Dauferulins A-L, daucane-type sesquiterpenes from the roots of Ferula communis: Their structures and biological activities.

Phytochemical study on the roots of a medicinal plant Ferula communis L. (Apiaceae) resulted in the isolation of 20 sesquiterpenes including 12 previously undescribed compounds, dauferulins A-L (1-12). The detailed spectroscopic analysis revealed 1-12 to be daucane-type sesquiterpenes with a p-methoxybenzoyloxy group at C-6. The absolute configurations of 1-12 were deduced by analysis of the ECD spectra. Dauferulins A-L (1-12), known sesquiterpenes (13-20), and analogues (14a-14l) derived from 6-O-p-methoxybenzoyl-10α-angeloyloxy-jeaschkeanadiol (14) were evaluated for their effects on AMPK phosphorylation in human hepatoma HepG2 cells as well as inhibitory activities against erastin-induced ferroptosis on human hepatoma Hep3B cells and IL-1ß production from LPS-treated murine microglial cells.

Meroterpenes and prenylated benzoylphloroglucinol from the flowers of Hypericum formosanum.

Formohyperins A-F, previously undescribed meroterpenes, and grandone, a prenylated benzoylphloroglucinol being considered to be one of their biogenetic precursors, were isolated from the flowers of a Hypericaceous plant, Hypericum formosanum Maxim. Detailed spectroscopic analyses showed that formohyperins A-D were meroterpenes with an enolized 3-phenylpropane-1,3-dione moiety. Formohyperins E and F were elucidated as meroterpenes having a 4-benzoyl-5-hydroxycyclopent-4-ene-1,3-dione moiety. Formohyperins A-C and E were optically active, and their absolute configurations were deduced by comparison of the experimental and TDDFT calculated ECD spectra. In contrast, formohyperin D was concluded to be a racemate. Formohyperins A-F and grandone were found to show inhibitory activities against LPS-stimulated IL-1ß production from murine microglial cells with EC50 values of 13.2, 6.6, 8.5, 24.3, 4.1, 10.9, and 3.0 µM, respectively.

Acylated iridoid glucoside and xanthones from Canscora lucidissima: Their structures and ferroptosis inhibitory activity.

Phytochemical study on the whole plants of a Gentianaceous medicinal plant, Canscora lucidissima, gave one new acylated iridoid glucoside, canscorin A (1), and two new xanthone glycosides (2 and 3) together with 17 known compounds including five xanthones, eight xanthone glycosides, two benzophenone glucosides, caffeic acid, and loganic acid. Canscorin A (1) was assigned as a loganic acid derivative having a hydroxyterephthalic acid moiety by spectroscopic analysis together with chemical evidence, while 2 and 3 were elucidated to be a rutinosylxanthone and a glucosylxanthone, respectively. The absolute configurations of the sugar moieties of 2 and 3 were determined by HPLC analysis. The isolated compounds were evaluated for their inhibitory activities against erastin-induced ferroptosis on human hepatoma Hep3B cells and LPS-stimulated IL-1ß production from murine microglial cells.

Heat shock-induced heme oxygenase-1 expression in a mouse hepatoma cell line is dependent on HSF1 and modified by NRF2 and BACH1.

The induction mechanism of heme oxygenase-1 (HO-1) by heat shock (HS) is still unknown. Here, we discovered that HS activates the HO-1 expression in a mouse hepatoma cell line (Hepa 1-6). Knockdown experiments showed that the HS-induced HO-1 expression was dependent on HS factor 1 (HSF1). A chromatin immunoprecipitation (ChIP) assay demonstrated that the HS-activated HSF1 bound to the HS elements (HSEs) in the upstream enhancer 1 region (E1). Unexpectedly, HS also facilitates the BTB and CNC homology 1 (BACH1) binding to the Maf recognition elements (MAREs) in E1. We examined the effects of a catalytically inactive CRISPR-associated 9 nucleases (dCas9) with short guide RNAs (sgRNAs), and demonstrated that the HSF1 binding to HSEs in E1 was indispensable for the HS-induced HO-1 expression. Heme treatment (HA) dissociates BACH1 from MAREs and facilitated the binding of nuclear factor-erythroid-2-related factor 2 (NRF2) to MAREs. Following treatment with both HS and HA, the HO-1 induction and the HSF1 binding to HSEs in E1 were most notably observed. These results indicate that the HS-induced HO-1 expression is dependent on the HSF1 binding to HSEs in E1, although modulated by the BACH1 and NRF2 binding to MAREs within the same E1.

Role of Heme Oxygenase in Gastrointestinal Epithelial Cells.

The gastrointestinal tract is a unique organ containing both vascular and luminal routes lined by epithelial cells forming the mucosa, which play an important role in the entry of nutrients and act as a selective barrier, excluding potentially harmful agents. Mucosal surfaces establish a selective barrier between hostile external environments and the internal milieu. Heme is a major nutritional source of iron and is a pro-oxidant that causes oxidative stress. Heme oxygenases (HOs) catalyze the rate-limiting step in heme degradation, resulting in the formation of iron, carbon monoxide, and biliverdin, which are subsequently converted to bilirubin by biliverdin reductase. In gastrointestinal pathogenesis, HO-1, an inducible isoform of HO, is markedly induced in epithelial cells and plays an important role in protecting mucosal cells. Recent studies have focused on the biological effects of the products of this enzymatic reaction, which have antioxidant, anti-inflammatory, and cytoprotective functions. In this review, the essential roles of HO in the gastrointestinal tract are summarized, focusing on nutrient absorption, protection against cellular stresses, and the maintenance and regulation of tight junction proteins, emphasizing the potential therapeutic implications. The biochemical basis of the potential therapeutic implications of glutamine for HO-1 induction in gastrointestinal injury is also discussed.

Heme oxygenase-1 induction by heat shock in rat hepatoma cell line is regulated by the coordinated function of HSF1, NRF2 and BACH1.

The mechanism of heme oxygenase-1 (HO-1) induction by heat shock (HS) loading remains unclear. Here, we investigated the contribution of transcription factors to HS-induced HO-1 expression, using a rat hepatoma cell line (H-4-II-E). Our results demonstrated that HS treatment resulted in a marked induction of HO-1. Immunohistochemical analysis showed a slight mismatch in the expression levels of HO-1 and HSP70 by HS among cells, suggesting a conflict between multiple induction mechanisms. We observed HS-induced nuclear localization of, not only phosphorylated HSF1 but also NRF2, which is a typical transcription factor activated by oxidative stress. HSF1 knockdown in H-4-II-E markedly reduced HO-1 induction by HS, while NRF2 knockdown resulted in a partial effect. The chromatin immunoprecipitation assay demonstrated that HS loading resulted in significant binding of HSF1 to the HSE in the promoter proximal region of HO-1 gene and another HSE located close to the Maf recognition element (MARE) in the -4 kb upstream enhancer region 1, where NRF2 also bound, together with basic leucine zipper transcription factor 1, a negative transcription factor of HO-1. These observations indicate that HO-1 induction by HS is mainly mediated by HSF1 binding to the proximal HSE. NRF2 binding to MARE by HS is predominantly suppressed by an increased binding of BACH1.

Role of heme oxygenase-1 in human placenta on iron supply to fetus.

INTRODUCTION: To date, details on how iron is supplied from the mother to the fetus through the placenta have remained unclear. Recently, increasing evidence has shown that heme oxygenase (HO)-1, which is an inducible isoform of the rate-limiting enzyme in the heme degradation pathway, may be involved in the effective reutilization of iron. In this study, we examined the distribution and gene expression of HO-1 in the villous tissue of human placenta at various periods of pregnancy. METHODS: Using the placenta of 38 samples for which consent was obtained, chronological changes in the localization of HO-1 protein were examined by histological examination. RT-PCR was also performed to examine the expression of HO-1, transferrin receptor-1, and ferroportin 1. Ferric iron in the tissues was analyzed by Prussian blue staining. RESULTS: Immunohistochemical studies showed that HO-1 protein was exclusively expressed in trophoblastic cells throughout gestation. In the miscarriage placenta in the first trimester, ho-1 mRNA levels were significantly higher than normal. Placenta with fetal death (miscarriage) in the first and second trimester indicate significantly higher ratio of ho-1 gene for iron production to the fpn-1 gene for iron excretion than normal. These suggest that the role of HO-1 with various physiological functions is changing throughout pregnancy. DISCUSSION: These findings suggest that HO-1 in placenta plays an important role in iron supplying system in the second trimester to support fetal development.

Visualization of the Redox Status of Cytosolic Glutathione Using the Organelle- and Cytoskeleton-Targeted Redox Sensors.

Glutathione is a small thiol-containing peptide that plays a central role in maintaining cellular redox homeostasis. Glutathione serves as a physiologic redox buffer by providing thiol electrons for catabolizing harmful oxidants and reversing oxidative effects on biomolecules. Recent evidence suggests that the balance of reduced and oxidized glutathione (GSH/GSSG) defines the redox states of Cys residues in proteins and fine-tunes their stabilities and functions. To elucidate the redox balance of cellular glutathione at subcellular resolution, a number of redox-sensitive green fluorescent protein (roGFP) variants have been developed. In this study, we constructed and functionally validated organelle- and cytoskeleton-targeted roGFP and elucidated the redox status of the cytosolic glutathione at a subcellular resolution. These new redox sensors firmly established a highly reduced redox equilibrium of cytosolic glutathione, wherein significant deviation was observed among cells. By targeting the sensor to the cytosolic and lumen sides of the Golgi membrane, we identified a prominent redox gradient across the biological membrane at the Golgi body. The results demonstrated that organelle- and cytoskeleton-targeted sensors enable the assessment of glutathione oxidation near the cytosolic surfaces of different organelle membranes.

Antitumor effect of palmitic acid-conjugated DsiRNA for colon cancer in a mouse subcutaneous tumor model.

In this study, we synthesized Dicer-substrate siRNA conjugated with palmitic acid at the 5'-end of the sense strand (C16-DsiRNA), and examined its RNAi effect on ß-catenin as a target gene in a colon cancer cell line, HT29Luc, both in vitro and in vivo. We examined the in vitro RNAi effect in HT29Luc cells and found that C16-DsiRNA strongly inhibited expression of the ß-catenin gene in comparison with non-modified DsiRNA. Also, high membrane permeability of C16-DsiRNA was exhibited, and it was confirmed that most of the C16-DsiRNA was localized in cytoplasm of HT29Luc cells. In regard to the in vivo RNAi effect, C16-DsiRNA complexed with Invivofectamine targeting the ß-catenin gene was locally administered to a subcutaneous tumor formed by implantation of HT29Luc cells into the subcutis of nude mice; we evaluated the effect by measuring the bioluminescence increase, which reflects tumor growth, using an in vivo imaging system. As a result, C16-DsiRNA strongly inhibited the growth of tumors formed in subcutis of nude mice compared with non-modified DsiRNA, and this in vivo RNAi effect lasted up to 15 days. Our results suggest that C16-DsiRNA should be vigorously pursued as a novel nucleic acid medicine for clinical treatment of cancer.

NRF2 and HSF1 coordinately regulate heme oxygenase-1 expression.

Heme oxygenase-1 (HO-1) is an inducible enzyme responding to various stresses and has cytoprotective activities. Although HO-1 has been referred to as heat shock protein (HSP) 32, the heat-mediated induction of HO-1 varies among different species and cell lines. We examined the effects of heat shock on HO-1 expression in mouse embryonic fibroblast (MEF) cells deficient in heat shock factor 1 (HSF1) or nuclear factor-erythroid-2-related factor 2 (NRF2). Heme-induced expression of HO-1 was 2-fold higher in Hsf1-/- cells than in the wild-type cells at both mRNA and protein levels. In Nrf2-/- cells, heme-induced expression of HO-1 was not detected. In contrast, HO-1 expression was markedly induced by heat shock at 40-42 °C in Nrf2-/- cells while the wild-type cells were not responsive. The heat-induced expression of HO-1 in Nrf2-/- cells were almost completely diminished by transfection of siRNA against Hsf1 gene. These results suggest that HSF1 and NRF2 suppress heme-induced and heat-induced HO-1 expression, respectively.

Local redox environment beneath biological membranes probed by palmitoylated-roGFP.

Production of reactive oxygen species (ROS) and consequent glutathione oxidation are associated with various physiological processes and diseases, including cell differentiation, senescence, and inflammation. GFP-based redox sensors provide a straight-forward approach to monitor ROS levels and glutathione oxidation within a living cell at the subcellular resolution. We utilized palmitoylated versions of cytosolic glutathione and hydrogen peroxide sensors (Grx1-roGFP2 and roGFP2-Orp1, respectively) and demonstrated a unique redox environment near biological membranes. In HeLa cells, cytosolic glutathione was practically completely reduced (EGSH/GSSG = - 333mV) and hydrogen peroxide level was under the detectable range. In contrast, the cytoplasmic milieu near membranes of intracellular vesicles exhibited significant glutathione oxidation (EGSH/GSSG > - 256mV) and relatively high H2O2 production, which was not observed for the plasma membrane. These vesicles colocalized with internalized EGFR, suggesting that H2O2 production and glutathione oxidation are characteristics of cytoplasmic surfaces of the endocytosed vesicles. The results visually illustrate local redox heterogeneity within the cytosol for the first time.

Thiol-based copper handling by the copper chaperone Atox1.

Human antioxidant protein 1 (Atox1) plays a crucial role in cellular copper homeostasis. Atox1 captures cytosolic copper for subsequent transfer to copper pumps in trans Golgi network, thereby facilitating copper supply to various copper-dependent oxidereductases matured within the secretory vesicles. Atox1 and other copper chaperones handle cytosolic copper using Cys thiols which are ideal ligands for coordinating Cu(I). Recent studies demonstrated reversible oxidation of these Cys residues in copper chaperones, linking cellular redox state to copper homeostasis. Highlighted in this review are unique redox properties of Atox1 and other copper chaperones. Also, summarized are the redox nodes in the cytosol which potentially play dominant roles in the redox regulation of copper chaperones. © 2016 IUBMB Life, 69(4):246-254, 2017.

Prevention of Barrier Disruption by Heme Oxygenase-1 in Intestinal Bleeding Model.

In this study we investigated the effect of free heme, the local level of which was increased by bleeding, on the intestinal barrier function, using human epithelial colorectal adenocarcinoma cells (Caco-2). Our results show that the addition of hemin to the culture medium markedly disrupted the barrier function, which was significantly improved by glutamine supplementation. Although hemin treatment caused the increased expression of heme oxygenase (HO)-1, the inhibition of HO activity resulted in the aggravation of hemin-induced barrier dysfunction. Up-regulation of HO-1 by pretreatment with a low concentration of hemin almost completely prevented hemin-induced barrier dysfunction. Taken together, these observations indicate that an abnormally high level of intracellular free heme causes barrier dysfunction, probably through the modulation of proteins forming tight junctions.

Nonpeptide neurotrophic agents useful in the treatment of neurodegenerative diseases such as Alzheimer's disease.

Developed regions, including Japan, have become "aged societies," and the number of adults with senile dementias, such as Alzheimer's disease (AD), Parkinson's disease, and Huntington's disease, has also increased in such regions. Neurotrophins (NTs) may play a role in the treatment of AD because endogenous neurotrophic factors (NFs) prevent neuronal death. However, peptidyl compounds have been unable to cross the blood-brain barrier in clinical studies. Thus, small molecules, which can mimic the functions of NFs, might be promising alternatives for the treatment of neurodegenerative diseases. Natural products, such as or nutraceuticals or those used in traditional medicine, can potentially be used to develop new therapeutic agents against neurodegenerative diseases. In this review, we introduced the neurotrophic activities of polyphenols honokiol and magnolol, which are the main constituents of Magnolia obovata Thunb, and methanol extracts from Zingiber purpureum (BANGLE), which may have potential therapeutic applications in various neurodegenerative disorders.

Glutamine protects intestinal barrier function of colon epithelial cells from ethanol by modulating Hsp70 expression.

In this study, we investigated the protective effect of glutamine on barrier dysfunction induced by ethanol, by using human epithelial colorectal adenocarcinoma cells (Caco-2). Our results show that addition of glutamine to culture medium significantly improved the disruption of integrity caused by ethanol, which was associated with increased expression of heat shock protein 70 (Hsp70). Ethanol exposure moderately activates heat shock factor 1 (HSF1), which was characterized by increased DNA-binding activity and phosphorylation status of HSF1. Remarkably, glutamine treatment enhanced ethanol-mediated expression of Hsp70 and activation of HSF1. Up-regulation of Hsp70 by pretreatment with heat stress also promoted recovery from the ethanol-induced barrier dysfunction. Taken together, these observations indicate that glutamine protects the intestinal barrier function in Caco-2 cells, in part by modulating HSF1-mediated Hsp70 expression.

Heme and non-heme iron transporters in non-polarized and polarized cells.

BACKGROUND: Heme and non-heme iron from diet, and recycled iron from hemoglobin are important products of the synthesis of iron-containing molecules. In excess, iron is potentially toxic because it can produce reactive oxygen species through the Fenton reaction. Humans can absorb, transport, store, and recycle iron without an excretory system to remove excess iron. Two candidate heme transporters and two iron transporters have been reported thus far. Heme incorporated into cells is degraded by heme oxygenases (HOs), and the iron product is reutilized by the body. To specify the processes of heme uptake and degradation, and the reutilization of iron, we determined the subcellular localizations of these transporters and HOs. RESULTS: In this study, we analyzed the subcellular localizations of 2 isoenzymes of HOs, 4 isoforms of divalent metal transporter 1 (DMT1), and 2 candidate heme transporters--heme carrier protein 1 (HCP1) and heme responsive gene-1 (HRG-1)--in non-polarized and polarized cells. In non-polarized cells, HCP1, HRG-1, and DMT1A-I are located in the plasma membrane. In polarized cells, they show distinct localizations: HCP1 and DMT1A-I are located in the apical membrane, whereas HRG-1 is located in the basolateral membrane and lysosome. 16Leu at DMT1A-I N-terminal cytosolic domain was found to be crucial for plasma membrane localization. HOs are located in smooth endoplasmic reticulum and colocalize with NADPH-cytochrome P450 reductase. CONCLUSIONS: HCP1 and DMT1A-I are localized to the apical membrane, and HRG-1 to the basolateral membrane and lysosome. These findings suggest that HCP1 and DMT1A-I have functions in the uptake of dietary heme and non-heme iron. HRG-1 can transport endocytosed heme from the lysosome into the cytosol. These localization studies support a model in which cytosolic heme can be degraded by HOs, and the resulting iron is exported into tissue fluids via the iron transporter ferroportin 1, which is expressed in the basolateral membrane in enterocytes or in the plasma membrane in macrophages. The liberated iron is transported by transferrin and reutilized for hemoglobin synthesis in the erythroid system.

Heme Oxygenase-1 is an Essential Cytoprotective Component in Oxidative Tissue Injury Induced by Hemorrhagic Shock.

Hemorrhagic shock causes oxidative stress that leads to tissue injuries in various organs including the lung, liver, kidney and intestine. Excess amounts of free heme released from destabilized hemoproteins under oxidative conditions might constitute a major threat because it can catalyze the formation of reactive oxygen species. Cells counteract this by rapidly inducing the rate-limiting enzyme in heme breakdown, heme oxygenase-1 (HO-1), which is a low-molecular-weight stress protein. The enzymatic HO-1 reaction removes heme. As such, endogenous HO-1 induction by hemorrhagic shock protects tissues from further degeneration by oxidant stimuli. In addition, prior pharmacological induction of HO-1 ameliorates oxidative tissue injuries induced by hemorrhagic shock. In contrast, the deletion of HO-1 expression, or the chemical inhibition of increased HO activity ablated the beneficial effect of HO-1 induction, and exacerbates tissue damage. Thus, HO-1 constitutes an essential cytoprotective component in hemorrhagic shock-induced oxidative tissue injures. This article reviews recent advances in understanding of the essential role of HO-1 in experimental models of hemorrhagic shock-induced oxidative tissue injuries with emphasis on the role of its induction in tissue defense.

Prevention of hemorrhagic shock-induced intestinal tissue injury by glutamine via heme oxygenase-1 induction.

Hemorrhagic shock (HS) is an oxidative stress that causes intestinal tissue injury. Heme oxygenase 1 (HO-1) is induced by oxidative stress and is thought to play an important role in the protection of tissues from oxidative injury. We previously reported the ileum to be the most susceptible to HS-induced tissue injury site in the intestine because HO-1 induction is the lowest at this site. We also previously demonstrated that glutamine (GLN) significantly induced HO-1 in the lower intestinal tract. In the present study, we investigated whether GLN pretreatment improves HS-induced intestinal tissue injury in the ileum by HO-1 induction. Treatment of rats with GLN (0.75 g/kg, i.v.) markedly induced functional HO-1 protein in mucosal epithelial cells in the ileum. Glutamine treatment before HS (MAP of 30 mmHg for 60 min) significantly ameliorated HS-induced mucosal inflammation and apoptotic cell death in the ileum, as judged by significant decreases in gene expression of TNF-alpha, iNOS, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1, myeloperoxidase activity, the number of infiltrated neutrophils, DNA fragmentation by in situ oligo ligation assay, and activated caspase-3 expression, and by increases in gene expression of IL-10 and Bcl-2. In contrast, treatment with tin mesoporphyrin, a specific inhibitor of HO activity, abolished the beneficial effect of GLN pretreatment. These findings indicate that GLN pretreatment significantly ameliorated tissue injury in the ileum after HS by inducing HO-1. Glutamine treatment may thus protect mucosal cells from HS-induced oxidative damage via the anti-inflammatory and antiapoptotic properties of HO-1.

Site-specific induction of intestinal hypoxia-inducible factor-1α after hemorrhagic shock.

The intestine is a major target organ in hemorrhagic shock (HS)-induced tissue injury. Hypoxia-inducible factor (HIF)-1α is the primary transcription factor responsible for regulating cellular response to changes in oxygen tension. Since HS is an acute hypoxic insult, the present study examined changes in the gene expression of HIF-1α in various regions of the intestine, as well as the distribution of HIF-1α protein in the intestinal cells of a rat model of HS. Levels of HIF-1α mRNA were marginally detectable in the intestine of sham-operated control animals, but obviously induced following HS. Duodenal, jejunal and colonic levels of HIF-1α mRNA robustly increased and reached a maximum during the ischemic phase of HS, followed by a rapid decrease almost to control levels during the early phase of resuscitation. The induction of HIF-1α mRNA was maximal in the duodenum. In contrast to the duodenum, jejunum and colon, in the ileum the HIF-1α mRNA level did not increase after HS. Consistent with enhanced HIF-1α gene expression, HIF-1α protein was expressed in the mucosal cells of the duodenum, jejunum and colon, but not in the ileum following HS. These findings indicate that intestinal HIF-1α expression was up-regulated at both the transcriptional and protein level in a site-specific manner in this rat model of HS. Differential regulation of HIF-1α expression along the longitudinal axes of the intestine might be a determinant of the adaptive response to HS-induced intestinal damage.