Cytoglobin functions as a redox regulator of melanogenesis in normal epidermal melanocytes.

Epidermal melanocytes are continuously exposed to sunlight-induced reactive oxygen species (ROS) and oxidative stress generated during the synthesis of melanin. Therefore, they have developed mechanisms that maintain normal redox homeostasis. Cytoglobin (CYGB), a ubiquitously expressed intracellular iron hexacoordinated globin, exhibits antioxidant activity and regulates the redox state of mammalian cells through its activities as peroxidase and nitric oxide (NO) dioxygenase. We postulated that CYGB functions in the melanogenic process as a regulator that maintains oxidative stress within a physiological level. This was examined by characterizing normal human melanocytes with the knockdown (KD) of CYGB using morphological and molecular biological criteria. CYGB-KD cells were larger, had more dendrites, and generated more melanin granules in the advanced stages of melanogenesis than control cells. The expression levels of major melanogenesis-associated genes and proteins were higher in CYGB-KD melanocytes than in wild type (WT) cells. As expected, CYGB-KD melanocytes generated more ROS and NO than WT cells. In conclusion, CYGB physiologically contributes to maintaining redox homeostasis in the melanogenic activity of normal melanocytes by controlling the intracellular levels of ROS and NO.

Transcriptomics of a cytoglobin knockout mouse: Insights from hepatic stellate cells and brain.

The vertebrate respiratory protein cytoglobin (Cygb) is thought to exert multiple cellular functions. Here we studied the phenotypic effects of a Cygb knockout (KO) in mouse on the transcriptome level. RNA sequencing (RNA-Seq) was performed for the first time on sites of major endogenous Cygb expression, i.e. quiescent and activated hepatic stellate cells (HSCs) and two brain regions, hippocampus and hypothalamus. The data recapitulated the up-regulation of Cygb during HSC activation and its expression in the brain. Differential gene expression analyses suggested a role of Cygb in the response to inflammation in HSCs and its involvement in retinoid metabolism, retinoid X receptor (RXR) activation-induced xenobiotics metabolism, and RXR activation-induced lipid metabolism and signaling in activated cells. Unexpectedly, only minor effects of the Cygb KO were detected in the transcriptional profiles in hippocampus and hypothalamus, precluding any enrichment analyses. Furthermore, the transcriptome data pointed at a previously undescribed potential of the Cygb- knockout allele to produce cis-acting effects, necessitating future verification studies.

Insights into the function of cytoglobin.

Since its discovery in 2001, the function of cytoglobin has remained elusive. Through extensive in vitro and in vivo research, a range of potential physiological and pathological mechanisms has emerged for this multifunctional member of the hemoglobin family. Currently, over 200 research publications have examined different aspects of cytoglobin structure, redox chemistry and potential roles in cell signalling pathways. This research is wide ranging, but common themes have emerged throughout the research. This review examines the current structural, biochemical and in vivo knowledge of cytoglobin published over the past two decades. Radical scavenging, nitric oxide homeostasis, lipid binding and oxidation and the role of an intramolecular disulfide bond on the redox chemistry are examined, together with aspects and roles for Cygb in cancer progression and liver fibrosis.

The knockout of cytoglobin 1 in zebrafish (Danio rerio) alters lipid metabolism, iron homeostasis and oxidative stress response.

Cytoglobin (Cygb) is an evolutionary ancient heme protein with yet unclear physiological function(s). Mammalian Cygb is ubiquitously expressed in all tissues and is proposed to be involved in reactive oxygen species (ROS) detoxification, nitric oxide (NO) metabolism and lipid-based signaling processes. Loss-of-function studies in mouse associate Cygb with apoptosis, inflammation, fibrosis, cardiovascular dysfunction or oncogenesis. In zebrafish (Danio rerio), two cygb genes exist, cytoglobin 1 (cygb1) and cytoglobin 2 (cygb2). Both have different coordination states and distinct expression sites within zebrafish tissues. The biological roles of the cygb paralogs are largely uncharacterized. We used a CRISPR/Cas9 genome editing approach and generated a knockout of the penta-coordinated cygb1 for in vivo analysis. Adult male cygb1 knockouts develop phenotypic abnormalities, including weight loss. To identify the molecular mechanisms underlying the occurrence of these phenotypes and differentiate between function and effect of the knockout we compared the transcriptomes of cygb1 knockout at different ages to age-matched wild-type zebrafish. We found that immune regulatory and cell cycle regulatory transcripts (e.g. tp53) were up-regulated in the cygb1 knockout liver. Additionally, the expression of transcripts involved in lipid metabolism and transport, the antioxidative defense and iron homeostasis was affected in the cygb1 knockout. Cygb1 may function as an anti-inflammatory and cytoprotective factor in zebrafish liver, and may be involved in lipid-, iron-, and ROS-dependent signaling.

Lansoprazole attenuates cyclophosphamide-induced cardiopulmonary injury by modulating redox-sensitive pathways and inflammation.

Cyclophosphamide (CPA) is a classical chemotherapeutic drug widely used as an anticancer and immunosuppressive agent. However, it is frequently associated with significant toxicities to the normal cells of different organs, including the lung and heart. Lansoprazole (LPZ), a proton pump inhibitor (PPI), possesses antioxidant and anti-inflammatory properties. The current study investigated how LPZ protects against CPA-induced cardiac and pulmonary damage, focusing on PPARγ, Nrf2, HO-1, cytoglobin, PI3K/AKT, and NF-κB signaling. Animals were randomly assigned into four groups: normal control group (received vehicle), LPZ only group (Rats received LPZ at a dose of 50 mg/kg/day P.O. for 10 days), CPA group (CPA was administered (200 mg/kg) as a single i.p. injection on the 7th day), and cotreatment group (LPZ plus CPA). Histopathological and biochemical analyses were conducted. Our results revealed that LPZ treatment revoked CPA-induced heart and lung histopathological alterations. Also, LPZ potently mitigated CPA-induced cardiac and pulmonary oxidative stress through the activation of PPARγ, Nrf2/HO-1, cytoglobin, and PI3K/AKT signaling pathways. Also, LPZ effectively suppressed inflammatory response as evidenced by down-regulating the inflammatory strategic controller NF-κB, MPO, and pro-inflammatory cytokines. The present findings could provide a mechanistic basis for understanding LPZ's role in CPA-induced cardiopulmonary injury through the alleviation of oxidative stress and inflammatory burden.

Nitric Oxide Derived from Cytoglobin-Deficient Hepatic Stellate Cells Causes Suppression of Cytochrome c Oxidase Activity in Hepatocytes.

Aims: Cell-cell interactions between hepatocytes (Hep) and other liver cells are key to maintaining liver homeostasis. Cytoglobin (CYGB), expressed exclusively by hepatic stellate cells (HSC), is essential in mitigating mitochondrial oxidative stress. CYGB absence causes Hep dysfunction and evokes hepatocarcinogenesis through an elusive mechanism. CYGB deficiency is speculated to hinder nitric oxide dioxygenase (NOD) activity, resulting in the elevated formation and release of nitric oxide (NO). Hence, we hypothesized that NO accumulation induced by the loss of NOD activity in CYGB-deficient HSC could adversely affect mitochondrial function in Hep, leading to disease progression. Results: NO, a membrane-permeable gas metabolite overproduced by CYGB-deficient HSC, diffuses into the neighboring Hep to reversibly inhibit cytochrome c oxidase (CcO), resulting in the suppression of respiratory function in an electron transport chain (ETC). The binding of NO to CcO is proved using purified CcO fractions from Cygb knockout (Cygb-/-) mouse liver mitochondria. Its inhibitory action toward CcO-specific activity is fully reversed by the external administration of oxyhemoglobin chasing away the bound NO. Thus, these findings indicate that the attenuation of respiratory function in ETC causes liver damage through the formation of excessive reactive oxygen species. Treating Cygb-/- mice with an NO synthase inhibitor successfully relieved NO-induced inhibition of CcO activity in vivo. Innovation and Conclusion: Our findings provide a biochemical link between CYGB-absence in HSC and neighboring Hep dysfunction; mechanistically the absence of CYGB in HSC causes mitochondrial dysfunction of Hep via the inhibition of CcO activity by HSC-derived NO. Antioxid. Redox Signal. 38, 463-479.

The involvement of Nrf2/HO-1/cytoglobin and Ang-II/NF-κB signals in the cardioprotective mechanism of lansoprazole against cisplatin-induced heart injury.

Cardiac toxicity is a serious adverse effect of cisplatin (CIS). Lansoprazole (LPZ) is a proton pump inhibitor with promising cardioprotective effects. Our study planned to examine the cardioprotective effect of LPZ against CIS-induced cardiac injury. To achieve this goal, 32 male rats were randomly allocated into four groups. CIS, 7 mg/kg, was injected i.p. on the fifth day of the experiment. LPZ was administered via oral gavage at a dose of 50 mg/kg. The present study revealed that CIS injection induced a remarkable cardiac injury evidenced by an increase in serum ALP, AST, CK-MB, LDH, and troponin-I levels. The cardiac oxidative damage was also observed after CIS injection and mediated by downregulation of GSH, SOD, GST, Nrf2, HO-1, PPAR-γ, and cytoglobin levels associated with the upregulation of MDA content. Besides, CIS injection caused a significant inflammatory reaction mediated by alteration of cardiac NF-κB, STAT-3, p-STAT-3, and IκB expressions. Additionally, cardiac Ang-II expression was significantly increased in CIS control rats, while Ang 1-7 expression was significantly reduced relative to normal rats. In contrast, LPZ administration remarkably ameliorated these changes in the heart of CIS-intoxicated rats. Collectively, LPZ potently attenuated cardiac toxicity induced by CIS via regulation of Nrf2/HO-1, PPAR-γ, cytoglobin, IκB/NF-κB/STAT-3, and Ang-II/Ang 1-7 signals.

Redox and spectroscopic properties of mammalian nitrite reductase-like hemoproteins.

Besides the canonical pathway of L-arginine oxidation to produce nitric oxide (NO) in vivo, the nitrate-nitrite-NO pathway has been widely accepted as another source for circulating NO in mammals, especially under hypoxia. To date, there have been at least ten heme-containing nitrite reductase-like proteins discovered in mammals with activities mainly identified in vitro, including four globins (hemoglobin, myoglobin, neuroglobin (Ngb), cytoglobin (Cygb)), three mitochondrial respiratory chain enzymes (cytochrome c oxidase, cytochrome bc1, cytochrome c), and three other heme proteins (endothelial nitric oxide synthase, cytochrome P450 and indoleamine 2,3-dioxygenase 1 (IDO1)). The pathophysiological functions of these proteins are closely related to their redox and spectroscopic properties, as well as their protein structure, although the physiological roles of Ngb, Cygb and IDO1 remain unclear. So far, comprehensive summaries of the redox and spectroscopic properties of these nitrite reductase-like hemoproteins are still lacking. In this review, we have mainly summarized the published data on the application of ultraviolet-visible, electron paramagnetic resonance, circular dichroism and resonance Raman spectroscopies, and X-ray crystallography in studying nitrite reductase-like activity of these 10 proteins, in order to sort out the relationships among enzymatic function, structure and spectroscopic characterization, which might help in understanding their roles in redox biology and medicine.

Role of cytoglobin in cigarette smoke constituent-induced loss of nitric oxide bioavailability in vascular smooth muscle cells.

Cytoglobin (Cygb) has been identified as the major nitric oxide (NO) metabolizing protein in vascular smooth muscle cells (VSMCs) and is crucial for the regulation of vascular tone. In the presence of its requisite cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism through the oxygen-dependent process of NO dioxygenation. Tobacco cigarette smoking (TCS) induces vascular dysfunction; however, the role of Cygb in the pathophysiology of TCS-induced cardiovascular disease has not been previously investigated. While TCS impairs NO biosynthesis, its effect on NO metabolism remains unclear. Therefore, we performed studies in aortic VSMCs with tobacco smoke extract (TSE) exposure to investigate the effects of cigarette smoke constituents on the rates of NO decay, with focus on the alterations that occur in the process of Cygb-mediated NO metabolism. TSE greatly enhanced the rates of NO metabolism by VSMCs. An initial increase in superoxide-mediated NO degradation was seen at 4 h of exposure. This was followed by much larger progressive increases at 24 and 48 h, accompanied by parallel increases in the expression of Cygb and B5/B5R. siRNA-mediated Cygb knockdown greatly decreased these TSE-induced elevations in NO decay rates. Therefore, upregulation of the levels of Cygb and its reducing system accounted for the large increase in NO metabolism rate seen after 24 h of TSE exposure. Thus, increased Cygb-mediated NO degradation would contribute to TCS-induced vascular dysfunction and partial inhibition of Cygb expression or its NO dioxygenase function could be a promising therapeutic target to prevent secondary cardiovascular disease.

The role of globins in cardiovascular physiology.

Globin proteins exist in every cell type of the vasculature, from erythrocytes to endothelial cells, vascular smooth muscle cells, and peripheral nerve cells. Many globin subtypes are also expressed in muscle tissues (including cardiac and skeletal muscle), in other organ-specific cell types, and in cells of the central nervous system (CNS). The ability of each of these globins to interact with molecular oxygen (O2) and nitric oxide (NO) is preserved across these contexts. Endothelial α-globin is an example of extraerythrocytic globin expression. Other globins, including myoglobin, cytoglobin, and neuroglobin, are observed in other vascular tissues. Myoglobin is observed primarily in skeletal muscle and smooth muscle cells surrounding the aorta or other large arteries. Cytoglobin is found in vascular smooth muscle but can also be expressed in nonvascular cell types, especially in oxidative stress conditions after ischemic insult. Neuroglobin was first observed in neuronal cells, and its expression appears to be restricted mainly to the CNS and the peripheral nervous system. Brain and CNS neurons expressing neuroglobin are positioned close to many arteries within the brain parenchyma and can control smooth muscle contraction and thus tissue perfusion and vascular reactivity. Overall, reactions between NO and globin heme iron contribute to vascular homeostasis by regulating vasodilatory NO signals and scavenging reactive species in cells of the mammalian vascular system. Here, we discuss how globin proteins affect vascular physiology, with a focus on NO biology, and offer perspectives for future study of these functions.

Cytoglobin has potent superoxide dismutase function.

Cytoglobin (Cygb) was discovered as a novel type of globin that is expressed in mammals; however, its functions remain uncertain. While Cygb protects against oxidant stress, the basis for this is unclear, and the effect of Cygb on superoxide metabolism is unknown. From dose-dependent studies of the effect of Cygb on superoxide catabolism, we identify that Cygb has potent superoxide dismutase (SOD) function. Initial assays using cytochrome c showed that Cygb exhibits a high rate of superoxide dismutation on the order of 108 M-1 ⋅ s-1 Spin-trapping studies also demonstrated that the rate of Cygb-mediated superoxide dismutation (1.6 × 108 M-1 ⋅ s-1) was only ∼10-fold less than Cu,Zn-SOD. Stopped-flow experiments confirmed that Cygb rapidly dismutates superoxide with rates within an order of magnitude of Cu,Zn-SOD or Mn-SOD. The SOD function of Cygb was inhibited by cyanide and CO that coordinate to Fe3+-Cygb and Fe2+-Cygb, respectively, suggesting that dismutation involves iron redox cycling, and this was confirmed by spectrophotometric titrations. In control smooth-muscle cells and cells with siRNA-mediated Cygb knockdown subjected to extracellular superoxide stress from xanthine/xanthine oxidase or intracellular superoxide stress triggered by the uncoupler, menadione, Cygb had a prominent role in superoxide metabolism and protected against superoxide-mediated death. Similar experiments in vessels showed higher levels of superoxide in Cygb-/- mice than wild type. Thus, Cygb has potent SOD function and can rapidly dismutate superoxide in cells, conferring protection against oxidant injury. In view of its ubiquitous cellular expression at micromolar concentrations in smooth-muscle and other cells, Cygb can play an important role in cellular superoxide metabolism.

A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma.

Cytoglobin (CYGB) is a ubiquitously expressed protein with a protective role against oxidative stress, fibrosis and tumor growth, shown to be transcriptionally regulated under hypoxic conditions. Hypoxia-inducible CYGB expression is observed in several cancer cell lines and particularly in various melanoma-derived cell lines. However, reliable detection of hypoxia-inducible mRNA levels by qPCR depends on the critical choice of suitable reference genes for accurate normalization. Limited evidence exists to support selection of the commonly used reference genes in hypoxic models of melanoma. This study aimed to select the optimal reference genes to study CYGB expression levels in melanoma cell lines exposed to hypoxic conditions (0.2% O2) and to the HIF prolyl hydroxylase inhibitor roxadustat (FG-4592). The expression levels of candidate genes were assessed by qPCR and the stability of genes was evaluated using the geNorm and NormFinder algorithms. Our results display that B2M and YWHAZ represent the most optimal reference genes to reliably quantify hypoxia-inducible CYGB expression in melanoma cell lines. We further validate hypoxia-inducible CYGB expression on protein level and by using CYGB promoter-driven luciferase reporter assays in melanoma cell lines.

Cytoglobin promotes sensitivity to ferroptosis by regulating p53-YAP1 axis in colon cancer cells.

Ferroptosis is an iron-dependent mode of non-apoptotic cell death characterized by accumulation of lipid reactive oxygen species (ROS). As a regulator of ROS, cytoglobin (CYGB) plays an important role in oxygen homeostasis and acts as a tumour suppressor. However, the mechanism by which CYGB regulates cell death is largely unknown. Here, we show that CYGB overexpression increased ROS accumulation and disrupted mitochondrial function as determined by the oxygen consumption rate and membrane potential. Importantly, ferroptotic features with accumulated lipid ROS and malondialdehyde were observed in CYGB-overexpressing colorectal cancer cells. Moreover, CYGB significantly increased the sensitivity of cancer cells to RSL3- and erastin-induced ferroptotic cell death. Mechanically, both YAP1 and p53 were significantly increased based on the RNA sequencing. The knock-down of YAP1 alleviated production of lipid ROS and sensitivity to ferroptosis in CYGB overexpressed cells. Furthermore, YAP1 was identified to be inhibited by p53 knock-down. Finally, high expression level of CYGB had the close correlation with key genes YAP1 and ACSL4 in ferroptosis pathway in colon cancer based on analysis from TCGA data. Collectively, our results demonstrated a novel tumour suppressor role of CYGB through p53-YAP1 axis in regulating ferroptosis and suggested a potential therapeutic approach for colon cancer.

Hexa Histidine-Tagged Recombinant Human Cytoglobin Deactivates Hepatic Stellate Cells and Inhibits Liver Fibrosis by Scavenging Reactive Oxygen Species.

BACKGROUND AND AIMS: Antifibrotic therapy remains an unmet medical need in human chronic liver disease. We report the antifibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. APPROACH AND RESULTS: Cygb-deficient mice that had bile duct ligation-induced liver cholestasis or choline-deficient amino acid-defined diet-induced steatohepatitis significantly exacerbated liver damage, fibrosis, and reactive oxygen species (ROS) formation. All of these manifestations were attenuated in Cygb-overexpressing mice. We produced hexa histidine-tagged recombinant human CYGB (His-CYGB), traced its biodistribution, and assessed its function in HSCs or in mice with advanced liver cirrhosis using thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes through a clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of collagen type 1 alpha 1 production and α-smooth muscle actin expression. Replacement the iron center of the heme group with cobalt nullified the effect of His-CYGB. In addition, His-CYGB induced interferon-ß secretion by HSCs that partly contributed to its antifibrotic function. Momelotinib incompletely reversed the effect of His-CYGB. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis, and oxidative cell damage in mice administered TAA or DDC mice without adverse effects. RNA-sequencing analysis revealed the down-regulation of inflammation- and fibrosis-related genes and the up-regulation of antioxidant genes in both cell culture and liver tissues. The injected His-CYGB predominantly localized to HSCs but not to macrophages, suggesting specific targeting effects. His-CYGB exhibited no toxicity in chimeric mice with humanized livers. CONCLUSIONS: His-CYGB could have antifibrotic clinical applications for human chronic liver diseases.

Nephroprotective effect of umbelliferone against cisplatin-induced kidney damage is mediated by regulation of NRF2, cytoglobin, SIRT1/FOXO-3, and NF- kB-p65 signaling pathways.

Cisplatin (Cis) is one of the most potent and effective broad-spectrum antitumor drugs, but its use is limited due to nephrotoxicity. The current study investigated the renoprotective effect of umbelliferone (UMB) on Cis-induced nephrotoxicity in rats. Renal injury was induced by a single injection of Cis (7 mg/kg, ip). Our results exhibited that the injection of Cis significantly disrupted renal function biomarkers as well as KIM-1 expression. The expressions of TNF-α, IL-1ß, NF-kB-p65, and IKKß were elevated along with downregulation of IkBα expression. Also, Cis disrupted cellular oxidant/antioxidant balance through the reduction of glutathione (GSH), glutathione-S-transferase (GST), and superoxide dismutase (SOD) levels and elevation of malondialdehyde (MDA) content. On the contrary, the levels of renal function biomarkers, cytokines, NF-kB-p65, IkBα, IKKß, and oxidant/antioxidant status have been improved after UMB treatment. Mechanistically, rats administered Cis only exhibited a significant decrease in NRF2 and cytoglobin expressions as well as the CREB, SIRT1, FOXO-3, and PPAR-γ genes. Treatment with UMB significantly upregulated NRF2 and cytoglobin proteins, as well as effectively increased the expression of CREB, SIRT1, FOXO-3, PPAR-γ, and NRF2 genes. Histopathological findings strongly supported our biochemical results, as evidenced by attenuation of renal hemorrhage, cast diffusion, and inflammatory cell infiltration. Interestingly, UMB significantly enhanced Cis cytotoxicity in both HL-60 and HeLa cells in a dose-dependent manner. Together, our results demonstrated that UMB can protect against Cis-induced nephrotoxicity in normal rats along with the enhancement of its in vitro antitumor activity. These findings suggested that UMB could be used as a potential adjuvant therapy in Cis chemotherapeutic protocols.

Defining the reducing system of the NO dioxygenase cytoglobin in vascular smooth muscle cells and its critical role in regulating cellular NO decay.

In smooth muscle, cytoglobin (Cygb) functions as a potent nitric oxide (NO) dioxygenase and regulates NO metabolism and vascular tone. Major questions remain regarding which cellular reducing systems regulate Cygb-mediated NO metabolism. To better define the Cygb-mediated NO dioxygenation process in vascular smooth muscle cells (SMCs), and the requisite reducing systems that regulate cellular NO decay, we assessed the intracellular concentrations of Cygb and its putative reducing systems and examined their roles in the process of NO decay. Cygb and the reducing systems, cytochrome b5 (B5)/cytochrome b5 reductase (B5R) and cytochrome P450 reductase (CPR) were measured in aortic SMCs. Intracellular Cygb concentration was estimated as 3.5 µM, while B5R, B5, and CPR were 0.88, 0.38, and 0.15 µM, respectively. NO decay in SMCs was measured following bolus addition of NO to air-equilibrated cells. siRNA-mediated knockdown experiments indicated that âˆ¼78% of NO metabolism in SMCs is Cygb-dependent. Of this, ∼87% was B5R- and B5-dependent. CPR knockdown resulted in a small decrease in the NO dioxygenation rate (VNO), while depletion of ascorbate had no effect. Kinetic analysis of VNO for the B5/B5R/Cygb system with variation of B5 or B5R concentrations from their SMC levels showed that VNO exhibits apparent Michaelis-Menten behavior for B5 and B5R. In contrast, linear variation was seen with change in Cygb concentration. Overall, B5/B5R was demonstrated to be the major reducing system supporting Cygb-mediated NO metabolism in SMCs with changes in cellular B5/B5R levels modulating the process of NO decay.

The Impact of Royal Jelly against Hepatic Ischemia/Reperfusion-Induced Hepatocyte Damage in Rats: The Role of Cytoglobin, Nrf-2/HO-1/COX-4, and P38-MAPK/NF-κB-p65/TNF-α Signaling Pathways.

OBJECTIVE: The present study was conducted to elucidate the underlying molecular mechanism as well as the potential hepatoprotective effects of royal jelly (RJ) against hepatic ischemia/ reperfusion (IR) injury. METHODS: Rats were assigned into four groups; sham (received vehicle), IR (30 minutes ischemia and 45 minutes reperfusion), sham pretreated with RJ (200 mg/kg P.O.), and IR pretreated with RJ (200 mg/kg P.O.). The experiment lasted for 28 days. RESULTS: Hepatic IR significantly induced hepatic dysfunctions, as manifested by elevation of serum transaminases, ALP and LDH levels. Moreover, hepatic IR caused a significant up-regulation of P38-MAPK, NF-κB-p65, TNF-α and MDA levels along with marked down-regulation of Nrf-2, HO-1, COX-4, cytoglobin, IκBa, IL-10, GSH, GST and SOD levels. Additionally, marked histopathological changes were observed after hepatic IR injury. On the contrary, pretreatment with RJ significantly improved hepatic functions along with the alleviation of histopathological changes. Moreover, RJ restored oxidant/antioxidant balance as well as hepatic expressions of Nrf- 2, HO-1, COX-4, and cytoglobin. Simultaneously, RJ significantly mitigated the inflammatory response by down-regulation of P38-MAPK, NF-κB-p65, TNF-α expression. CONCLUSION: The present results revealed that RJ has successfully protected the liver against hepatic IR injury through modulation of cytoglobin, Nrf-2/HO-1/COX-4, and P38-MAPK/NF-κB-p65/TNF- α signaling pathways.

A globin-family protein, Cytoglobin 1, is involved in the development of neural crest-derived tissues and organs in zebrafish.

The zebrafish is an excellent model animal that is amenable to forward genetics approaches. To uncover unknown developmental regulatory mechanisms in vertebrates, we conducted chemical mutagenesis screening and identified a novel mutation, kanazutsi (kzt). This mutation is recessive, and its homozygotes are embryonic lethal. Mutant embryos suffered from a variety of morphological defects, such as head flattening, pericardial edema, circulation defects, disrupted patterns of melanophore distribution, dwarf eyes, a defective jaw, and extensive apoptosis in the head, which indicates that the main affected tissues are derived from neural crest cells (NCCs). The expression of tissue-specific markers in kzt mutants showed that the early specification of NCCs was normal, but their later differentiation was severely affected. The mutation was mapped to chromosome 3 by linkage analyses, near cytoglobin 1 (cygb1), the product of which is a globin-family respiratory protein. cygb1 expression was activated during somitogenesis in somites and cranial NCCs in wild-type embryos but was significantly downregulated in mutant embryos, despite the normal primary structure of the gene product. The kzt mutation was phenocopied by cygb1 knockdown with low-dose morpholino oligos and was partially rescued by cygb1 overexpression. Both severe knockdown and null mutation of cygb1, established by the CRISPR/Cas9 technique, resulted in far more severe defects at early stages. Thus, it is highly likely that the downregulation of cygb1 is responsible for many, if not all, of the phenotypes of the kzt mutation. These results reveal a requirement for globin family proteins in vertebrate embryos, particularly in the differentiation and subsequent development of NCCs.

Redox sensor properties of human cytoglobin allosterically regulate heme pocket reactivity.

Cytoglobin is a conserved hemoprotein ubiquitously expressed in mammalian tissues, which conducts electron transfer reactions with proposed signaling functions in nitric oxide (NO) and lipid metabolism. Cytoglobin has an E7 distal histidine (His81), which unlike related globins such as myoglobin and hemoglobin, is in equilibrium between a bound, hexacoordinate state and an unbound, pentacoordinate state. The His81 binding equilibrium appears to be allosterically modulated by the presence of an intramolecular disulfide between two cysteines (Cys38 and Cys83). The formation of this disulfide bridge regulates nitrite reductase activity and lipid binding. Herein, we attempt to clarify the effects of defined thiol oxidation states on small molecule binding of cytoglobin heme, using cyanide binding to probe the ferric state. Cyanide binding kinetics to wild-type cytoglobin reveal at least two kinetically distinct subpopulations, depending on thiol oxidation states. Experiments with covalent thiol modification by NEM, glutathione, and amino acid substitutions (C38S, C83S and H81A), indicate that subpopulations ranging from fully reduced thiols, single thiol oxidation, and intramolecular disulfide formation determine heme binding properties by modulating the histidine-heme affinity and ligand binding. The redox modulation of ligand binding is sensitive to physiological levels of hydrogen peroxide, with a functional midpoint redox potential for the native cytoglobin intramolecular disulfide bond of -189 ± 4 mV, a value within the boundaries of intracellular redox potentials. These results support the hypothesis that Cys38 and Cys83 on cytoglobin serve as sensitive redox sensors that modulate the cytoglobin distal heme pocket reactivity and ligand binding.

Intranasal administration of Cytoglobin modifies human umbilical cord­derived mesenchymal stem cells and improves hypoxic­ischemia brain damage in neonatal rats by modulating p38 MAPK signaling­mediated apoptosis.

Neonatal hypoxic­ischemic brain damage (HIBD) is a common clinical syndrome in newborns. Hypothermia is the only approved therapy for the clinical treatment; however, the therapeutic window of hypothermia is confined to 6 h after birth and even then, >40% of the infants either die or survive with various impairments, including cerebral palsy, seizure disorder and intellectual disability following hypothermic treatment. The aim of the present study was to determine whether nasal transplantation of Cytoglobin (CYGB) genetically modified human umbilical cord­derived mesenchymal stem cells (CYGB­HuMSCs) exhibited protective effects in neonatal rats with HIBD compared with those treated without genetically modified CYGB. A total of 120 neonatal Sprague­Dawley rats (postnatal day 7) were assigned to either a Sham, HIBD, HuMSCs or CYGB­HuMSCs group (n = 30 rats/group). For HIBD modeling, rats underwent left carotid artery ligation and were exposed to 8% oxygen for 2.5 h. A total of 30 min after HI, HuMSCs (or CYGB­HuMSCs) labeled with enhanced­green fluorescent protein (eGFP) were intranasally administered. After modeling for 3, 14 and 29 days, five randomly selected rats were sacrificed in each group, and the expression levels of CYGB, ERK, JNK and p38 in brain tissues were determined. Nissl staining of the cortex and hippocampal Cornu Ammonis 1 area of rats in each group were compared after 3 days of modeling. TUNEL assay and immunofluorescence were performed 3 days after modeling. Long term memory in rats was assessed using a Morris­water maze 29 days after modeling. The HIBD group demonstrated significant deficiencies compared with the Sham group based on Nissl staining, TUNEL assay and the Morris­water maze test. HuMSC treated rats exhibited improvement on in all the tests, and CYGB­HuMSCs treatment resulted in further improvements. PCR and western blotting results indicated that the CYGB mRNA and protein levels were increased from day 3 to day 29 after transplantation of CYGB­HuMSCs. Furthermore, it was identified that CYGB­HuMSC transplantation suppressed p38 signaling at all experimental time points. Immunofluorescence indicated the scattered presence of HuMSCs or CYGB­HuMSCs in damaged brain tissue. No eGFP and glial fibrillary acidic protein or eGFP and neuron­specific enolase double­stained positive cells were found in the brain tissues. Therefore, CYGB­HuMSCs may serve as a gene transporter, as well as exert a neuroprotective and antiapoptotic effect in HIBD, potentially via the p38 mitogen­activated protein kinase signaling pathway.