Hepcidin Promoted Ferroptosis through Iron Metabolism which Is Associated with DMT1 Signaling Activation in Early Brain Injury following Subarachnoid Hemorrhage.

Iron metabolism disturbances play an important role in early brain injury (EBI) after subarachnoid hemorrhage (SAH), and hepcidin largely influences iron metabolism. Importantly, iron metabolism may be associated with ferroptosis, recently a nonapoptotic iron-dependent form of cell death that may have a great impact on brain injury after SAH. We investigated hepcidin on iron metabolism and ferroptosis involving divalent metal transporter 1 (DMT1), and ferroportin-1 (FPN1) in a rat model of SAH. Male Sprague-Dawley rats were subjected to the endovascular perforation to induce SAH, and treated with heparin (inhibitor of hepcidin), or oncostatin M (OSM, inducer of hepcidin), or ebselen (inhibitor of DMT1) by intracerebroventricular injections. Hepcidin, DMT1, FPN1 and glutathione peroxidase 4 (GPX4), were detected by western blot and immunofluorescence. Iron metabolism was detected through Perl's iron staining and iron content assay. Ferroptosis, the ROS production, lipid peroxidation (LPO) was evaluated by monitoring methane dicarboxylic aldehyde (MDA), glutathione (GSH), glutathione peroxidase 4 (GPX4) activity, and transmission electron microscopy. Neurological deficit scores, Evans blue staining and brain water content were also determined to detect EBI 72 h after SAH. Our results showed that inhibition of DMT1 by ebselen could suppress iron accumulation and lipid peroxidation, and thereby alleviate ferroptosis and EBI in SAH rats. Heparin downregulated the expression of hepcidin and DMT1, increased FPN1, and exerted protective effects that were equivalent to those of ebselen on ferroptosis and EBI. In addition, OSM increased the expression of hepcidin and DMT1, decreased FPN1, and aggravated ferroptosis and EBI, while the effect on ferroptosis was reversed by ebselen. Therefore, the study revealed that hepcidin could regulate iron metabolism and contribute to ferroptosis via DMT1 signaling activation in rats with EBI after SAH.

Evaluation of serum extracellular vesicles as noninvasive diagnostic markers of glioma.

Rationale: Glioma is the most common malignant primary brain tumor in the central nervous system (CNS). The lack of reliable noninvasive diagnostic and prognostic methods is one of the main reasons for the high mortality of glioma. Serum has become a useful biomarker for the diagnosis and prognosis prediction of glioma because extracellular vesicles (EVs) carry molecular components from their parental cells. Methods: To detect EVs and perform molecular analysis of serum EVs, we established and optimized a microbead-assisted method based on flow cytometry and estimated the efficacy of EGFR protein expression and NLGN3 and PTTG1 mRNA in serum EVs from glioma patients (n=23) and healthy individuals (n=12). We evaluated the ability of EGFR+ EVs to differentiate high-grade and low-grade glioma patients and checked the correlation between EGFR in EVs and the ki-67 labeling index (LI) in the tumor tissue. Results: We demonstrated that EGFR+ EVs are effective diagnostic and prognostic markers of glioma. The expression of EGFR in serum EVs can accurately differentiate high-grade and low-grade glioma patients, and EGFR in EVs positively correlates with ki-67 LI in the tumor tissue. We also showed the potential of NLGN3 and PTTG1 mRNA in EVs for detecting glioma patients. Conclusions: We demonstrate that the protein expression of EGFR in serum EVs is an effective diagnostic marker of glioma. EGFR in EVs highly correlates with the malignancy of glioma. We also show the potential of NLGN3 and PTTG1 in EVs for detecting glioma. The optimized flow cytometry with the aid of microbead-based EV enrichment show its potential as a noninvasive method for the detection of glioma and will be beneficial to the management of glioma.

Thioredoxin-interacting protein mediates mitochondrion-dependent apoptosis in early brain injury after subarachnoid hemorrhage.

Early brain injury (EBI) was reported to be the primary cause of high mortality and poor outcomes in subarachnoid hemorrhage (SAH) patients, and apoptosis is regarded as the most important physiopathologic mechanism during EBI. Recently, our team found that thioredoxin-interacting protein (TXNIP) links endoplasmic reticulum stress (ER stress) to neuronal apoptosis and aggravates EBI. However, the other underlying mechanisms remain unknown. Mitochondria are considered to be the central points in integrating apoptotic cell death. However, whether crosstalk between TXNIP and the mitochondria-mediated intrinsic apoptotic pathway is effective on EBI has not been previously reported. Therefore, we created an endovascular perforation SAH model in Sprague-Dawley rats to determine the possible mechanism. We found that TXNIP expression in apoptotic neurons significantly increased in the SAH group compared with the sham group. In addition, increased TXNIP expression was accompanied by remarkable changes in mitochondrial-related antiapoptotic and proapoptotic factors. Furthermore, resveratrol (RES, a TXNIP inhibitor) administration significantly downregulated the expression of TXNIP and mitochondria-related proapoptotic factors. Additionally, it attenuated SAH prognostic indicators, such as brain edema, blood-brain barrier permeability, and neurological deficits. Therefore, our study further confirms that TXNIP may participate in neuronal apoptosis through the mitochondrial signaling pathway and that TXNIP may be a target for SAH treatment.

CHOP induces apoptosis by affecting brain iron metabolism in rats with subarachnoid hemorrhage.

The endoplasmic reticulum stress-related factor CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) aggravates early brain injury (EBI) in rats after subarachnoid hemorrhage (SAH). Our research aims to investigate the role of CHOP-mediated iron metabolism in EBI after SAH and the underlying mechanism. Male Sprague-Dawley rats were used to establish SAH models. Tunicamycin (Tm) was employed to excite CHOP expression, and two CHOP small interfering RNAs (siRNAs) were used to inhibit CHOP expression. Neurological scores, brain water content, and blood-brain barrier (BBB) permeability were evaluated at 24h after SAH. Western blotting and immunofluorescence were implemented for the quantification and localization of GRP78 (glucoseregulated protein78), CHOP, C/EBPα (CCAAT/enhancer binding proteinα) and hepcidin. Apoptotic cells were detected by TUNEL staining, and the brain iron content was measured via Perls' staining. The expression of CHOP and hepcidin increased and the expression of C/EBPα decreased after SAH. Knockdown of CHOP decreased the brain water content, reduced Evans blue extravasation, and improved neurological functions. CHOP significantly increased hepcidin levels and significantly decreased C/EBPα levels after SAH. Hepcidin is expressed in the nuclei of neurons and is widely co-localized with TUNEL-positive cells both in the hippocampus and cortex. Along with increased hepcidin expression, the iron content in brain tissue and the apoptosis rate were increased. Thus, CHOP promotes hepcidin expression by regulating C/EBPα activity, which increases the brain iron content, induces apoptosis and is involved in the development of EBI after SAH.

Thioredoxin-interacting protein links endoplasmic reticulum stress to inflammatory brain injury and apoptosis after subarachnoid haemorrhage.

BACKGROUND: Early brain injury (EBI) is considered a major contributor to the high morbidity and mortality associated with subarachnoid haemorrhage (SAH). Both of sterile inflammation and apoptosis are considered the important causes of EBI. Recently, it was confirmed that thioredoxin-interacting protein (TXNIP) not only participates in inflammatory amplification but also stimulates the apoptosis signalling cascade pathway. However, whether the effects of TXNIP influence the pathogenesis of SAH remains unclear. Here, we hypothesize that TXNIP activity induced by endoplasmic reticulum stress (ER stress) may contribute to the pathogenesis of EBI through pro-inflammatory and pro-apoptotic mechanisms. METHODS: A total of 299 male Sprague-Dawley rats were used to create SAH models. Resveratrol (RES, 60 mg/kg) and two TXNIP small interfering RNA (siRNA) were used to inhibit TXNIP expression. The specific inhibitors of ER stress sensors were used to disrupt the link between TXNIP and ER stress. SAH grade, neurological deficits, brain water content and blood-brain barrier (BBB) permeability were evaluated simultaneously as prognostic indicators. Fluorescent double-labelling was employed to detect the location of TXNIP in cerebral cells. Western blot and TUNEL were performed to study the mechanisms of TXNIP and EBI. RESULTS: We found that TXNIP expression significantly increased after SAH, peaking at 48 h (0.48 ± 0.04, up to 3.2-fold) and decreasing at 72 h after surgery. This process was accompanied by the generation of inflammation-associated factors. TXNIP was expressed in the cytoplasm of neurons and was widely co-localized with TUNEL-positive cells in both the hippocampus and the cortex of SAH rats. We discovered for the first time that TXNIP was co-localized in neural immunocytes (microglia and astrocytes). After administration of RES, TXNIP siRNA and ER stress inhibitors, TXNIP expression was significantly reduced and the crosstalk between TXNIP and ER stress was disrupted; this was accompanied by a reduction in inflammatory and apoptotic factors, as well as attenuation of the prognostic indices. CONCLUSIONS: These results may represent the critical evidence to support the pro-inflammatory and pro-apoptotic effects of TXNIP after SAH. Our data suggest that TXNIP participates in EBI after SAH by mediating inflammation and apoptosis; these pathways may represent a potential therapeutic strategy for SAH treatment.

Thioredoxin-Interacting Protein Mediates Apoptosis in Early Brain Injury after Subarachnoid Haemorrhage.

Early brain injury (EBI) is considered to be the major factor associated with high morbidity and mortality after subarachnoid haemorrhage (SAH). Apoptosis is the major pathological mechanism of EBI, and its pathogenesis has not been fully clarified. Here, we report that thioredoxin-interacting protein (TXNIP), which is induced by protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK), participates in EBI by promoting apoptosis. By using adult male Sprague-Dawley rats to establish SAH models, as well as Terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining, immunofluorescence, and western blot, we found that TXNIP expression significantly increased after SAH in comparison to the sham group and peaked at 48 h (up to 3.2-fold). Meanwhile, TXNIP was widely expressed in neurons and colocalized with TUNEL-positive cells in the hippocampus and cortex of SAH rats. After administration of TXNIP inhibitor-resveratrol (60 mg/kg), TXNIP small interfering RNA (siRNA) and the PERK inhibitor GSK2656157, TXNIP expression was significantly reduced, accompanied by an attenuation of apoptosis and prognostic indicators, including SAH grade, neurological deficits, brain water content, and blood-brain barrier (BBB) permeability. Collectively, these results suggest that TXNIP may participate in EBI after SAH by mediating apoptosis. The blockage of TXNIP induced by PERK could be a potential therapeutic strategy for SAH treatment.