable of acting as a free radical scavenger due to its chemical similarities to quercetin. Our results show that OH-VPA induces nuclear to cytoplasmic translocation of HMGB1, as demonstrated by confocal microscopy observations and infrared spectra that revealed high quantities of acetylated HMGB1 in HeLa cells. Cells treated with 0.8 mM OH-VA exhibited decreased viability and increased ROS levels compared with the lower OH-VPA concentrations tested. Therefore, the antiproliferative mechanism of OH-VPA may be related to histone deacetylase (HDAC) inhibition, as is the case for VPA, which promotes high HMBG1 acetylation, which alters its subcellular localization. In addition, OH-VPA generates an imbalance in cellular ROS levels due to its biochemical activity.

Paclitaxel-induced HMGB1 release from macrophages and its implication for peripheral neuropathy in mice: Evidence for a neuroimmune crosstalk

Given our recent evidence for the role of high mobility group box 1 (HMGB1) in chemotherapy-induced peripheral neuropathy (CIPN) in rats, we examined the origin of HMGB1 and the upstream and downstream mechanisms of HMGB1 release involved in paclitaxel-induced neuropathy in mice. Paclitaxel treatment developed mechanical allodynia in mice, as assessed by von Frey test, which was prevented by an anti-HMGB1-neutralizing antibody or thrombomodulin alfa capable of inactivating HMGB1. RAGE or CXCR4 antagonists, ethyl pyruvate or minocycline, known to inhibit HMGB1 release from macrophages, and liposomal clodronate, a macrophage depletor, prevented the paclitaxel-induced allodynia. Paclitaxel caused upregulation of RAGE and CXCR4 in the dorsal root ganglia and macrophage accumulation in the sciatic nerve. In macrophage-like RAW264.7 cells, paclitaxel evoked cytoplasmic translocation of nuclear HMGB1 followed by its extracellular release, and overexpression of CBP and PCAF, histone acetyltransferases (HATs), known to cause acetylation and cytoplasmic translocation of HMGB1, which were suppressed by ethyl pyruvate, N-acetyl-l-cysteine, an anti-oxidant, and SB203580 and PDTC, inhibitors of p38 MAP kinase (p38MAPK) and NF-κB, respectively. Paclitaxel increased accumulation of reactive oxygen species (ROS) and phosphorylation of p38MAPK, NF-κB p65 and I-κB in RAW264.7 cells. In mice, N-acetyl-l-cysteine or PDTC prevented the paclitaxel-induced allodynia. Co-culture of neuron-like NG108-15 cells or stimulation with their conditioned medium promoted paclitaxel-induced HMGB1 release from RAW264.7 cells. Our data indicate that HMGB1 released from macrophages through the ROS/p38MAPK/NF-κB/HAT pathway participates in the paclitaxel-induced peripheral neuropathy in mice, and unveils an emerging therapeutic avenue targeting a neuroimmune crosstalk in CIPN.

Regulation of HMGB1 in Hepatocytes by MyD88 and Type‐I interferon (IFN‐I) During Ehrlichia‐induced acute liver injury.

BackgroundLiver is the major target organ in Human Monocytic ehrlichiosis (HME), an undifferentiated febrile illness that is life‐threatening and caused by intracellular bacterial pathogen, Ehrlichia, that target liver and infect macrophages, hepatocytes and endothelial cells. Severe Hepatic inflammation is the common cause of acute liver injury following systemic infection with Ehrlichia. Our previous studies have shown that Myeloid differentiation primary response gene 88 (MYD88) and type I IFN (IFN‐I) promotes excessive inflammation and liver damage following fatal Ehrlichia infection. In this study, we explored the role of the MyD88 and IFN‐I in liver inflammation.ResultsIn this study, we provide evidence to support that high mobility group box 1 (HMGB1), a chromosomal protein and damage‐associated molecular patterns (DAMPs), plays a role in the regulation of innate responses and autophagy during Ehrlichia infection. Wild‐type (WT) mice infected with virulent Ehrlichia develop sepsis and liver injury marked by excessive systemic production of pro‐inflammatory cytokines and chemokines as well as HMGB1. To determine the cellular source of HMGB1 and regulatory mechanisms, we infected hepatocytes (HC) from WT and MYD88−/− knockout mice with virulent Ehrlichia. To mimic paracrine effect of IFN‐I cytokines produced by immune cells on HC response to Ehrlichia infection, we cultured infected HC in the presence or absence of IFN‐β (100 IU/mL). Our data indicate that in‐vitro infected HC produces low levels of IFN‐beta compared to uninfected cells. Exogenous stimulation of infected HC with IFN‐β induced elevated expression and cytoplasmic translocation of HMGB1 in WT‐HC. This was associated with increased autophagy and higher expression of IL‐23R, IFNAR, and heightened JAK/STAT signaling. Importantly, the effect of high dose IFN‐I on autophagy and HMGB1 was MYD88‐dependent. Interestingly, infection of MYD88−/− HC with Ehrlichia, in the absence of exogenous IFN‐β, enhanced autophagy induction and cytosolic translocation of HMGB1, suggesting that MYD88 negatively regulate HMGB1 and autophagy in HC in the absence of the paracrine effect of IFN‐β.ConclusionTogether, our data suggest that autophagy regulation and HMGB1 response in hepatocytes during Ehrlichia‐induced sepsis is controlled by the strength of IFN‐I signaling and dependent on interplay between MYD88 and IFN‐I pathways.Support or Funding InformationThis work was supported by grants from NIAMS, NIGMD, and NIAID (AR68317 CCY, RO1‐GM102146 MS, and R56A1097679 NI)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibiting the cytoplasmic location of HMGB1 reverses cisplatin resistance in human cervical cancer cells.

Cervical cancer is the fourth most common malignancy in women worldwide, and resistance to chemotherapy drugs is the biggest obstacle in the treatment of cervical cancers. In the present study, the molecular mechanisms underlying cisplatin resistance in human cervical cancer cells were investigated. When human cervical cancer cells were treated with 10µg/ml of cisplatin for 24 and 48h, high mobility group box1 (HMGB1) protein expression levels significantly increased in a time‑dependent manner. Comparisons between cisplatin‑sensitive HeLa cells and cisplatin‑resistant HeLa/DDP cells revealed higher levels of HMGB1 in HeLa/DDP cells than in HeLa cells. Additionally, the half maximal inhibitory concentration (IC50) value for cisplatin in HeLa/DDP cells was 5.3‑fold that in HeLa cells. Analysis of the distribution of cellular components revealed that HMGB1 translocation from the nucleus to cytoplasm contributed to cisplatin resistance. This was further confirmed by demonstration that ethyl pyruvate treatment suppressed the cytoplasmic translocation of HMGB1, resulting in inhibition of HeLa cell proliferation. Furthermore, endogenous HMGB1 was inhibited with HMGB1‑specific short hairpin (sh)RNA, and MTT assay results showed that interference with HMGB1 expression reduced cell viability and potentially reversed cisplatin resistance in HeLa cells. Transfection with HMGB1 shRNA was demonstrated to induce cell apoptosis in HeLa cells, as detected by FACS analysis. In addition, administration of recombinant HMGB1 protein in HeLa cells promoted cell autophagy, mediated by the phosphorylation of extracellular signal‑regulated kinase1/2. Thus, cytoplasmic HMGB1 translocation and HMGB1‑induced cell autophagy are proposed to contribute to cisplatin resistance by inhibiting apoptosis of cervical cancer cells. HMGB1 could, therefore, represent a novel therapeutic target for, and a diagnostic marker of, chemotherapy resistant cervical cancers.

Quercetin attenuates the activation of hepatic stellate cells and liver fibrosis in mice through modulation of HMGB1-TLR2/4-NF-κB signaling pathways

This study aimed to investigate the effects of quercetin on liver fibrogenesis in mice and to elucidate the underlying molecular mechanisms. Mice were administered with carbon tetrachloride (CCl4) for eight weeks to induce liver fibrosis and concomitantly orally treated with quercetin (50mgkg−1day−1). Here, we demonstrated that quercetin dramatically ameliorated liver injury, inflammation, and hepatic fibrogenesis induced by CCl4. Quercetin also inhibited the activation of hepatic stellate cells (HSC) in vivo and in vitro, as evaluated by α-smooth muscle actin (α-SMA) expression, which is a specific marker of HSC activation. Moreover, reduced fibrosis was associated with decreased high-mobility group box 1 (HMGB1), toll like receptor (TLR) 2 and TLR4 genes, and protein expression. Quercetin also inhibited the cytoplasmic translocation of HMGB1 in hepatocytes of fibrotic livers. Further investigation demonstrated that quercetin treatment significantly attenuated CCl4-induced nuclear translocation of the nuclear factor-κB (NF-κB) p65 and inhibited degradation of IκBα (an inhibitor of NF-κB) expression in the liver compared with vehicle-treated fibrotic mice. Considered together, our data indicate that quercetin has hepatoprotective and anti-fibrotic effects in animal models of liver fibrosis, the mechanism of which may be involved in modulating the HMGB1-TLR2/4-NF-κB signaling pathways.

Cytoplasmic translocation of high-mobility group box-1 protein is induced by diabetes and high glucose in retinal pericytes.

The aim of the present study was to assess the involvement of the high-mobility group box-1 (HMGB1) protein, receptor for advanced glycation end products (RAGE) and nuclear factor (NF)-κB signaling pathway in the development of diabetic retinopathy. Rat primary retinal pericytes were exposed to 25 mmol/l D-glucose for 48 h. Diabetic retinal vessels were prepared from streptozotocin-induced diabetic rats 12 weeks following the induction of diabetes. The expression of HMGB1 was detected using immunofluorescence staining. The expression of RAGE and the activity of NF-κB were analyzed using western blot and electrophoretic mobility shift assays, respectively. The results showed that HMGB1 was translocated to the cytoplasm of the high glucose-treated pericytes and diabetic retinal pericytes, whereas, in the control cells and the normal retinas, HMGB1 was expressed in the cell nuclei only. The expression of RAGE, a potential receptor for HMGB1, and the activity of NF-κB were also increased in the high glucose-treated pericytes, compared with the normal control cells. In addition, high glucose increased the binding of NF-κB to the RAGE promoter. These findings suggested that the cytoplasmic translocation of HMGB1 may be caused by diabetes and high glucose in retinal pericytes, and that the pathogenic role of HMGB1 may be dependent on the expression of RAGE and activation of NF-κB.

Association between protective effect of Liuwei Wuling tablets against acute liver injury and its inhibitory effect on cytoplasmic translocation of high-mobility group box-1 in hepatocytes in mice

To investigate the effect of Liuwei Wuling tablets on the cytoplasmic translocation and release of high-mobility group box-1 (HMGB1) in hepatocytes in mice with acute live injury induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). A Balb/c mouse model of acute liver injury was established by intraperitoneal injection of D-GalN (400 mg/kg) and LPS (5 ug/kg). A total of 24 healthy mice were randomly and equally divided into acute liver injury control group and Liuwei Wuling tablet treatment group. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in both groups at each time point within one week. Liver tissues were collected at 36 hours to perform pathological examination. The serum levels of HMGB1, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), complement 3a (C3a), and complement 5a (C5a) were measured. Immunohistochemistry was used to determine the expression and cytoplasmic translocation of HMGB1 in hepatocytes. At 6, 12, and 24 hours, the Liuwei Wuling tablet treatment group had significantly lower serum levels of ALT than the control group (225.33±181.64 U/L vs 471.17±174.72 U/L, t = 3.38, P < 0.01; 1509.53±182.51 U/L vs 7149.52±734.25 U/L, t = 25.82, P < 0.01; 162.89±86.51 U/L vs 1318.16±557.71 U/L, t = 7.09, P < 0.01), as well as significantly lower serum levels of AST than the control group (179.22±94.57 U/L vs 561.91±209.6 U/L, t = 5.76, P < 0.01; 590.92±190.92 U/L vs 2266.48±705.64 U/L, t = 7.94, P < 0.01; 231.24±87.7 U/L vs 444.32±117.01 U/L, t = 5.05, P < 0.01). The treatment group had significantly lower levels of HMGB1 than the control group at 6 and 12 hours (54.21±11.89 ng/ml vs 72.07±13.65 ng/ml, t = 3.41, P < 0.01; 49.23±5.97 ng/ml vs 68.71±13.07 ng/ml, t = 4.70, P < 0.01). The treatment group had significantly lower levels of TNF-α, IL-1β, and IL-6 than the control group at 12 hours (163.62±9.12 pg/ml vs 237.09±51.47 pg/ml, t = 4.86, P < 0.01; 15.66±13.13 pg/ml vs 37.43±18.68 pg/ml, t = 3.30, P < 0.01; 7.10±3.06 pg/ml vs 21.42±8.23 pg/ml, t = 5.65, P < 0.01). The treatment group had significantly lower levels of C3a and C5a than the control group at 12 hours (2.52±1.27 pg/ml vs 9.83±2.96 ng/ml, t = 7.86, P < 0.01; 2.16±1.03 ng/ml vs 7.23±1.55 ng/ml, t = 9.67, P < 0.01). Compared with the control group, the treatment group had significantly reduced liver inflammation and necrosis, and a significantly lower cytoplasmic translocation rate of HMGB1 in hepatocytes (38.76%±7.37% vs 8.15%±2.11%, P < 0.01). Liuwei Wuling tablets can reduce the cytoplasmic translocation of HMGB1 in hepatocytes and relieve liver inflammation in mice with acute liver injury.

Poly-ADP-ribosylation of HMGB1 regulates TNFSF10/TRAIL resistance through autophagy

Both apoptosis ("self-killing") and autophagy ("self-eating") are evolutionarily conserved processes, and their crosstalk influences anticancer drug sensitivity and cell death. However, the underlying mechanism remains unclear. Here, we demonstrated that HMGB1 (high mobility group box 1), normally a nuclear protein, is a crucial regulator of TNFSF10/TRAIL (tumor necrosis factor [ligand] superfamily, member 10)-induced cancer cell death. Activation of PARP1 (poly [ADP-ribose] polymerase 1) was required for TNFSF10-induced ADP-ribosylation of HMGB1 in cancer cells. Moreover, pharmacological inhibition of PARP1 activity or knockdown of PARP1 gene expression significantly inhibited TNFSF10-induced HMGB1 cytoplasmic translocation and subsequent HMGB1-BECN1 complex formation. Furthermore, suppression of the PARP1-HMGB1 pathway diminished autophagy, increased apoptosis, and enhanced the anticancer activity of TNFSF10 in vitro and in a subcutaneous tumor model. These results indicate that PARP1 acts as a prominent upstream regulator of HMGB1-mediated autophagy and maintains a homeostatic balance between apoptosis and autophagy, which provides new insight into the mechanism of TNFSF10 resistance.

Inhibition of sphingosine kinase 1 ameliorates acute liver failure by reducing high-mobility group box 1 cytoplasmic translocation in liver cells.

To determine the therapeutic potential of sphingosine kinase 1 (Sphk1) inhibition and its underlying mechanism in a well-characterized mouse model of D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced acute liver failure (ALF). Balb/c mice were randomly assigned to different groups, with ALF induced by intraperitoneal injection of D-GaIN (600 mg/kg) and LPS (10 μg/kg). The Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at different time points within one week were determined using a multi-parametric analyzer. Serum high-mobility group box 1 (HMGB1), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-10, and sphingosine-1-phosphate were detected by enzyme-linked immunosorbent assay. Hepatic morphological changes at 36 h after acute liver injury induction were assessed by hematoxylin and eosin staining. HMGB1 expression in hepatocytes and cytoplasmic translocation were detected by immunohistochemistry. Expression of Sphk1 in liver tissue and peripheral blood mononuclear cells (PBMCs) was analyzed by Western blot. The expression of Sphk1 in liver tissue and PBMCs was upregulated in GalN/LPS-induced ALF. Upregulated Sphk1 expression in liver tissue was mainly caused by Kupffer cells, the resident macrophages of the liver. The survival rates of mice in the N,N-dimethylsphingosine (DMS, a specific inhibitor of SphK1) treatment group were significantly higher than that of the control group (P < 0.001). DMS treatment significantly decreased the levels of serum ALT and AST at 6, 12, and 24 h compared with that of the control group (P < 0.01 for all). Serum HMGB1 levels at 6, 12, and 24 h, as well as serum TNF-α, IL-6, and IL-1β levels at 12 h, were significantly lower in the DMS treatment group than in the control group (P < 0.01 for all). Furthermore, hepatic inflammation, necrosis, and HMGB1 cytoplasm translocation in liver cells were significantly decreased in the DMS treatment group compared to the control group (43.72% ± 5.51% vs 3.57% ± 0.83%, χ(2) = 12.81, P < 0.01). Inhibition of SphK1 ameliorates ALF by reducing HMGB1 cytoplasmic translocation in liver cells, and so might be a potential therapeutic strategy for this disease.

Role of high mobility group box 1 (HMGB1) in SCA17 pathogenesis.

Spinocerebellar ataxia type 17 (SCA17) involves the expression of a polyglutamine (polyQ) expanded TATA-binding protein (TBP), a general transcription initiation factor. TBP interacts with other protein factors, including high mobility group box 1 (HMGB1), to regulate gene expression. Previously, our proteomic analysis of soluble proteins prepared from mutant TBP (TBP/Q61) expressing cells revealed a reduced concentration of HMGB1. Here, we show that HMGB1 can be incorporated into mutant TBP aggregates, which leads to reduced soluble HMGB1 levels in TBP/Q61∼79 expressing cells. HMGB1 overexpression reduced mutant TBP aggregation. HMGB1 cDNA and siRNA co-transfection, as well as an HSPA5 immunoblot and luciferase reporter assay demonstrated the important role of HMGB1 in the regulation of HSPA5 transcription. In starvation-stressed TBP/Q36 and TBP/Q79 cells, increased reactive oxygen species generation accelerated the cytoplasmic translocation of HMGB1, which accompanied autophagy activation. However, TBP/Q79 cells displayed a decrease in autophagy activation as a result of the reduction in the cytoplasmic HMGB1 level. In neuronal SH-SY5Y cells with induced TBP/Q61∼79 expression, HMGB1 expression was reduced and accompanied by a significant reduction in the total outgrowth and branches in the TBP/Q61∼79 expressing cells compared with the non-induced cells. The decreased soluble HMGB1 and impaired starvation-induced autophagy in cells suggest that HMGB1 may be a critical modulator of polyQ disease pathology and may represent a target for drug development.

Novel mechanisms involving chemically modified tetracycline 3 cytotoxicity.

Chemically modified tetracycline 3 (CMT-3) is a potential anticancer drug because of its retained matrix metalloproteinases inhibitory property. In the present study,we showed that CMT-3 significantly inhibited the growth and proliferation of human hepatocellular carcinoma HepG2 cells. Novel mechanisms including increased intracellular autophagy level and high-mobility group box 1 (HMGB1)release were involved. In addition, a major Danshen ingredient, tanshinone IIA sodium sulfonate (TSN-SS),significantly increased the cytotoxic effects of CMT-3 in HepG2 cells. Combining CMT-3 with TSN-SS led to enhanced accumulation of endogenous LC3-II, but reduced HMGB1 cytoplasmic translocation. Altogether, these findings suggest that autophagy and HMGB1 release may play important roles in the anticancer effect of CMT-3. As an ovel candidate for cancer therapy, CMT-3 may be used in combination with TSN-SS, which possibly facilitates the execution of a death signal (e.g. autophagy) and prevents the survival of an inducer (e.g. HMGB1 cytoplasmic translocation), thus improving its therapeutic effect.

HIGH-MOBILITY GROUP BOX 1 (HMGB1) EXPRESSION IS INCREASED IN THE NORMAL-APPEARING BRAIN TISSUE OF MULTIPLE SCLEROSIS (MS) PATIENTS VS. CONTROLS

<h3>Background</h3> MS is characterised pathologically by focal inflammatory, demyelinating plaques. More recently, normal-appearing white and grey matter (NAWM/ NAGM) distal to these lesions have also been shown to be altered in MS, suggesting more widespread changes affecting MS brain tissue. High-mobility group box 1 (HMGB1) acts as a damage-associated pattern recognition molecule (DAMP) released by activated macrophages, in addition to sensing sterile damage and propagating subsequent inflammatory responses. Whilst increased macrophage/microglial HMGB1 expression has been demonstrated previously in active lesions, we extended these findings, including analysis of non-lesional brain tissue in MS patients vs. non-MS controls. <h3>Results</h3> Semi-quantitative immunoreactivity (IR) analysis showed increased HMGB1 expression in active WM lesions in MS vs. control WM tissue, predominantly affecting macrophages/ activated microglial cells. However, IR was also significantly increased in non-lesional brain tissue in MS patients vs. controls, with the changes consistently marked in oligodendroglial cells in both grey and white matter. In addition, neuronal cells in non-lesional cortical regions demonstrated significantly greater cytoplasmic translocation of HMGB1. These changes were most marked in regions distal to active lesional areas (‘active lesion’ tissue blocks) but were also noted in the majority of tissue blocks without an active lesion (‘NAWM’ tissue block). Both chronic white matter and cortical demyelinating regions demonstrated reduced expression of HMGB1. <h3>Conclusion</h3> Increased expression of HMGB1 in CNS-resident cells supports the notion of pervasive tissue alteration in MS brain tissue. Whether this expression pattern reflects a pro-inflammatory, destructive process or a protective response to surrounding cellular stress or indeed a balance between the two, is yet to be determined.

Baicalin Inhibits High-Mobility Group Box 1 Release and Improves Survival in Experimental Sepsis

Baicalin, as the main active ingredient of the root from Scutellaria, is usually used in the treatment of inflammatory diseases. In our study, we found that baicalin improved survival in septic mice in vivo and attenuated high-mobility group box 1 (HMGB1) and cytokine release from macrophages in vitro. The experiments in vitro showed that baicalin inhibited both viability of macrophages and the cell's secretion of HMGB1, tumor necrosis factor α, interleukin 6 (IL-6), and IL-1β induced by lipopolysaccharide. Meanwhile, experiments in vitro also indicated that baicalin did not affect the transcription and translation of HMGB1 but inhibit the cytoplasmic translocation of HMGB1 induced by lipopolysaccharide. We found that baicalin improved survival and tissue injury of septic mice in vivo. It also decreased serum HMGB1, tumor necrosis factor α, IL-6, and IL-1β in septic mice. In conclusion, baicalin inhibits the release of HMGB1 from macrophages and may be a potential therapeutic strategy for sepsis-related diseases.