ß-Cell-selective regulation of gene expression by nitric oxide.

Nitric oxide is produced at low micromolar levels following the induction of inducible nitric oxide synthase (iNOS) and is responsible for mediating the inhibitory actions of cytokines on glucose-stimulated insulin secretion by islets of Langerhans. It is through the inhibition of mitochondrial oxidative metabolism, specifically aconitase and complex 4 of the electron transport chain, that nitric oxide inhibits insulin secretion. Nitric oxide also attenuates protein synthesis, induces DNA damage, activates DNA repair pathways, and stimulates stress responses (unfolded protein and heat shock) in ß-cells. In this report, the time- and concentration-dependent effects of nitric oxide on the expression of six genes known to participate in the response of ß-cells to this free radical were examined. The genes included Gadd45α (DNA repair), Puma (apoptosis), Hmox1 (antioxidant defense), Hsp70 (heat shock), Chop (UPR), and Ppargc1α (mitochondrial biogenesis). We show that nitric oxide stimulates ß-cell gene expression in a narrow concentration range of ∼0.5-1 µM or levels corresponding to iNOS-derived nitric oxide. At concentrations greater than 1 µM, nitric oxide fails to stimulate gene expression in ß-cells, and this is associated with the inhibition of mitochondrial oxidative metabolism. This narrow concentration range of responses is ß-cell selective, as the actions of nitric oxide in non-ß-cells (α-cells, mouse embryonic fibroblasts, and macrophages) are concentration dependent. Our findings suggest that ß-cells respond to a narrow concentration range of nitric oxide that is consistent with the levels produced following iNOS induction, and that these concentration-dependent actions are selective for insulin-containing cells.

Melatonin activates Nrf2/HO-1 signalling pathway to antagonizes oxidative stress-induced injury via melatonin receptor 1 (MT1) in cryopreserved mice ovarian tissue.

Our previous research has shown that melatonin (MLT) can reduce cryopreserved ovarian damage in mice. Yet, the molecular mechanism of MLT protection is still unclear. Some studies have shown that melatonin receptor 1 (MT1) is very important for animal reproductive system. To evaluate whether MLT exerts its protective effect on cryopreserved mice ovarian tissue via MT1, we added antagonist of MT1/MT2 (Luzindor) or antagonist of MT2 (4P-PDOT) to the freezing solution, followed by cryopreservation and thawing of ovarian tissue. The levels of total superoxide dismutase (T-SOD), catalase (CAT), nitric oxide (NO) and malondialdehyde (MDA) were detected. Besides, by using RT-PCR and Western blotting, the expression of Bcl-2, Bax and Nrf2/HO-1 signalling pathway-related proteins was detected. These findings demonstrated that compared with the melatonin group, the addition of Luzindor increased apoptosis, NO and MDA activities, decreased CAT and T-SOD activities and inhibited Nrf2/HO-1 signalling pathway. In conclusion, melatonin can play a protective role in cryopreserved ovarian tissue of mice through MT1 receptor.

Heme oxygenase-2 (HO-2) binds and buffers labile ferric heme in human embryonic kidney cells.

Heme oxygenases (HOs) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms of HO: the inducible HO-1, which is upregulated in response to excess heme and other stressors, and the constitutive HO-2. Much is known about the regulation and physiological function of HO-1, whereas comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or overexpressed HO-2, as well as various HO-2 mutant alleles, we found that endogenous heme is too limiting a substrate to observe HO-2-dependent heme degradation. Rather, we discovered a novel role for HO-2 in the binding and buffering of heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor that controls heme bioavailability. When heme is in excess, HO-1 is induced, and both HO-2 and HO-1 can provide protection from heme toxicity via enzymatic degradation. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with labile heme being oxidized, thereby providing new insights into heme trafficking and signaling.

Heme catabolism mediated by heme oxygenase in uninfected interstitial cells enables efficient symbiotic nitrogen fixation in Lotus japonicus nodules.

Legume nodules produce large quantities of heme required for the synthesis of leghemoglobin (Lb) and other hemoproteins. Despite the crucial function of Lb in nitrogen fixation and the toxicity of free heme, the mechanisms of heme homeostasis remain elusive. Biochemical, cellular, and genetic approaches were used to study the role of heme oxygenases (HOs) in heme degradation in the model legume Lotus japonicus. Heme and biliverdin were quantified and localized, HOs were characterized, and knockout LORE1 and CRISPR/Cas9 mutants for LjHO1 were generated and phenotyped. We show that LjHO1, but not the LjHO2 isoform, is responsible for heme catabolism in nodules and identify biliverdin as the in vivo product of the enzyme in senescing green nodules. Spatiotemporal expression analysis revealed that LjHO1 expression and biliverdin production are restricted to the plastids of uninfected interstitial cells. The nodules of ho1 mutants showed decreased nitrogen fixation, and the development of brown, rather than green, nodules during senescence. Increased superoxide production was observed in ho1 nodules, underscoring the importance of LjHO1 in antioxidant defense. We conclude that LjHO1 plays an essential role in degradation of Lb heme, uncovering a novel function of nodule plastids and uninfected interstitial cells in nitrogen fixation.

The heme-binding protein PhuS transcriptionally regulates the Pseudomonas aeruginosa tandem sRNA prrF1,F2 locus.

Pseudomonas aeruginosa is an opportunistic pathogen requiring iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the nonredundant heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme transported by either the heme assimilation system or Phu system is sequestered by the cytoplasmic protein PhuS. Furthermore, PhuS has been shown to specifically transfer heme to the iron-regulated heme oxygenase HemO. As the PhuS homolog ShuS from Shigella dysenteriae was observed to bind DNA as a function of its heme status, we sought to further determine if PhuS, in addition to its role in regulating heme flux through HemO, functions as a DNA-binding protein. Herein, through a combination of chromatin immunoprecipitation-PCR, EMSA, and fluorescence anisotropy, we show that apo-PhuS but not holo-PhuS binds upstream of the tandem iron-responsive sRNAs prrF1,F2. Previous studies have shown the PrrF sRNAs are required for sparing iron for essential proteins during iron starvation. Furthermore, under certain conditions, a heme-dependent read through of the prrF1 terminator yields the longer PrrH transcript. Quantitative PCR analysis of P. aeruginosa WT and ΔphuS strains shows that loss of PhuS abrogates the heme-dependent regulation of PrrF and PrrH levels. Taken together, our data show that PhuS, in addition to its role in extracellular heme metabolism, also functions as a transcriptional regulator by modulating PrrF and PrrH levels in response to heme. This dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network that is critical for P. aeruginosa adaptation and virulence within the host.

Using genetically encoded heme sensors to probe the mechanisms of heme uptake and homeostasis in Candida albicans.

Candida albicans is a major fungal pathogen that can utilise hemin and haemoglobin as iron sources in the iron-scarce host environment. While C. albicans is a heme prototroph, we show here that it can also efficiently utilise external heme as a cellular heme source. Using genetically encoded ratiometric fluorescent heme sensors, we show that heme extracted from haemoglobin and free hemin enter the cells with different kinetics. Heme supplied as haemoglobin is taken up via the Common in Fungal Extracellular Membrane (CFEM) hemophore cascade, and reaches the cytoplasm over several hours, whereas entry of free hemin via CFEM-dependent and independent pathways is much faster, less than an hour. To prevent an influx of extracellular heme from reaching toxic levels in the cytoplasm, the cells deploy Hmx1, a heme oxygenase. Hmx1 was previously suggested to be involved in utilisation of haemoglobin and hemin as iron sources, but we find that it is primarily required to prevent heme toxicity. Taken together, the combination of novel heme sensors with genetic analysis revealed new details of the fungal mechanisms of heme import and homeostasis, necessary to balance the uses of heme as essential cofactor and potential iron source against its toxicity.

Polyherbal Formulation Ameliorates Diabetic Cardiomyopathy Through Attenuation of Cardiac Inflammation and Oxidative Stress Via NF-κB/Nrf-2/HO-1 Pathway in Diabetic Rats.

ABSTRACT: The present study was intended to evaluate the effect of polyherbal formulation (PHF) made with 3 nutraceuticals, such as Piper nigrum, Terminalia paniculata, and Bauhinia purpurea on inflammation and oxidative stress in diabetic cardiomyopathy (DCM), which is induced by streptozotocin and nicotinamide administration in rats. We supplemented DCM rats with PHF (250 and 500 mg/kg/BW) for 45 days and evaluated their effects on oxidative stress markers, proinflammatory cytokines, and messenger RNA expressions of the nuclear factor erythroid 2-related factor-2 (Nrf-2) and its linked genes [heme oxygenase-1 (HO-1), superoxide dismutase, catalase] along with inflammatory genes [tumour necrosis factor α and nuclear factor kappa B (NF-κB)]. Our study demonstrated that PHF successfully attenuated inflammation and oxidative stress via messenger RNA upregulation of Nrf-2, HO-1, superoxide dismutase, and catalase and concomitantly with downregulation of tumour necrosis factor α and NF-κB. Conversely, PHF also protected hyperglycemia-mediated cardiac damage, which was confirmed with histopathological and scanning electron microscopy analysis. In conclusion, our results suggested that PHF successfully ameliorated hyperglycemia-mediated inflammation and oxidative stress via regulation of NF-κB/Nrf-2/HO-1 pathway. Therefore, these results recommend that PHF may be a prospective therapeutic agent for DCM.

Influence of Heme Oxygenase-1 on Rats with Diabetic Retinopathy Through ERK1/2 Signaling Pathway.

The study aimed to investigate the influence of heme oxygenase-1 (HO-1) on rats with diabetic retinopathy (DR) through the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway. 40 rats were selected and divided into Control group (n=10), diabetes mellitus (DM) group (n=10), cobalt protoporphyrin (CoPP) group (n=10) and zinc protoporphyrin (ZnPP) group (n=10) according to weight. Streptozotocin (STZ) was intraperitoneally injected to establish the DM model in DM, CoPP and ZnPP groups, and CoPP and ZnPP solution was intraperitoneally injected in CoPP and ZnPP groups, respectively. Blood was drawn to determine fasting blood glucose. The changes in the protein and messenger ribonucleic acid (mRNA) levels were evaluated via Western blotting and polymerase chain reaction (qRT-PCR), respectively. Enzyme-linked immunosorbent assay (ELISA) was performed to measure antioxidant capacity and the levels of total reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase (GPx). The weight of rats was notably higher in the CoPP group and lower inZnPP group than in the DM group (p<0.05). After induction of DM, compared with those in the DM group, the protein expression levels of Nrf2 and pERK were considerably elevated in the CoPP group (p<0.05) but declined remarkably in the ZnPP group (p<0.05). The levels of total ROS and MDA were notably elevated (p<0.05) in DM and ZnPP groups, with a lowered level of GPx and distinctly elevated levels of MDA and total ROS (p<0.05). Moreover, the mRNA expression level of HO-1 in the retinas of rats was remarkably raised in the DM group and CoPP group (p<0.05), but it declined markedly in the ZnPP group (p<0.05). The red fluorescent aggregation of Nrf2 and pERK proteins was overtly less in the ZnPP group than that in the DM group (p<0.05). HO-1 can affect the level of oxidative stress and intervene in retinopathy in DM rats through the Nrf2/ERK pathway.

Identification of bacteriophage-encoded anti-sRNAs in pathogenic Escherichia coli.

In bacteria, Hfq is a core RNA chaperone that catalyzes the interaction of mRNAs with regulatory small RNAs (sRNAs). To determine in vivo RNA sequence requirements for Hfq interactions, and to study riboregulation in a bacterial pathogen, Hfq was UV crosslinked to RNAs in enterohemorrhagic Escherichia coli (EHEC). Hfq bound repeated trinucleotide motifs of A-R-N (A-A/G-any nucleotide) often associated with the Shine-Dalgarno translation initiation sequence in mRNAs. These motifs overlapped or were adjacent to the mRNA sequences bound by sRNAs. In consequence, sRNA-mRNA duplex formation will displace Hfq, promoting recycling. Fifty-five sRNAs were identified within bacteriophage-derived regions of the EHEC genome, including some of the most abundant Hfq-interacting sRNAs. One of these (AgvB) antagonized the function of the core genome regulatory sRNA, GcvB, by mimicking its mRNA substrate sequence. This bacteriophage-encoded "anti-sRNA" provided EHEC with a growth advantage specifically in bovine rectal mucus recovered from its primary colonization site in cattle.

Phylogeny of the HO family in cyprinus carpio and the response of the HO-1 gene to adding Bacillus coagulans in feed under Cd2+ stress.

The heavy metal cadmium (Cd2+) is an environmental pollutant that poses serious health hazards. Due to the increasing contamination of aquatic systems with Cd2+, the increased accumulation of Cd2+ in fish has become a food safety and public health concern. Heme oxygenase (HO) is an important antioxidant enzyme that plays a key role in defending the body against oxidative damage, but little research has been done in common carp. In this study, 6 HO genes were identified in the common carp genome database. Comparative genomics analysis showed considerable expansion of the HO genes and verified the four-round whole genome duplication (WGD) event in common carp. Phylogenetic analysis revealed that all HO genes of common carp were clustered into orthologous groups, indicating high conservation during evolution. In addition, the tissue distribution results showed that most HO genes had extensive tissue distribution and showed tissue-specific expression patterns. Exposure to 0.5 mg/L Cd2+ significantly reduced the expression of TGF-ß and IL-10 in common carp, which may indicate that Cd2+ exposure can destroy the physical barrier function of the intestine, inhibit intestinal immune defense and induce intestinal inflammation. To find a suitable concentration of Bacillus coagulans that could activate HO-1 genes and the immunity of the organism, we investigated the changes in HO-1 gene expression levels in the intestinal tract of common carp under Cd2+ stress at 30 days and 60 days by adding different concentrations of B. coagulans to the feed. Compared with the Cd2+ stress group without supplementation, the expression levels of the HO-1 gene in the gut of three different concentrations of B. coagulans were almost increased. And B. coagulans with L2 concentrations had better activation effect on the HO-1 gene. Similarly, compared to the Cd2+ stressed group, adding B. coagulans to the diet can almost cause the early upregulation of IL-10 and TGF-ß genes. Therefore, the addition of appropriate concentrations of B. coagulans may be a good way to activate HO-1, IL-10, and TGF-ß genes, reduce oxidative damage, and encourage the immune.

Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site.

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme. We find that, under heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting that heme plays a direct role in HO2 regulation. HO2 has three heme binding sites: one at its catalytic site and the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2's role in maintaining heme homeostasis, paving the way for future investigation into HO2's pathophysiological role in heme deficiency response.

GRP78 effectively protect hypoxia/reperfusion-induced myocardial apoptosis via promotion of the Nrf2/HO-1 signaling pathway.

Myocardial infarction is a major cause of death worldwide. Despite our understanding of the pathophysiology of myocardial infarction and the therapeutic options for treatment have improved substantially, acute myocardial infarction remains a leading cause of morbidity and mortality. Recent findings revealed that GRP78 could protect myocardial cells against ischemia reperfusion injury-induced apoptosis, but the exact function and molecular mechanism remains unclear. In this study, we aimed to explore the effects of GRP78 on hypoxia/reperfusion (H/R)-induced cardiomyocyte injury. Intriguingly, we first observed that GRP78 overexpression significantly protected myocytes from H/R-induced apoptosis. On mechanism, our work revealed that GRP78 protected myocardial cells from hypoxia/reperfusion-induced apoptosis via the activation of the Nrf2/HO-1 signaling pathway. We observed the enhanced expression of Nrf2/HO-1 in GRP78 overexpressed H9c2 cell, while GRP78 deficiency dramatically antagonized the expression of Nrf2/HO-1. Furthermore, we found that blocked the Nrf2/HO-1 signaling by the HO-1 inhibitor zinc protoporphyrin IX (Znpp) significantly retrieved H9c2 cells apoptosis that inhibited by GRP78 overexpression. Taken together, our findings revealed a new mechanism by which GRP78 alleviated H/R-induced cardiomyocyte apoptosis in H9c2 cells via the promotion of the Nrf2/HO-1 signaling pathway.

Role of olfactory receptor78 in carotid body-dependent sympathetic activation and hypertension in murine models of chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) is a hallmark manifestation of obstructive sleep apnea (OSA), a widespread breathing disorder. CIH-treated rodents exhibit activation of the sympathetic nervous system and hypertension. Heightened carotid body (CB) activity has been implicated in CIH-induced hypertension. CB expresses high abundance of olfactory receptor (Olfr) 78, a G-protein coupled receptor. Olfr 78 null mice exhibit impaired CB sensory nerve response to acute hypoxia. Present study examined whether Olfr78 participates in CB-dependent activation of the sympathetic nervous system and hypertension in CIH-treated mice and in hemeoxygenase (HO)-2 null mice experiencing CIH as a consequence of naturally occurring OSA. CIH-treated wild-type (WT) mice showed hypertension, biomarkers of sympathetic nerve activation, and enhanced CB sensory nerve response to hypoxia and sensory long-term facilitation (sLTF), and these responses were absent in CIH-treated Olfr78 null mice. HO-2 null mice showed higher apnea index (AI) (58 ± 1.2 apneas/h) than WT mice (AI = 8 ± 0.8 apneas/h) and exhibited elevated blood pressure (BP), elevated plasma norepinephrine (NE) levels, and heightened CB sensory nerve response to hypoxia and sLTF. The magnitude of hypertension correlated with AI in HO-2 null mice. In contrast, HO-2/Olfr78 double null mice showed absence of elevated BP and plasma NE levels and augmented CB response to hypoxia and sLTF. These results demonstrate that Olfr78 participates in sympathetic nerve activation and hypertension and heightened CB activity in two murine models of CIH.NEW & NOTEWORTHY Carotid body (CB) sensory nerve activation is essential for sympathetic nerve excitation and hypertension in rodents treated with chronic intermittent hypoxia (CIH) simulating blood O2 profiles during obstructive sleep apnea (OSA). Here, we report that CIH-treated mice and hemeoxygenase (HO)-2-deficient mice, which show OSA phenotype, exhibit sympathetic excitation, hypertension, and CB activation. These effects are absent in Olfr78 null and Olfr78/HO-2 double null mice.

Brain-targeted hypoxia-inducible factor stabilization reduces neonatal hypoxic-ischemic brain injury.

Hypoxia-inducible factor-1α (HIF1α) is a major regulator of cellular adaptation to hypoxia and oxidative stress, and recent advances of prolyl-4-hydroxylase (P4H) inhibitors have produced powerful tools to stabilize HIF1α for clinical applications. However, whether HIF1α provokes or resists neonatal hypoxic-ischemic (HI) brain injury has not been established in previous studies. We hypothesize that systemic and brain-targeted HIF1α stabilization may have divergent effects. To test this notion, herein we compared the effects of GSK360A, a potent P4H inhibitor, in in-vitro oxygen-glucose deprivation (OGD) and in in-vivo neonatal HI via intracerebroventricular (ICV), intraperitoneal (IP), and intranasal (IN) drug-application routes. We found that GSK360A increased the erythropoietin (EPO), heme oxygenase-1 (HO1) and glucose transporter 1 (Glut1) transcripts, all HIF1α target-genes, and promoted the survival of neurons and oligodendrocytes after OGD. Neonatal HI insult stabilized HIF1α in the ipsilateral hemisphere for up to 24 h, and either ICV or IN delivery of GSK360A after HI increased the HIF1α target-gene transcripts and decreased brain damage. In contrast, IP-injection of GSK360A failed to reduce HI brain damage, but elevated the risk of mortality at high doses, which may relate to an increase of the kidney and plasma EPO, leukocytosis, and abundant vascular endothelial growth factor (VEGF) mRNAs in the brain. These results suggest that brain-targeted HIF1α-stabilization is a potential treatment of neonatal HI brain injury, while systemic P4H-inhibition may provoke unwanted adverse effects.

NLRC5 protects neurons from oxygen-glucose deprivation-induced injury through activating the Nrf2/HO-1 pathway.

NLRC5 is a member of the Nod-like receptor (NLR) family that has been found to be associated with the hepatic ischemia/reperfusion (I/R) injury. However, the role of NLRC5 in cerebral I/R has not been fully understood. The aim of the current study was to evaluate the effects of NLRC5 on primary hippocampal neuronal cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R). Our results showed that the mRNA and protein levels of NLRC5 were significantly decreased in OGD/R-induced neurons. Overexpression of NLRC5 caused significant increase in cell viability, as well as decrease in ROS level. The bax expression was significantly decreased, while bcl-2 expression was increased in NLRC5-overexpressing neurons. Furthermore, increased nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression levels were observed in neurons transfected with pcDNA3.0-NLRC5. The mRNA levels of HO-1, NAD(P)H:quinone oxidoreductase 1 (NQO-1) and glutathione peroxidase 3 (GPx-3) were induced by NLRC5 overexpression in OGD/R-induced hippocampal neurons. Additionally, inhibition of Nrf2/HO-1 pathway abolished the protective effect of NLRC5 on cerebral I/R injury. In conclusion, these results indicated that NLRC5 protected hippocampal neurons from OGD/R-induced injury. The protective effects of NLRC5 were mediated by the Nrf2/HO-1 pathway. Thus, NLRC5 might serve as an effective target for the treatment of cerebral I/R injury.

Mangiferin mitigates di-(2-ethylhexyl) phthalate-induced testicular injury in rats by modulating oxidative stress-mediated signals, inflammatory cascades, apoptotic pathways, and steroidogenesis.

Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine disruptor that causes reproductive defects in male animal models. This study was conducted to explore the plausible modulatory effects of mangiferin (MF) against DEHP-induced testicular injury in rats. Thirty-two adult male albino rats were allocated into four groups. Two groups were given DEHP (2 g/kg/day, p.o) for 14 days. One of these groups was treated with MF (20 mg/kg/day, i.p) for 7 days before and 14 days after DEHP administration. A vehicle-treated control was included, and another group of rats was given MF only. Results revealed that MF treatment suppressed oxidative testicular injury by amplifying the mRNA expression of nuclear factor-erythroid 2 related factor-2 (Nrf2) and increasing hemoxygenase-1 (HO-1), glutathione, and total antioxidant capacity (TAC) levels. This treatment also enhanced superoxide dismutase activity, but it decreased malondialdehyde and nitric oxide levels. MF had an anti-inflammatory characteristic, as demonstrated by the downregulation of the mRNA of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The content of tumor necrosis factor-alpha also decreased. MF modulated the apoptotic pathway by suppressing the mRNA of cytochrome c (Cyt c), Fas ligand content, Bax IHC expression, caspase-3 activity and cleaved caspase-3 IHC expression. It also upregulated the expression levels of heat-shock protein 70 (HSP70) and B-cell lymphoma 2. Moreover, MF upregulated the mRNA expression levels of HSP70 and c-kit and enriched the content of steroidogenic acute regulatory (StAR) protein, which were reflected in serum testosterone levels. This result indicated that MF played crucial roles in steroidogenesis and spermatogenesis. Besides, the activities of testicular marker enzymes, namely, acid and alkaline phosphatases, and lactate dehydrogenase, significantly increased. Histopathological observations provided evidence supporting the biochemical and molecular measurements. In conclusion, MF provided protective mechanisms against the DEHP-mediated deterioration of testicular functions partially through its antioxidant, anti-inflammatory, and anti-apoptotic properties. It also involved the restoration of steroidogenesis and spermatogenesis through the modulation of Nrf2/HO-1, NF-κB/Cyt c/HSP70, and c-Kit signaling cascades.

Oat polyphenol avenanthramide-2c confers protection from oxidative stress by regulating the Nrf2-ARE signaling pathway in PC12 cells.

Accumulating evidence has demonstrated that cellular antioxidant systems play essential roles in retarding oxidative stress-related diseases, such as Parkinson's disease. Because nuclear factor erythroid 2-related factor 2 (Nrf2) is a chief regulator of cellular antioxidant systems, small molecules with Nrf2-activating ability may be promising neuroprotective agents. Avenanthramide-2c (Aven-2c), avenanthramide-2f (Aven-2f) and avenanthramide-2p (Aven-2p) are the most abundant avenanthramides in oats, and they have been documented to possess multiple pharmacological benefits. In this work, we synthesized these three compounds and evaluated their cytoprotective effect against oxidative stress-induced PC12 cell injuries. Aven-2c displayed the best protective potency among them. Aven-2c conferred protection on PC12 cells by scavenging free radicals and activating the Nrf2-ARE signaling pathway. Pretreatment of PC12 cells with Aven-2c efficiently enhanced Nrf2 nuclear accumulation and evoked the expression of a set of cytoprotective molecules. The mechanistic study also supports that Nrf2 activation is the molecular basis for the cellular action of Aven-2c. Collectively, this study demonstrates that Aven-2c is a potent Nrf2 agonist, shedding light on the potential usage of Aven-2c in the treatment of neuroprotective diseases.

Ischemic postconditioning attenuates acute kidney injury following intestinal ischemia-reperfusion through Nrf2-regulated autophagy, anti-oxidation, and anti-inflammation in mice.

Intestinal ischemia-reperfusion (IIR) often occurs during and following major cardiovascular or gut surgery and causes significant organ including kidney injuries. This study was to investigate the protective effect of intestinal ischemic postconditioning (IPo) on IIR-induced acute kidney injury (AKI) and the underling cellular signaling mechanisms with focus on the Nrf2/HO-1. Adult C57BL/6J mice were subjected to IIR with or without IPo. IIR was established by clamping the superior mesenteric artery (SMA) for 45 minutes followed by 120 minutes reperfusion. Outcome measures were: (i) Intestinal and renal histopathology; (ii) Renal function; (iii) Cellular signaling changes; (iv) Oxidative stress and inflammatory responses. IPo significantly attenuated IIR-induced kidney injury. Furthermore, IPo significantly increased both nuclear Nrf2 and HO-1 expression in the kidney, upregulated autophagic flux, inhibited IIR-induced inflammation and reduced oxidative stress. The protective effect of IPo was abolished by the administration of Nrf2 inhibitor (Brusatol) or Nrf2 siRNA. Conversely, a Nrf2 activator t-BHQ has a similar protective effect to that of IPo. Our data indicate that IPo protects the kidney injury induced by IIR, which was likely mediated through the Nrf2/HO-1 cellular signaling activation.

Investigation of Cyanide Ligand as an Active Site Probe of Human Heme Oxygenase.

Human heme oxygenase (hHO-1) is a physiologically important enzyme responsible for free heme catabolism. The enzyme's high regiospecificity is controlled by the distal site hydrogen bond network that involves water molecules and the D140 amino acid residue. In this work, we probe the active site environment of the wild-type (WT) hHO-1 and its D140 mutants using resonance Raman (rR) spectroscopy. Cyanide ligands are more stable than dioxygen adducts and are an effective probe of active site environment of heme proteins. The inherently linear geometry of the Fe-C-N fragment can be altered by the steric, electrostatic, and H-bonding interactions imposed by the amino acid residues present in the heme distal site, resulting in a tilted or bent configuration. The WT hHO-1 and its D140A, D140N, and D140E mutants were studied in the presence of natural abundance CN- and its isotopic analogues (13CN-, C15N-, and 13C15N-). Deconvolution of spectral data revealed that the ν(Fe-CN) stretching and δ(Fe-CN) bending modes are present at 454 and 376 cm-1, respectively. The rR spectral patterns of the CN- adducts of WT revealed that the Fe-C-N fragment adopts a tilted conformation, with a larger bending contribution for the D140A, D140N, and D140E mutants. These studies suggest that the FeCN fragment in hHO-1 is tilted more strongly toward the porphyrin macrocycle compared to other histidine-ligated proteins, reflecting the propensity of the exogenous hHO-l ligands to position toward the α-meso-carbon, which is crucial for the HO reactivity and essential for regioselectivity.

Resistance exercise affects catheter-related thrombosis in rats through miR-92a-3p, oxidative stress and the MAPK/NF-κB pathway.

BACKGROUND: MiR-92a-3p and oxidative stress are associated with catheter-related thrombosis (CRT). As a kind of physical intervention, resistance exercise can effectively promote blood circulation. In this study, we investigated the roles of miR-92a-3p, oxidative stress and the P38 mitogen-activated protein kinase/nuclear factor-κB (MAPK/NF-κB) pathway in CRT during resistance exercise. METHODS: The rat CRT model was used for resistance exercise intervention. Moreover, pathological changes from the right jugular vein to the right auricle were observed under an electron microscope. In addition, reactive oxygen species (ROS) production, malondialdehyde (MDA) activity and heme oxygenase (HO-1) level in rat serum were detected via ELISA. The expression levels of miR-92A-3p and HO-1 in the vascular tissues of the rats were determined via real-time quantitative PCR. Additionally, the expression levels of HO-1, NF-κB P65, p38MAPK and IκBa in the venous tissues of the rats were analysed by Western blot analysis. RESULTS: The pathological results showed that the thrombosis incidence rate in the CRT + RE group was lower than that in the CRT group. In the CRT group, the expression levels of ROS and MDA, which are markers related to oxidative stress in serum, significantly increased whilst the expression of HO-1 decreased. In the venous tissue, the expression of miR-92a-3p increased, the level of HO-1 decreased, the levels of p38MAPK and NF-κB p65 significantly increased but that of P-IκBa and IκBa significantly decreased. In the CRT + RE group, after administering the resistance exercise intervention, ROS production and MDA activity in serum significantly decreased, the expression level of HO-1 increased and the expression level of miR-92a-3p in the venous tissues significantly decreased and was negatively correlated with that of HO-1. The levels of p38MAPK and NF-κB p65 significantly decreased but that of P- IκBa and IκBa significantly increased. CONCLUSION: Resistance exercise intervention downregulated miR-92a-3p expression, repaired oxidative stress injury and prevented CRT formation.