Topological Relationships Cytoskeleton-Membrane Nanosurface-Morphology as a Basic Mechanism of Total Disorders of RBC Structures.

The state of red blood cells (RBCs) and their functional possibilities depend on the structural organization of the membranes. Cell morphology and membrane nanostructure are compositionally and functionally related to the cytoskeleton network. In this work, the influence of agents (hemin, endogenous oxidation during storage of packed RBCs, ultraviolet (UV) radiation, temperature, and potential of hydrogen (pH) changes) on the relationships between cytoskeleton destruction, membrane nanostructure, and RBC morphology was observed by atomic force microscope. It was shown that the influence of factors of a physical and biochemical nature causes structural rearrangements in RBCs at all levels of organization, forming a unified mechanism of disturbances in relationships "cytoskeleton-membrane nanosurface-cell morphology". Filament ruptures and, consequently, large cytoskeleton pores appeared. The pores caused membrane topological defects in the form of separate grain domains. Increasing loading doses led to an increase in the number of large cytoskeleton pores and defects and their fusion at the membrane nanosurfaces. This caused the changes in RBC morphology. Our results can be used in molecular cell biology, membrane biophysics, and in fundamental and practical medicine.

Human Milk Oligosaccharide 2'-Fucosyllactose Induces Neuroprotection from Intracerebral Hemorrhage Stroke.

Intracerebral hemorrhage (ICH) occurs when brain blood vessels rupture, causing inflammation and cell death. 2-Fucosyllactose (2FL), a human milk oligosaccharide, has potent antiapoptotic and anti-inflammatory effects. The purpose of this study was to examine the protective effect of 2FL in cellular and rodent models of ICH. Hemin was added to a primary rat cortical neuronal and BV2 microglia coculture to simulate ICH in vitro. IBA1 and MAP2 immunoreactivities were used to determine inflammation and neuronal survival. Hemin significantly increased IBA1, while it reduced MAP2 immunoreactivity. 2FL significantly antagonized both responses. The protective effect of 2FL was next examined in a rat ICH model. Intracerebral administration of type VII collagenase reduced open-field locomotor activity. Early post-treatment with 2FL significantly improved locomotor activity. Brain tissues were collected for immunohistochemistry and qRT-PCR analysis. 2FL reduced IBA1 and CD4 immunoreactivity in the lesioned striatum. 2FL downregulated the expression of ER stress markers (PERK and CHOP), while it upregulated M2 macrophage markers (CD206 and TGFß) in the lesioned brain. Taken together, our data support that 2FL has a neuroprotective effect against ICH through the inhibition of neuroinflammation and ER stress. 2FL may have clinical implications for the treatment of ICH.

Perampanel, an AMPAR antagonist, alleviates experimental intracerebral hemorrhage­induced brain injury via necroptosis and neuroinflammation.

Spontaneous intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and morbidity due to the lack of effective therapies. The alpha­amino­3­hydroxy­5­methyl­4­isoxazolepropionic acid receptor antagonist perampanel has been reported to alleviate early brain injury following subarachnoid hemorrhage and traumatic brain injury by reducing reactive oxygen species, apoptosis, autophagy, and necroptosis. Necroptosis is a caspase­independent programmed cell death mechanism that serves a vital role in neuronal cell death following ICH. However, the precise role of necroptosis in perampanel­mediated neuroprotection following ICH has not been confirmed. The present study aimed to investigate the neuroprotective effects and potential molecular mechanisms of perampanel in ICH­induced early brain injury by regulating neural necroptosis in C57BL/6 mice and in a hemin­induced neuron damage cell culture model. Mortality, neurological score, brain water content, and neuronal death were evaluated. The results demonstrated that perampanel treatment increased the survival rate and neurological score, and increased neuron survival. In addition, perampanel treatment downregulated the protein expression levels of receptor interacting serine/threonine kinase (RIP) 1, RIP3, and mixed lineage kinase domain like pseudokinase, and of the cytokines IL­1ß, IL­6, TNF­α, and NF­κB. These results indicated that perampanel­mediated inhibition of necroptosis and neuroinflammation ameliorated neuronal death in vitro and in vivo following ICH. The neuroprotective capacity of perampanel was partly dependent on the PTEN pathway. Taken together, the results of the present study demonstrated that perampanel improved neurological outcomes in mice and reduced neuronal death by protecting against neural necroptosis and neuroinflammation.

Atorvastatin ameliorates early brain injury after subarachnoid hemorrhage via inhibition of pyroptosis and neuroinflammation.

Subarachnoid hemorrhage (SAH) is a subtype of stroke with high mortality and morbidity due to the lack of effective therapy. Atorvastatin has been reported to alleviate early brain injury (EBI) following subarachnoid hemorrhage (SAH) via reducing reactive oxygen species, antiapoptosis, regulated autophagy, and neuroinflammation. Which was the related to the pyroptosis? Pyroptosis can be defined as a highly specific inflammatory programmed cell death, distinct from classical apoptosis and necrosis. However, the precise role of pyroptosis in atorvastatin-mediated neuroprotection following SAH has not been confirmed. The present study aimed to investigate the neuroprotection and potential molecular mechanisms of atorvastatin in the SAH-induced EBI via regulating neural pyroptosis using the filament perforation model of SAH in male C57BL/6 mice, and the hemin-induced neuron damage model in HT-22. Atorvastatin or vehicle was administrated 2 h after SAH and hemin-induced neuron damage. The mortality, neurological score, brain water content, and neuronal death were evaluated. The results show that the atorvastatin treatment markedly increased survival rate, neurological score, greater survival of neurons, downregulated the protein expression of NLRP1, cleaved caspase-1, interleukin-1ß (IL-1ß), and IL-18, which indicated that atorvastatin-inhibited pyroptosis and neuroinflammation, ameliorated neuron death in vivo/vitro subjected to SAH. Taken together, this study demonstrates that atorvastatin improved the neurological outcome in rats and reduced the neuron death by against neural pyroptosis and neuroinflammation.

A Selenium Nanocomposite Protects the Mouse Brain from Oxidative Injury Following Intracerebral Hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is a common neurological crisis leading to high mortality and morbidity. Oxidative stress-induced secondary injury plays a critical role in neurological deterioration. Previously, we synthesized a porous Se@SiO2 nanocomposite and identified their therapeutic role in osteonecrosis of the femoral head. Whether this nanocomposite is neuroprotective remains to be elucidated. METHODS: A porous Se@SiO2 nanocomposite was synthesized, and its biosafety was determined using a CCK-8 assay. The neuroprotective effect was evaluated by TUNEL staining, and intracellular ROS were detected with a DCFH-DA probe in SH-SY5Y cells exposed to hemin. Furthermore, the effect of the nanocomposite on cell apoptosis, brain edema and blood-brain barrier permeability were evaluated in a collagenase-induced ICH mouse model. The potential mechanism was also explored. RESULTS: The results demonstrated that Se@SiO2 treatment significantly improved neurological function, increased glutathione peroxidase activity and downregulated malonaldehyde levels. The proportion of apoptotic cells, brain edema and blood-brain barrier permeability were reduced significantly in ICH mice treated with Se@SiO2 compared to vehicle-treated mice. In vitro, Se@SiO2 protected SH-SY5Y cells from hemin-induced apoptosis by preventing intracellular reactive oxygen species accumulation. CONCLUSION: These results suggested that the porous Se@SiO2 nanocomposite exerted neuroprotection by suppressing oxidative stress. Se@SiO2 may be a potential candidate for the clinical treatment of ICH and oxidative stress-related brain injuries.

Activation of KEAP1/NRF2 stress signaling involved in the molecular basis of hemin-induced cytotoxicity in human pro-erythroid K562 cells.

During hemolysis, free heme released from damaged RBCs impairs adjacent cells. As a response, heme induces its metabolic degradation via heme oxygenase-1 (HO-1), activated by NF-E2-related factor 2 (NRF2), the master stress response transcription factor. Heme is well considered a signaling molecule, but how heme does activate NRF2 is not well understood. K562, human pro-erythroid cells responding to hemin (ferric chloride heme), were employed to uncover the major role of Kelch-like ECH-associated protein 1 (KEAP1)/NRF2 stress response signaling, embedded in hemin-induced cytotoxicity (HIC), at ≥50 µM. The intracellular pools of hemin were found to determine the progression from the reversible cell growth inhibition to non-apoptotic cell death. Hemin-induced accumulation of both reactive oxygen species (ROS) and ubiquitinated proteins provoked disturbed cellular proteostasis. Immediate accumulation and nuclear translocation of NRF2 were recorded as defensive adaptation. The NRF2-driven genes encoding glutamate-cysteine ligase (GCLC) and cystine/glutamate antiporter (xCT) were substantially activated. Hemin orchestrated a defensive pathway involving the management of cellular non-protein thiols, via an increase in GSH levels and secretion of cysteine. Mechanistically, hemin stabilized NRF2 protein levels selectively by inhibiting the KEAP1-driven ubiquitination of NRF2, while allowing KEAP1 ubiquitination. High-molecular-weight ubiquitinated KEAP1 variants formed in hemin-treated cells degraded in proteasomes, while a portion of them translocated into the nucleus. The KEAP1/NRF2 system can be revealed as a basic homeostatic mechanism, activated in cells encountering free heme, both in healthy and diseased state. Its activation provides a multi-target cytoprotective platform to develop agents preventing heme toxicity.

Dietary hemin promotes colonic preneoplastic lesions and DNA damage but not tumor development in a medium-term model of colon carcinogenesis in rats.

Red and processed meat consumption has been strongly related to increase the risk of colorectal cancer (CRC), although its impact is largely unknown. Hemin, an iron-containing porphyrin, is acknowledged as a putative factor of red and processed meat pro-carcinogenic effects. The aim of this study was to investigate the effects of high dietary hemin on the promotion/progression stages of 1,2-dimethylhydrazine (1,2-DMH)-induced colon carcinogenesis. Twenty-four Wistar male rats were given four subcutaneous 1,2-DMH injections and received either balanced diet or balanced diet supplemented with hemin 0.5 mmol/kg for 23 weeks. Colon specimens were analyzed for aberrant crypt foci (ACF) and tumor development. Dietary hemin significantly increased ACF number and fecal water cytotoxicity/genotoxicity in Caco-2 cells when compared to 1,2-DMH control group. However, tumor incidence, multiplicity and cell proliferation did not differ between 1,2-DMH + hemin and 1,2-DMH control group. Gene expression analysis of 91 target-genes revealed that only three genes (Figf, Pik3r5 and Tgfbr2) were down-regulated in the tumors from hemin-fed rats compared to those from 1,2-DMH control group. Therefore, the findings of this study show that high hemin intake promotes mainly DNA damage and ACF development and but does not change the number nor incidence of colon tumors induced by 1,2-DMH in male rats.

Toxicity of metamizole on differentiating HL60 cells and human neutrophil granulocytes.

Metamizole is an analgesic and antipyretic with a superior analgesic efficacy than paracetamol. Since metamizole can cause neutropenia and agranulocytosis, it is currently used in only few countries. In a previous study, we have shown that N-methyl-4-aminoantipyrine (MAA), the active metamizole metabolite, reacts with hemin and forms an electrophilic metabolite that is toxic for HL60 cells, but not for mature neutrophil granulocytes. In the current study, we investigated the toxicity of hemin (12.5 µM) and MAA (100 µM) on differentiating HL60 cells. In undifferentiated HL60 cells, hemin decreased the viability and this effect was significantly increased by MAA. Similarly, hemin/MAA was more toxic than hemin alone on human cord blood cells. At 3 days (metamyelocyte stage) and 5 days of differentiation (mature neutrophils), hemin/MAA was not toxic on HL60 cells, whereas hemin alone was still toxic. No toxicity was observed on freshly isolated human neutrophils. The protein expression of enzymes responsible for hemin metabolism increased with HL60 cell differentiation. Inhibition of heme oxygenase-1 or cytochrome P450 reductase increased the toxicity of hemin and hemin/MAA in undifferentiated, but only for hemin in differentiated HL60 cells. Similar to the enzymes involved in hemin metabolism, the protein expression of enzymes involved in antioxidative defense and the cellular glutathione pool increased with HL60 cell differentiation. In conclusion, HL60 cells become resistant to the toxicity of hemin/MAA and partly also of hemin during their differentiation. This resistance is associated with the development of heme metabolism and of the antioxidative defense system including the cellular glutathione pool.

Facile synthesis of novel carbon-dots/hemin nanoplatforms for synergistic photo-thermal and photo-dynamic therapies.

Due to the traditional therapies of cancer inducing huge pains to patients, the non-invasive photo-guided therapies are attracting massive attentions of researchers. Herein, the intelligent-designed carbon-dots/hemin nanoplatforms (HCDs NPs) were developed, owning high-authority photo-therapy for cancer. The fluorescence resonance energy transfer (FRET) effect enhanced the photo-thermal ability of HCDs NPs, endowing the synthesized nanoplatforms with photo-dynamic property simultaneously. Therefore, the obtained HCDs NPs could achieve synergetic photo-thermal and photo-dynamic therapies for cancer. Basing on the experimental results, the prepared HCDs NPs could induce the temperature enhancement high to ca 26 °C under laser irradiation, also with the outstanding photo-dynamic efficacy. More than 90% of cancer cells die after 10 min laser treatment. Thus, the dual-modal photo-therapeutic HCDs NPs are promising and excellent nanomaterials for potential application in synergistic cancer therapy.

N-acetylcysteine targets 5 lipoxygenase-derived, toxic lipids and can synergize with prostaglandin E2 to inhibit ferroptosis and improve outcomes following hemorrhagic stroke in mice.

OBJECTIVES: N-acetylcysteine (NAC) is a clinically approved thiol-containing redox modulatory compound currently in trials for many neurological and psychiatric disorders. Although generically labeled as an "antioxidant," poor understanding of its site(s) of action is a barrier to its use in neurological practice. Here, we examined the efficacy and mechanism of action of NAC in rodent models of hemorrhagic stroke. METHODS: Hemin was used to model ferroptosis and hemorrhagic stroke in cultured neurons. Striatal infusion of collagenase was used to model intracerebral hemorrhage (ICH) in mice and rats. Chemical biology, targeted lipidomics, arachidonate 5-lipoxygenase (ALOX5) knockout mice, and viral-gene transfer were used to gain insight into the pharmacological targets and mechanism of action of NAC. RESULTS: NAC prevented hemin-induced ferroptosis by neutralizing toxic lipids generated by arachidonate-dependent ALOX5 activity. NAC efficacy required increases in glutathione and is correlated with suppression of reactive lipids by glutathione-dependent enzymes such as glutathione S-transferase. Accordingly, its protective effects were mimicked by chemical or molecular lipid peroxidation inhibitors. NAC delivered postinjury reduced neuronal death and improved functional recovery at least 7 days following ICH in mice and can synergize with clinically approved prostaglandin E2 (PGE2 ). INTERPRETATION: NAC is a promising, protective therapy for ICH, which acted to inhibit toxic arachidonic acid products of nuclear ALOX5 that synergized with exogenously delivered protective PGE2 in vitro and in vivo. The findings provide novel insight into a target for NAC, beyond the generic characterization as an antioxidant, resulting in neuroprotection and offer a feasible combinatorial strategy to optimize efficacy and safety in dosing of NAC for treatment of neurological disorders involving ferroptosis such as ICH. Ann Neurol 2018;84:854-872.