AHCCⓇ inhibited Hepatic Stellate Cells Activation by Regulation of Cytoglobin induction via TLR2-SAPK/JNK pathway and Collagen Production via TLR4-NFκB pathway.

[Introduction] Cirrhosis, which represents the end stage of liver fibrosis, remains a life-threatening condition without effective treatment. Therefore, prevention of the progression of liver fibrosis through lifestyle habits such as diet and exercise is crucial. The functional food AHCCⓇ has been reported to be effective in improving the pathophysiology of various liver diseases. In this study, the aim was to analyze the influence of AHCCⓇ on hepatic stellate cells, which are responsible for liver fibrosis. [Materials and Methods] Eight-week-old male C57BL6/j mice were induced liver fibrosis by intraperitoneal injection of carbon tetrachloride. Simultaneously, they were orally administered 3% AHCCⓇto investigate its impact on the progression of liver fibrosis. Using the human hepatic stellate cell line HHSteC, we analyzed the influence of AHCCⓇ on the expression of molecules related to hepatic stellate cell activation. [Results] The administration of AHCCⓇ resulted in reduced expression of collagen1a, alpha smooth muscle actin (αSMA), and Heat shock protein 47 in the liver. Furthermore, the expression of cytoglobin, a marker for quiescent hepatic stellate cells, was enhanced. In vitro study, it was confirmed that AHCCⓇ inhibited αSMA by induction of cytoglobin via upregulating the SAPK/JNK pathway through toll-like receptor (TLR) 2. In addition, AHCCⓇ suppressed collagen1a production by hepatic stellate cells through TLR4-NFκß pathway. [Conclusion] AHCCⓇ was suggested to suppress hepatic fibrosis by inhibition of hepatic stellate cells activation. Daily intake of AHCCⓇ from mild fibrotic stages may have the potential to prevent the progression of liver fibrosis.

SOX7 inhibits the malignant progression of bladder cancer via the DNMT3B/CYGB axis.

Bladder cancer (BCa) stands out as a highly prevalent malignant tumor affecting the urinary system. The Sex determining region Y-box protein family is recognized for its crucial role in BCa progression. However, the effect of Sex determining region Y-box 7 (SOX7) on BCa progression has not been fully elucidated. Herein, RNA-sequencing, western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF) and tissue microarray were utilized to assess SOX7 expression in vitro and in vivo. Additionally, SOX7 expression, prognosis, and SOX7 + cytoglobin (CYGB) score were analyzed using R software. In vitro and vivo experiments were performed with BCa cell lines to validate the effect of SOX7 knockdown and overexpression on the malignant progression of BCa. The results showed that SOX7 exhibits low expression in BCa. It functions in diverse capacities, inhibiting the proliferative, migratory, and invasive capabilities of BCa. In addition, the experimental database demonstrated that SOX7 binds to the promoter of DNA methyltransferase 3 beta (DNMT3B), leading to the transcriptional inhibition of DNMT3B. This subsequently results in a reduced methylation of CYGB promoter, ultimately inhibiting the tumor progression of BCa. SOX7 + CYGB scores were significantly linked to patient prognosis. In conclusion, SOX7 inhibits the malignant progression of BCa via the DNMT3B/CYGB axis. Additionally, the SOX7 + CYGB score is capable of predicting the prognostic outcomes of BCa patients. Therefore, SOX7 and CYGB may play an important role in the progression of bladder cancer, and they can be used as prognostic markers of bladder cancer patients.

Investigating Cytoglobin Expression in Colon Cancer: Clinicopathological Insights from Immunohistochemical Analysis.

BACKGROUND/AIM: Cytoglobin (Cygb), a protein involved in cellular oxygen metabolism and protection, has garnered attention owing to its potential role in the initiation and progression of cancer, particularly colon cancer (CC). This study investigated the expression and significance of Cygb in CC. PATIENTS AND METHODS: This study included 145 patients who underwent R0 surgery for CC (clinical stage II/III) at our institution between January 2007 and December 2014. Immunohistochemical analysis was performed to evaluate the Cygb expression patterns in CC tissues. Additionally, the correlation between Cygb expression levels and the clinicopathological characteristics of patients with CC was investigated. RESULTS: Colon cancer tissues were categorized into high-expression (95 cases) and low-expression (50 cases) groups. Cygb was highly expressed in well-differentiated cases, whereas its expression decreased in poorly differentiated cases. No significant differences in other clinicopathological factors were observed between the two groups. Cygb expression had no significant effect on recurrence-free survival or overall survival. CONCLUSION: This study contributes to the growing understanding of Cygb expression and its significance in CC. The expression of Cygb in CC was found to be unrelated to the recurrence rate and prognosis, but showed a correlation with differentiation status.

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.

Inhibition of ALA dehydratase activity in heme biosynthesis reduces cytoglobin expression which is related to the proliferation and viability of keloid fibroblasts.

The aim of this study was to analyze the effect of heme synthesis inhibition on cytoglobin expression and its correlation with keloid fibroblast viability and proliferation. The study was conducted on primary culture of keloid fibroblasts. Heme synthesis in keloid fibroblasts was inhibited using succinyl acetone. We measured amino levulinic acid dehydratase (ALAD) enzyme activity using a colorimetric method; cytoglobin mRNA expression using qRT-PCR, cytoglobin protein expression using ELISA and immunocytochemistry, fibroblast viability using the MTT test; and fibroblast proliferation using BrdU test. The results showed that the ALAD enzyme activity level was lower in the keloid fibroblasts treated with succinyl-acetone (SA, 1, 2.5, and 5 mM) than in the control. The cytoglobin mRNA and protein expressions level were significantly lower in the keloid fibroblasts cultured with 2.5 mM and 5 mM SA than in the control and 1 mM SA. The viability and proliferation of the keloid fibroblasts decreased when the SA concentration was increased. In conclusion, the use of succinyl acetone at a concentration of 1; 2.5; and 5 mM caused decrease ALAD enzyme activity which indicated the inhibition of the heme synthesis. Inhibition of heme synthesis can affect cytoglobin expression, which correlates with the viability and proliferation of keloid fibroblasts.

Comparison of Evolutionary Relationships between Branchiostoma floridae, Ciona intestinalis, and Homo sapiens Globins Provide Evidence of Gene Co-Option and Convergent Evolution.

Globins have been studied as model proteins to elucidate the principles of protein evolution. This was achieved by understanding the relationship between amino acid sequence, three-dimensional structure, physicochemical properties, and physiological function. Previous molecular phylogenies of chordate globin genes revealed the monophyletic evolution of urochordate globins and suggested convergent evolution. However, to provide evidence of convergent evolution, it is necessary to determine the physicochemical and functional similarities between vertebrates and urochordate globins. In this study, we determined the expression patterns of Ciona globin genes using real-time RT-PCR. Two genes (Gb-1 and Gb-2) were predominantly expressed in the branchial sac, heart, and hemocytes and were induced under hypoxia. Combined with the sequence analysis, our findings suggest that Gb-1/-2 correspond to vertebrate hemoglobin-α/-ß. However, we did not find a robust similarity between Gb-3, Gb-4, and vertebrate globins. These results suggested that, even though Ciona globins obtained their unique functions differently from vertebrate globins, the two of them shared some physicochemical features and physiological functions. Our findings offer a good example for understanding the molecular mechanisms underlying gene co-option and convergence, which could lead to evolutionary innovations.

Functional study of Cygb in the immune response to Vibrio harveyi disease in yellow drum (Nibea albiflora).

Cytoglobin (Cygb) is a 21-kDa heme-protein that belongs to the globin superfamily and is expressed in vertebrate tissues. It can participate in the oxidative stress response in organisms through the porphyrin ring. Previous studies have shown that this protein, also known as YdCygb, has potential immune abilities in the infection of Vibrio harveyi in yellow drum (Nibea albiflora). In this study, we report the role of Cygb in the immune response of teleost fish for the first time. Quantitative RT-PCR analysis indicated that YdCygb was highly expressed in the liver and intestine of yellow drum, and its expression can be upregulated by pathogenic attack. The cellular distribution of YdCygb-EGFP proteins was observed in cell membrane, cytoplasm, and nucleus in the kidney cells of N. albiflora. Furthermore, a comparative transcriptome analysis between the YdCygb overexpression group and control vector group identified 28 differentially expressed genes (DEGs). The analysis showed that ANPEP, CLDN5, ORM1/2, SERPINC1 and HPN and ITGAM might play important regulatory roles to Cygb in fish. Notably, using GST-pull down technology, we identified 3-phosphoglyceraldehyde dehydrogenase and intermediate filament protein as direct interactors with YdCygb, playing a role against V. harveyi. The molecular and functional characterization of YdCygb provides better understanding of the genetic basis of disease resistance traits in yellow drum and sheds new light on the functioning of Cygb and its potential regulatory signaling pathway as well.

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.

Regulation of DNA damage and transcriptional output in the vasculature through a cytoglobin-HMGB2 axis.

Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.

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.

Neuronal Cytoglobin in the Auditory Brainstem of Rat and Mouse: Distribution, Cochlear Projection, and Nitric Oxide Production.

Cytoglobin (Cygb), a hemoprotein of the globin family, is expressed in the supportive tissue cells of the fibroblast lineage and in distinct neuronal cell populations. The expression pattern and regulatory parameters of fibroblasts and related cells were studied in organs such as the kidney and liver in a variety of animal models. In contrast, knowledge about cytoglobin-expressing neurons is sparse. Only a few papers described the distribution in the brain as ubiquitous with a restricted number of neurons in focal regions. Although there is evidence for cytoglobin involvement in neuronal hypoxia tolerance, its presence in the auditory system was not studied despite high metabolism rates and oxygen demands of the cochlea and related brainstem centers. In a continuation of a previous study demonstrating Cygb-neurons in, inter alia, auditory regions of the mouse brain, we concentrated on the superior olivary complex (SOC) in the present study. We sought to investigate the distribution, projection pattern and neurochemistry of Cygb-neurons in the SOC. We conducted immunohistochemistry using a Cygb antibody and found that this brainstem region, functionally competent for bilateral hearing and providing cochlear hair cell innervation, contains a considerable number of Cygb-expressing neurons (averaging 2067 ± 211 making up 10 ±1% percent of total neuron number) in rats, and 514 ± 138 (6 ± 1%) in mice. They were observed in all regions of the SOC. Retrograde neuronal tract tracing with Fluorogold injected into the cochlea demonstrated that 1243 ± 100 (6 ± 1% of total neuron number in rat SOC)) were olivocochlear neurons. Approximately 56% of total Cygb neurons were retrogradely labelled, while the majority of olivocochlear neurons of both lateral and medial systems were Cygb-immunoreactive. We also conducted double immunofluorescence staining for Cygb and neuronal nitric oxide synthase (nNOS), the enzyme responsible for nitric oxide production, and observed that cytoglobin in the SOC frequently co-localized with nNOS. Our findings suggest that cytoglobin plays an important physiologic role in the oxygen homeostasis of the peripheral and central auditory nervous system. Further studies, also including transgenic animal models, are required to shed more light on the function(s) of Cygb in neurons, in particular of the auditory system.

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.

Ordered Motions in the Nitric-Oxide Dioxygenase Mechanism of Flavohemoglobin and Assorted Globins with Tightly Coupled Reductases.

Nitric-oxide dioxygenases (NODs) activate and combine O2 with NO to form nitrate. A variety of oxygen-binding hemoglobins with associated partner reductases or electron donors function as enzymatic NODs. Kinetic and structural investigations of the archetypal two-domain microbial flavohemoglobin-NOD have illuminated an allosteric mechanism that employs selective tunnels for O2 and NO, gates for NO and nitrate, transient O2 association with ferric heme, and an O2 and NO-triggered, ferric heme spin crossover-driven, motion-controlled, and dipole-regulated electron-transfer switch. The proposed mechanism facilitates radical-radical coupling of ferric-superoxide with NO to form nitrate while preventing suicidal ferrous-NO formation. Diverse globins display the structural and functional motifs necessary for a similar allosteric NOD mechanism. In silico docking simulations reveal monomeric erythrocyte hemoglobin alpha-chain and beta-chain intrinsically matched and tightly coupled with NADH-cytochrome b5 oxidoreductase and NADPH-cytochrome P450 oxidoreductase, respectively, forming membrane-bound flavohemoglobin-like mammalian NODs. The neuroprotective neuroglobin manifests a potential NOD role in a close-fitting ternary complex with membrane-bound NADH-cytochrome b5 oxidoreductase and cytochrome b5. Cytoglobin interfaces weakly with cytochrome b5 for O2 and NO-regulated electron-transfer and coupled NOD activity. The mechanistic model also provides insight into the evolution of O2 binding cooperativity in hemoglobin and a basis for the discovery of allosteric NOD inhibitors.

Modulating Nitric Oxide Dioxygenase and Nitrite Reductase of Cytoglobin through Point Mutations.

Cytoglobin is a hexacoordinate hemoglobin with physiological roles that are not clearly understood. Previously proposed physiological functions include nitric oxide regulation, oxygen sensing, or/and protection against oxidative stress under hypoxic/ischemic conditions. Like many globins, cytoglobin rapidly consumes nitric oxide under normoxic conditions. Under hypoxia, cytoglobin generates nitric oxide, which is strongly modulated by the oxidation state of the cysteines. This gives a plausible role for this biochemistry in controlling nitric oxide homeostasis. Mutations to control specific properties of hemoglobin and myoglobin, including nitric oxide binding/scavenging and the nitrite reductase activity of various globins, have been reported. We have mapped these key mutations onto cytoglobin, which represents the E7 distal ligand, B2/E9 disulfide, and B10 heme pocket residues, and examined the nitric oxide binding, nitric oxide dioxygenase activity, and nitrite reductase activity. The Leu46Trp mutation decreases the nitric oxide dioxygenase activity > 10,000-fold over wild type, an effect 1000 times greater than similar mutations with other globins. By understanding how particular mutations can affect specific reactivities, these mutations may be used to target specific cytoglobin activities in cell or animal models to help understand the precise role(s) of cytoglobin under physiological and pathophysiological conditions.

Cytoglobin Silencing Promotes Melanoma Malignancy but Sensitizes for Ferroptosis and Pyroptosis Therapy Response.

Despite recent advances in melanoma treatment, there are still patients that either do not respond or develop resistance. This unresponsiveness and/or acquired resistance to therapy could be explained by the fact that some melanoma cells reside in a dedifferentiated state. Interestingly, this dedifferentiated state is associated with greater sensitivity to ferroptosis, a lipid peroxidation-reliant, iron-dependent form of cell death. Cytoglobin (CYGB) is an iron hexacoordinated globin that is highly enriched in melanocytes and frequently downregulated during melanomagenesis. In this study, we investigated the potential effect of CYGB on the cellular sensitivity towards (1S, 3R)-RAS-selective lethal small molecule (RSL3)-mediated ferroptosis in the G361 melanoma cells with abundant endogenous expression. Our findings show that an increased basal ROS level and higher degree of lipid peroxidation upon RSL3 treatment contribute to the increased sensitivity of CYGB knockdown G361 cells to ferroptosis. Furthermore, transcriptome analysis demonstrates the enrichment of multiple cancer malignancy pathways upon CYGB knockdown, supporting a tumor-suppressive role for CYGB. Remarkably, CYGB knockdown also triggers activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and subsequent induction of pyroptosis target genes. Altogether, we show that silencing of CYGB expression modulates cancer therapy sensitivity via regulation of ferroptosis and pyroptosis cell death signaling pathways.

Cytoglobin inhibits non-thermal plasma-induced apoptosis in melanoma cells through regulation of the NRF2-mediated antioxidant response.

Melanoma arises from pigment-producing cells called melanocytes located in the basal layers of the epidermis of the skin. Cytoglobin (CYGB) is a ubiquitously expressed hexacoordinated globin that is highly enriched in melanocytes and frequently downregulated during melanomagenesis. Previously, we showed that non-thermal plasma (NTP)-produced reactive oxygen and nitrogen species (RONS) lead to the formation of an intramolecular disulfide bridge that would allow CYGB to function as a redox-sensitive protein. Here, we investigate the cytotoxic effect of indirect NTP treatment in two melanoma cell lines with divergent endogenous CYGB expression levels, and we explore the role of CYGB in determining treatment outcome. Our findings are consistent with previous studies supporting that NTP cytotoxicity is mediated through the production of RONS and leads to apoptotic cell death in melanoma cells. Furthermore, we show that NTP-treated solutions elicit an antioxidant response through the activation of nuclear factor erythroid 2-related factor 2 (NRF2). The knockdown and overexpression of CYGB respectively sensitizes and protects melanoma cells from RONS-induced apoptotic cell death. The presence of CYGB enhances heme-oxygenase 1 (HO-1) and NRF2 protein expression levels, whereas the absence impairs their expression. Moreover, analysis of the CYGB-dependent transcriptome demonstrates the tumor suppressor long non-coding RNA maternally expressed 3 (MEG3) as a hitherto undescribed link between CYGB and NRF2. Thus, the presence of CYGB, at least in melanoma cells, seems to play a central role in determining the therapeutic outcome of RONS-inducing anticancer therapies, like NTP-treated solutions, possessing both tumor-suppressive and oncogenic features. Hence, CYGB expression could be of interest either as a biomarker or as a candidate for future targeted therapies in melanoma.

Regulation of nitrite reductase and lipid binding properties of cytoglobin by surface and distal histidine mutations.

Cytoglobin is a hemoprotein widely expressed in fibroblasts and related cell lineages with yet undefined physiological function. Cytoglobin, as other heme proteins, can reduce nitrite to nitric oxide (NO) providing a route to generate NO in vivo in low oxygen conditions. In addition, cytoglobin can also bind lipids such as oleic acid and cardiolipin with high affinity. These two processes are potentially relevant to cytoglobin function. Little is known about how specific amino acids contribute to nitrite reduction and lipid binding. Here we investigate the role of the distal histidine His81 (E7) and several surface residues on the regulation of nitrite reduction and lipid binding. We observe that the replacement of His81 (E7) greatly increases heme reactivity towards nitrite, with nitrite reduction rate constants of up to 1100 M-1s-1 for the His81Ala mutant. His81 (E7) mutation causes a small decrease in lipid binding affinity, however experiments on the presence of imidazole indicate that His81 (E7) does not compete with the lipid for the binding site. Mutations of the surface residues Arg84 and Lys116 largely impair lipid binding. Our results suggest that dissociation of His81 (E7) from the heme mediates the formation of a hydrophobic cavity in the proximal heme side that can accommodate the lipid, with important contributions of the hydrophobic patch around residues Thr91, Val105, and Leu108, whereas the positive charges from Arg84 and Lys116 stabilize the carboxyl group of the fatty acid. Gain and loss-of-function mutations described here can serve as tools to study in vivo the physiological role of these putative cytoglobin functions.

Nitric Oxide Production and Regulation in the Teleost Cardiovascular System.

Nitric Oxide (NO) is a free radical with numerous critical signaling roles in vertebrate physiology. Similar to mammals, in the teleost system the generation of sufficient amounts of NO is critical for the physiological function of the cardiovascular system. At the same time, NO amounts are strictly controlled and kept within basal levels to protect cells from NO toxicity. Changes in oxygen tension highly influence NO bioavailability and can modulate the mechanisms involved in maintaining the NO balance. While NO production and signaling appears to have general similarities with mammalian systems, the wide range of environmental adaptations made by fish, particularly with regards to differing oxygen availabilities in aquatic habitats, creates a foundation for a variety of in vivo models characterized by different implications of NO production and signaling. In this review, we present the biology of NO in the teleost cardiovascular system and summarize the mechanisms of NO production and signaling with a special emphasis on the role of globin proteins in NO metabolism.

Protective role of Cytoglobin and Neuroglobin against the Lipopolysaccharide (LPS)-induced inflammation in Leydig cells ex vivo.

Leydig cells are responsible for testosterone production in male testis upon stimulation by luteinizing hormone. Inflammation and oxidative stress related Leydig cell dysfunction is one of the major causes of male infertility. Cytoglobin (CYGB) and Neuroglobin (NGB) are two globin family member proteins which protect cells against oxidative stress. In the current study, we established a Lipopolysaccharide (LPS)-induced inflammation model in TM3 Leydig cell culture to study the function of CYGB and NGB proteins under inflammatory conditions. CYGB and NGB were downregulated using siRNA and shRNA based experimental strategies. Overexpression was conducted using lentiviral pLenti-III-CYGB-2A-GFP, and pLenti-III-NGB-2A-GFP vector systems. As testicular macrophages regulate immune function upon inflammation and steroidogenesis of Leydig cells, we generated direct/indirect co-culture systems of TM3 and mouse macrophage (RAW264.7) cells ex vivo. Downregulation of CYGB and NGB induced nitride oxide (NO) release, blocked cell cycle progression, reduced testosterone production and increased inflammatory and apoptotic pathway gene expression in the presence and absence of LPS. On the other hand, CYGB and NGB overexpression reduced TNFα and COX-2 protein expressions and increased the expression of testosterone biogenesis pathway genes upon LPS stimulation. In addition, CYGB and NGB overexpression upregulated testosterone production. The present study successfully established an inflammatory interaction model of TM3 and RAW264.7 cells. Suppression of CYGB and NGB in TM3 cells changed macrophage morphology, enhanced macrophage cell number and NO release in co-culture experiments upon LPS exposure. In summary, these results demonstrate that globin family members might control LPS induced inflammation by regulating apoptotic mechanisms and macrophage response.

Bioinformatics analysis of differentially expressed proteins in alcoholic fatty liver disease treated with recombinant human cytoglobin.

Cytoglobin (Cygb) is a globin molecule that is ubiquitously expressed in all tissues and has a protective role under oxidative stress. It has also been demonstrated to be effective in the treatment of alcoholic fatty liver disease (AFLD). In order to study the molecular mechanisms underlying its beneficial effects for the treatment of alcoholic liver, two­dimensional electrophoresis and mass spectrometric analysis were performed on serum and liver tissues from an in vivo rat model of AFLD. A total of 26 differentially expressed proteins were identified in the serum and 20 differentially expressed proteins were identified in liver specimens. Using online bioinformatics tools, it was indicated that these differentially expressed proteins were primarily associated with pathways including binding and uptake of ligands by scavenger receptors, response to corticosteroid, plasma lipoprotein remodeling, regulation of complement cascade, hydrogen peroxide catabolic process, as well as response to nutrient and monosaccharide. The present results suggested that recombinant human Cygb exerts its role in the treatment of AFLD primarily through affecting nutrient metabolism, monocarboxylic acid biosynthesis, regulation of glutathione expression, plasma lipoprotein remodeling and removal of metabolic waste from the blood.