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Identification of Proteins Interacting with Cytoplasmic High-Mobility Group Box 1 during the Hepatocellular Response to Ischemia Reperfusion Injury.

ABSTRACT: Ischemia/reperfusion injury (IRI) occurs inevitably in liver transplantations and frequently during major resections, and can lead to liver dysfunction as well as systemic disorders. High-mobility group box 1 (HMGB1) plays a pathogenic role in hepatic IRI. In the normal liver, HMGB1 is located in the nucleus of hepatocytes; after ischemia reperfusion, it translocates to the cytoplasm and it is further released to the extracellular space. Unlike the well-explored functions of nuclear and extracellular HMGB1, the role of cytoplasmic HMGB1 in hepatic IRI remains elusive. We hypothesized that cytoplasmic HMGB1 interacts with binding proteins involved in the hepatocellular response to IRI. In this study, binding proteins of cytoplasmic HMGB1 during hepatic IRI were identified. Liver tissues from rats with warm ischemia reperfusion (WI/R) injury and from normal rats were subjected to cytoplasmic protein extraction. Co-immunoprecipitation using these protein extracts was performed to enrich HMGB1-protein complexes. To separate and identify the immunoprecipitated proteins in eluates, 2-dimensional electrophoresis and subsequent mass spectrometry detection were performed. Two of the identified proteins were verified using Western blotting: betaine-homocysteine S-methyltransferase 1 (BHMT) and cystathionine ?-lyase (CTH). Therefore, our results revealed the binding of HMGB1 to BHMT and CTH in cytoplasm during hepatic WI/R. This finding may help to better understand the cellular response to IRI in the liver and to identify novel molecular targets for reducing ischemic injury.

SUBMITTER: Zhang T

PROVIDER: S-EPMC5297800 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Identification of Proteins Interacting with Cytoplasmic High-Mobility Group Box 1 during the Hepatocellular Response to Ischemia Reperfusion Injury.

Zhang Tianjiao T Wei Weiwei W Dirsch Olaf O Krüger Thomas T Kan Chunyi C Xie Chichi C Kniemeyer Olaf O Fang Haoshu H Settmacher Utz U Dahmen Uta U

International journal of molecular sciences 20170116 1

Ischemia/reperfusion injury (IRI) occurs inevitably in liver transplantations and frequently during major resections, and can lead to liver dysfunction as well as systemic disorders. High-mobility group box 1 (HMGB1) plays a pathogenic role in hepatic IRI. In the normal liver, HMGB1 is located in the nucleus of hepatocytes; after ischemia reperfusion, it translocates to the cytoplasm and it is further released to the extracellular space. Unlike the well-explored functions of nuclear and extracel ...[more]

PMID: 28275217

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Project description:Myeloperoxidase (MPO) plays critical role in the pathology of cerebral ischemia-reperfusion (I/R) injury via producing hypochlorous acid (HOCl) and inducing oxidative modification of proteins. High-mobility group box 1 (HMGB1) oxidation, particularly disulfide HMGB1 formation, facilitates the secretion and release of HMGB1 and activates neuroinflammation, aggravating cerebral I/R injury. However, the cellular sources of MPO/HOCl in ischemic brain injury are unclear yet. Whether HOCl could promote HMGB1 secretion and release remains unknown. In the present study, we investigated the roles of microglia-derived MPO/HOCl in mediating HMGB1 translocation and secretion, and aggravating the brain damage and blood-brain barrier (BBB) disruption in cerebral I/R injury. In vitro, under the co-culture conditions with microglia BV cells but not the single culture conditions, oxygen-glucose deprivation/reoxygenation (OGD/R) significantly increased MPO/HOCl expression in PC12 cells. After the cells were exposed to OGD/R, MPO-containing exosomes derived from BV2 cells were released and transferred to PC12 cells, increasing MPO/HOCl in the PC12 cells. The HOCl promoted disulfide HMGB1 translocation and secretion and aggravated OGD/R-induced apoptosis. In vivo, SD rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) plus different periods of reperfusion. Increased MPO/HOCl production was observed at the reperfusion stage, accomplished with enlarged infarct volume, aggravated BBB disruption and neurological dysfunctions. Treatment of MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH) and HOCl scavenger taurine reversed those changes. HOCl was colocalized with cytoplasm transferred HMGB1, which was blocked by taurine in rat I/R-injured brain. We finally performed a clinical investigation and found that plasma HOCl concentration was positively correlated with infarct volume and neurological deficit scores in ischemic stroke patients. Taken together, we conclude that ischemia/hypoxia could activate microglia to release MPO-containing exosomes that transfer MPO to adjacent cells for HOCl production; Subsequently, the production of HOCl could mediate the translocation and secretion of disulfide HMGB1 that aggravates cerebral I/R injury. Furthermore, plasma HOCl level could be a novel biomarker for indexing brain damage in ischemic stroke patients.

Project description:BackgroundCerebral ischemia-reperfusion injury (CI/R) is among the most common diseases affecting the central nervous system. Due to the poor efficacy and adverse side effects of the drugs used to treat CI/R in clinical trials, a new treatment strategy is urgently needed. In this study, we aimed to investigate whether miR-103a-3p alleviates CI/R in vivo and vitro and to explore the relevant mechanisms.MethodsBV2 microglial cells underwent oxygen-glucose deprivation (OGD) treatment to imitate the pathophysiology of CI/R in vitro. A middle cerebral artery occlusion (MCAO) rat model was established to imitate the pathophysiology of CI/R in vivo. The expression levels of miR-103a-3p and HMGB1 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, enzyme-linked immunosorbent assay (ELISA), and hematoxylin and eosin (H&E) and Nissl staining were used to evaluated apoptosis, oxidative stress, inflammatory response, and histopathology, respectively.ResultsOGD-stimulated BV2 microglial cells and brain tissues with CI/R had low expression of miR-103a-3p but high expression of high mobility group box 1 (HMGB1). As expected, miR-103a-3p and HMGB1 had a targeting relationship. Overexpression of HMGB1 enhanced the the levels of interleukin (IL)-1 beta, tumor necrosis factor-alpha (TNF-α) and malondialdehyde (MDA), but reduced the content of superoxide dismutase (SOD), IL-4, and IL-10, in vitro. Moreover, high expression of HMGB1 aggravated the brain injury of the model rats, and increased the secretion of inflammatory factors, exacerbated oxidative stress, and further induced tissue apoptosis in the brain tissue. Importantly, these effects of HMGB1 overexpression were partly reversed by miR-103a-3p overexpression on HMGB1 interference.ConclusionsHMGB1 is targeted by miR-103a-3p, which may be a new strategy in the treatment of CI/R.

Dataset Information

Identification of Proteins Interacting with Cytoplasmic High-Mobility Group Box 1 during the Hepatocellular Response to Ischemia Reperfusion Injury.

Publications

Identification of Proteins Interacting with Cytoplasmic High-Mobility Group Box 1 during the Hepatocellular Response to Ischemia Reperfusion Injury.

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