Increased Heme Oxygenase-1 Expression Research Articles

Ginsenoside Rb1 targets to HO-1 to improve sepsis by inhibiting ferroptosis

Sepsis remains the leading cause of mortality among Intensive Care Unit (ICU) patients, with its pathogenesis and treatment not yet fully elucidated. Ferroptosis plays a critical role in sepsis, suggesting that ferroptosis-related genes may serve as potential therapeutic targets. This study aims to identify key ferroptosis-related genes in sepsis and explore targeted therapeutics. Through differential expression analysis of the GSE13940 and GSE26440 datasets, heme oxygenase-1 (HO-1) was identified as a hub gene associated with ferroptosis. Additionally, single-cell analysis of the GSE175453 dataset revealed a significant upregulation of HO-1 expression in monocyte lineages during sepsis. The cecal ligation and puncture (CLP) method was employed to induce sepsis in a mouse model, lung and intestinal tissues exhibited typical ferroptosis characteristics, with a significant increase in HO-1 expression. However, treatment with the HO-1 inhibitor zinc protoporphyrin (ZNPP) significantly ameliorated ferroptosis in CLP-induced lung and intestinal tissues, as well as in lipopolysaccharide (LPS)-induced THP-1 cells. Subsequently, molecular docking, surface plasmon resonance (SPR), and microscale thermophoresis (MST) experiments demonstrated that ginsenoside Rb1 specifically targets HO-1, identifying K18A as the key binding residue. Finally, experiments conducted both in vitro and in vivo verified that ginsenoside Rb1 significantly reduces HO-1 expression, inhibits ferroptosis in sepsis-induced lung, and intestinal tissues and THP-1 cells, and improves sepsis-induced pulmonary and intestinal damage. In conclusion, this study identifies HO-1 as a key ferroptosis target in sepsis and suggests ginsenoside Rb1 as a potential novel HO-1 inhibitor for the therapeutic approach of sepsis-induced organ dysfunction.

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction.

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.

Dapagliflozin prevents kidney podocytes pyroptosis via miR-155-5p/HO-1/NLRP3 axis modulation

Diabetic nephropathy (DN) is a significant clinical microvascular complication associated with diabetes mellitus (DM), and end-stage diabetes giving rise to kidney failure is developing into the major etiological factor of chronic kidney failure. Dapagliflozin is reported to limit podocyte damage in DM, which has proven to protect against renal failure. Mounting evidence has demonstrated that pyroptosis is associated with DM progression. Nevertheless, whether pyroptosis causes DN and the underlying molecular pathways remain obscure. In this study, we aimed to explore the antipyroptotic attributes of dapagliflozin and elucidate the underlying mechanisms of kidney damage in diabetes. In vivo, experiments were conducted in streptozotocin (STZ)-induced type 2 diabetic mice, which were administered dapagliflozin via gavage for 6 weeks. Subsequently, the specific organizational characteristics and expression of pyroptosis-related genes were evaluated. Intragastric dapagliflozin administration markedly reduced renal tissue injury. Meanwhile, dapagliflozin also attenuated the expression level of pyroptosis associated genes, including ASC, cleaved Caspase-1, GSDMD N-termini, NLRP3, IL-18, and IL-1β in renal tissue of dapagliflozin-treated animals. Similar antipyroptotic effects were observed in palmitic acid (PA)-treated mouse podocytes. We also found that heme oxygenase 1 (HO-1) enhanced the protection of mouse podocyte clone 5 cells (MPC5). Moreover, miR-155-5p inhibition increased pyroptosis in PA-treated MPC5 cells, suggesting that miR-155-5p acts as an endogenous stimulator that increases HO-1 expression and reduces pyroptosis. Hence, our findings imply that dapagliflozin inhibits podocyte pyroptosis via the miR-155-5p/HO-1/NLRP3 axis in DM. Furthermore, dapagliflozin substitution may be regarded as an effective strategy for preventing pyroptosis in the kidney, including a therapeutic option for treating pyroptosis-related DN.

Atorvastatin inhibits Lipopolysaccharide (LPS)-induced vascular inflammation to protect endothelium by inducing Heme Oxygenase-1 (HO-1) expression.

This study aimed to explore the differential effects of varying doses of atorvastatin on antagonizing lipopolysaccharide (LPS)-induced endothelial inflammation based on heme oxygenase 1 (HO-1) expression. Vascular endothelial inflammatory injury was induced in 40 Sprague-Dawley rats by intraperitoneal injection of LPS. These rats were randomly divided into control, low-dose atorvastatin, high-dose atorvastatin, and HO-1 blocking groups. Seven days after treatment, all rats were sacrificed, and heart-derived peripheral blood was collected to measure the serum concentrations of bilirubin, alanine aminotransferase (ALT), total cholesterol, malondialdehyde, endothelial cell protein C receptor, endothelin-1, von Willebrand factor, and soluble thrombomodulin. Meanwhile, the number of circulating endothelial cells was determined using flow cytometry. Vascular tissues from descending aorta of rats from each group were extracted to detect the expression level of HO-1. After different doses of atorvastatin intervention, the above inflammatory indices were decreased, and HO-1 expression and ALT concentration were increased in the atorvastatin-treated group of rats compared with the control group. These changes were more pronounced in the high-dose statin group (P < 0.05). Conversely, no significant decrease in the above inflammatory indices and no significant increase in HO-1 expression were observed in rats in the blocking group (P > 0.05). For LPS-induced vascular inflammation, high-dose atorvastatin exerts potent anti-inflammatory and vascular endothelial protection effects by inducing HO-1 expression.

Fibroblast growth factor 21 inhibits ferroptosis following spinal cord injury by regulating heme oxygenase-1.

Abstract JOURNAL/nrgr/04.03/01300535-202407000-00037/figure1/v/2023-11-20T171125Z/r/image-tiff Interfering with the ferroptosis pathway is a new strategy for the treatment of spinal cord injury. Fibroblast growth factor 21 can inhibit ferroptosis and promote neurofunctional recovery, while heme oxygenase-1 is a regulator of iron and reactive oxygen species homeostasis. The relationship between heme oxygenase-1 and ferroptosis remains controversial. In this study, we used a spinal cord injury rat model to show that the levels of fibroblast growth factor 21 in spinal cord tissue decreased after spinal cord injury. In addition, there was a significant aggravation of ferroptosis and a rapid increase in heme oxygenase-1 expression after spinal cord injury. Further, heme oxygenase-1 aggravated ferroptosis after spinal cord injury, while fibroblast growth factor 21 inhibited ferroptosis by downregulating heme oxygenase-1. Thus, the activation of fibroblast growth factor 21 may provide a potential treatment for spinal cord injury. These findings could provide a new potential mechanistic explanation for fibroblast growth factor 21 in the treatment of spinal cord injury.

Hydrogen regulates mitochondrial quality to protect glial cells and alleviates sepsis-associated encephalopathy by Nrf2/YY1 complex promoting HO-1 expression.

Sepsis-associated encephalopathy (SAE) is a complication of the central nervous system in patients with sepsis. Currently, no effective treatment for sepsis is available. Hydrogen plays a protective role in different diseases; however, the detailed mechanism of hydrogen-treated disease remains unclear. The purpose of this study was to investigate the effect of hydrogen on SAE in vitro and in vivo and the mechanism of hydrogen in mitochondrial dynamics and its function in astrocytes and microglia stimulated by lipopolysaccharides (LPSs). Animal models of SAE were generated by cecal ligation and puncture, and the SAE model was established by in vitro LPS stimulation. MTT, lactate dehydrogenase (LDH), reactive oxygen species (ROS), heme oxygenase-1 (HO-1) activity, mitochondrial membrane potential (MMP), and cell apoptosis assays were used to determine the effect of hydrogen on astrocytes and microglia stimulated by LPSs. The relationships between nuclear factor erythroid 2-related factor 2 (Nrf2), YY1, and HO-1 were examined by chromatin immunoprecipitation and co-immunoprecipitation. Mitochondrial homeostasis-related proteins in LPS-stimulated glial cells and brain tissues of SAE mice were detected by western blotting. The effects of hydrogen treatment in the SAE mouse model were investigated using Morris water maze and Y-maze analyses. After performing experiments with different concentrations of LPSs in vitro, we selected 1000ng/ml for subsequent experiments. Hydrogen attenuated the increase in ROS, LDH, and apoptosis and promoted decreases in cell activity and MMP, further promoting an increase in HO-1 expression induced by LPSs in astrocytes and microglia. Moreover, hydrogen further promoted the expression of Nrf2, HO-1, PGC-1α, TFAM, PARKIN, and PINK1, inhibited LPS-induced OPA1 and MFN2 expression in astrocytes and microglia, and downregulated the expression of DRP1 after LPS induction. Intriguingly, hydrogen treatment enhanced the binding between Nrf2 and YY1. However, silencing Nrf2 or YY1 abolished the protective effects of hydrogen on cell activity, LDH, ROS, and MMP; apoptosis; and regulation of Nrf2, HO-1, PGC-1α, TFAM, OPA1, DRP1, MFN2, PARKIN, and PINK1 in microglia. Finally, hydrogen treatment improved the results of behavioral detection, apoptosis, Nrf2, HO-1, PGC-1α, TFAM, OPA1, DRP1, MFN2, PARKIN, PINK1, and cytokines in SAE in vivo. Hydrogen improved cell injury and mitochondrial quality, which were associated with HO-1 expression promoted by the Nrf2/YY1 complex in vitro. Thus, hydrogen treatment may represent a novel therapeutic method for treating SAE.

Elevation of HO-1 expression protects the intestinal mucosal barrier in severe acute pancreatitis via inhibition of the MLCK/p-MLC signaling pathway

In severe acute pancreatitis (SAP), intestinal mucosal barrier damage can cause intestinal bacterial translocation and induce or aggravate systemic infections. Heme oxygenase-1 (HO-1) is a validated antioxidant and cytoprotective agent. This research aimed to investigate the effect and mechanism of HO-1 on SAP-induced intestinal barrier damage in SAP rats. Healthy adult male Sprague-Dawley rats were randomly separated into the sham-operated group, SAP group, SAP + Hemin group, and SAP + Znpp group. The rat model of SAP was established by retrograde injection of sodium taurocholate (5%) into the biliopancreatic duct. Hemin (a potent HO-1 activator) and Znpp (a competitive inhibitor of HO-1) were injected intraperitoneally in the selected groups 24 h before SAP. Serum and intestinal tissue samples were collected for analysis after 24 h in each group. Hemin pretreatment significantly reduced systemic inflammation, intestinal oxidative stress, and intestinal epithelial apoptosis in SAP by increasing HO-1 expression. Meanwhile, pretreatment with Hemin abolished the inhibitory effect on the expression of the tight junction proteins and significantly inhibited the activation of the MLCK/P-MLC signaling pathway. Conversely, ZnPP completely reversed these effects. Our study indicates that upregulation of HO-1 expression attenuates the intestinal mucosal barrier damage in SAP. The protective effect of HO-1 on the intestine is attributed to MLCK/p-MLC signaling pathway inhibition.

Withaferin A inhibits ferroptosis and protects against intracerebral hemorrhage.

Recent studies have indicated that suppressing oxidative stress and ferroptosis can considerably improve the prognosis of intracerebral hemorrhage (ICH). Withaferin A (WFA), a natural compound, exhibits a positive effect on a number of neurological diseases. However, the effects of WFA on oxidative stress and ferroptosis-mediated signaling pathways to ICH remain unknown. In this study, we investigated the neuroprotective effects and underlying mechanism for WFA in the regulation of ICH-induced oxidative stress and ferroptosis. We established a mouse model of ICH by injection of autologous tail artery blood into the caudate nucleus and an in vitro cell model of hemin-induced ICH. WFA was injected intracerebroventricularly at 0.1, 1 or 5 µg/kg once daily for 7 days, starting immediately after ICH operation. WFA markedly reduced brain tissue injury and iron deposition and improved neurological function in a dose-dependent manner 7 days after cerebral hemorrhage. Through in vitro experiments, cell viability test showed that WFA protected SH-SY5Y neuronal cells against hemin-induced cell injury. Enzyme-linked immunosorbent assays in vitro and in vivo showed that WFA markedly decreased the level of malondialdehyde, an oxidative stress marker, and increased the activities of anti-oxidative stress markers superoxide dismutase and glutathione peroxidase after ICH. Western blot assay, quantitative polymerase chain reaction and immunofluorescence results demonstrated that WFA activated the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling axis, promoted translocation of Nrf2 from the cytoplasm to nucleus, and increased HO-1 expression. Silencing Nrf2 with siRNA completely reversed HO-1 expression, oxidative stress and protective effects of WFA. Furthermore, WFA reduced hemin-induced ferroptosis. However, after treatment with an HO-1 inhibitor, the neuroprotective effects of WFA against hemin-induced ferroptosis were weakened. MTT test results showed that WFA combined with ferrostatin-1 reduced hemin-induced SH-SY5Y neuronal cell injury. Our findings reveal that WFA treatment alleviated ICH injury-induced ferroptosis and oxidative stress through activating the Nrf2/HO-1 pathway, which may highlight a potential role of WFA for the treatment of ICH.

Novel Evidence That Pineal Gland Hormone Melatonin Inhibits Migration of Hematopoietic Stem/Progenitor Cells (HSPCs) By Inhibiting Nlrp3 Inflammasome and Upregulating Heme Oxygenase-1 (HO-1) - Implications to Understand Better Diurnal Trafficking of HSPCs and Anti-Inflammatory Effects of Melatonin

Background. Hematopoietic stem progenitor cells (HSPCs) follow the diurnal circulation rhythm in peripheral blood (PB) with nadir during late night and pick at early morning hours. We have reported that the level of these cells in PB correlates with activation of innate immunity pathways, including complement cascade (ComC) (Stem Cell Rev Rep. 2018;14(5):677-685) that drives activation of Nlrp3 inflammasome (Stem Cell Rev Rep. 2020;16(2):335-343). Therefore, mice both in defective ComC activation as well as Nlrp3 inflammasome do not show typical changes in the diurnal level of circulating HSPCs. Cell migration is also inhibited by the anti-inflammatory enzyme heme oxygenase-1 (HO-1), which inhibits Nlrp3 inflammasome (Leukemia. 2022;36(1):23-32). It is well known that circadian rhythm is regulated by the release of melatonin from the pineal gland, which also has some anti-inflammatory effects (J. Pineal Res. 2018;65:e12525). Aim of the study.Since stem cell trafficking is driven by innate immunity-induced sterile inflammation and melatonin has an anti-inflammatory effect, we hypothesized that melatonin could negatively impact the release of HSPCs from bone marrow (BM) into PB by inhibiting Nlrp3 inflammasome activation. Materials and Methods. To test this hypothesis, first, we tested the expression of melatonin receptors in murine and human HSPCs. We employed Transwell system to see the effect of melatonin on the migration of human and murine HSPCs to the SDF-1 gradient. Next, we performed pharmacological mobilization of normal mice with G-CSF or AMD3100 in the presence of melatonin. Finally, employing Glow assay, we evaluated the effect of melatonin on activation of Nlrp3 inflammasome in HSPCs stimulated by BM chemotactic factors and the effect of melatonin on expression of migration inhibitory HO-1. Since membrane lipid rafts (MLRs) are crucial for the proper response of cells to chemoattractants, we also evaluated the effect of melatonin on the expression of enzymes (SREBP2, HMGCs, and HMGCR) involved in the synthesis of cholesterol and sphingolipids that are major constituents of MLRs. Results. We noticed that HSPCs express melatonin receptors, and melatonin inhibits in vitro Transwell assays migration of HSPCs and negatively affects mobilization of these cells into PB in response to G-CSF and AMD3100 administration. We also observed that melatonin inhibits Nlrp3 inflammasome activation and the formation of MLRs. This is explained by a decrease in the expression of enzymes such as SREBP2, HMGCs, and HMGCR involved in cholesterol synthesis leading to impaired formation of MLRs. Moreover, melatonin increases in HSPCs expression of heme oxygenase-1 (HO-1), that is a known inhibitor of Nlrp3 inflammasome and a negative regulator of cell migration. Conclusions. We provide for the first-time evidence that melatonin being a ''sleep regulating pineal hormone'' directly inhibits migration of HSPCs in vitro assays and in vivo their release from BM in response to G-CSF and AMD3100. This is correlated with inhibition of cholesterol synthesis, which results in defective assembly of MLRs, as well as a decrease in Nlrp3 inflammasome activity and an increase in expression of HO-1. Since melatonin is a commonly used shelf drug, this should be considered while preparing a patient for the procedure of HSPCs mobilization. More importantly, our studies shed more light on molecular mechanisms of melatonin involvement in the diurnal circulation of HSPCs.

HO-1/autophagic flux axis alleviated sepsis-induced acute lung injury via inhibiting NLRP3 inflammasome

Among the multiple organ injuries induced by sepsis, acute lung injury (ALI) triggered by an excessive inflammatory response is one of the main causes contributing to patient death, and inhibition of the inflammation cascade is the key therapeutic strategy to improve prognosis. The NLRP3 inflammasome complex is considered an intracellular signaling molecule closely associated with the uncontrolled inflammatory response in sepsis-induced ALI. Therefore, exploring new targets to repress its activation is regarded as a potential therapeutic strategy. Growing evidence demonstrated that heme oxygenase-1 (HO-1) contributed to general anti-inflammation and exerted a protective role in ALI, but its underlying mechanisms have not been clarified completely. Herein, we investigated HO-1 was elevated in alveolar macrophages isolated from bronchoalveolar lavage fluid (BALF) of sepsis mice. HO-1 abundance suppressed NLRP3 inflammasome complex activation and attenuated pro-inflammatory cytokines release, thereby alleviating sepsis-induced ALI. Whereas inhibition of HO-1 reached the opposite effect. Meanwhile, HO-1 is an effective and functionally relevant regulator of autophagic flux. HO-1 activator decreased the expression of P62 and enhanced the LC3 II/LC3 I ratio, resulting in autophagic flux activation. In addition, the protective effects HO-1 exerted in sepsis-induced ALI could be abolished by autophagic flux inhibitor. Autophagic flux activator could suppress NLRP3 inflammasome activation and attenuate ALI, while autophagic flux inhibitor had the opposite effect. In conclusion, our study revealed increased HO-1 expression inhibited the level of NLRP3 inflammasome via regulating the activation of autophagic flux, thus attenuating inflammatory response and alleviating sepsis-induced ALI.

Protective Effect of Anwulignan on Gastric Injury Induced by Indomethacin in Mice.

Anwulignan (AN) is a monomer lignan from Schisandra sphenanthera Rehd. et Wits (Schisandra sphenanthera fructus, Schisandra sphenanthera). The protective effect of AN against the indomethacin (IND)-induced gastric injury to mice and the related mechanism of action was investigated in this study. The effect of AN was mainly assessed by observing the gastric tissue morphology, gastric ulcer index (GUI), ulcer inhibition rate (UIR), gastric juice volume (GJV) and pH value. Chemical colorimetry, immunofluorescence, ELISA, and Western blot were used to detect related factors in the gastric tissue. The results showed that AN reduced the GUI, increased the UIR, inhibited the GJV, and increased the gastric pH value. AN significantly increased cyclooxygenase-1, cyclooxygenase-2, and prostaglandin E2 expression levels in the gastric tissue, activated nuclear factor (erythroid-derived 2)-like 2 (Nrf2), increased heme oxygenase-1 expression, enhanced the activity of superoxide dismutase and glutathione peroxidase, and decreased the malondialdehyde content. AN reduced the phosphorylation of nuclear factor-κ gene binding (NF-κB) p65 and its nuclear translocation, the key protein of NF-κB signaling pathway in the gastric tissue, and the content of the pathway downstream signaling molecules, including interleukin-6, interleukin-1β, and tumor necrosis factor-α, to play an anti-inflammatory role. AN inhibited the downstream signals B-cell lymphoma 2-associated x protein and cleaved caspase-3 in gastric tissue, and activated B-cell lymphoma 2, to play an antiapoptotic role, which were further verified by Hoechst staining. Therefore, AN has a significant protection against the gastric injury induced by IND in mice, and the mechanism may be concerned in its activation of Nrf2, inhibition of NF-κB signaling pathway, and anti-apoptotic effect. SIGNIFICANCE STATEMENT: Anwulignan (AN) significantly reduced the indomethacin-induced gastric injury in mice, and its antioxidation, anti-inflammation, and antiapoptosis were considered to be involve in the effect, suggesting that AN should be a potential drug or food supplement for gastric injury induced by indomethacin.

Eriodictyol Attenuates Cholangiocarcinoma Malignancy by Regulating HMOX1 Expression: An In Vitro Study.

Cholangiocarcinoma remains one of the most dangerous types of cancer. Eriodictyol is a well-known flavonoid having effective bioactivity against various malignant tumor types. However, the anticancer effect of eriodictyol against cholangiocarcinoma remains ambiguous. Thus, the aim of the present study was to investigate the effects of eriodictyol on human cholangiocarcinoma. The biological effects of eriodictyol were validated by viability assay, colony formation and western blot analysis. The significance of heme oxygenase 1 (HMOX1) expression in cholangio-carcinoma was demonstrated using bioinformatics analysis and knockdown of HMOX1 by transfection with short interfering (si)-RNA. Eriodictyol highly reduced the in vitro viability of SNU-308, SNU-478, SNU-1079, and SNU-1196 cholangiocarcinoma cells compared with that of 293T cells, in a dose-dependent manner. The anticancer effect of eriodictyol was achieved by caspase-3-mediated apoptosis. In particular, eriodictyol increased HMOX1 expression, which resulted in attenuation of cholangiocarcinoma cell proliferation. In contrast, ablating HMOX1 expression by si-RNA transfection against HMOX1 made cholangiocarcinoma cells insensitive to the antiproliferative effect of eriodictyol treatment. These results collectively indicate that eriodictyol acts as an anticancer agent via regulation of HMOX1 expression against human cholangiocarcinoma.

Zingerone stimulates osteoblast differentiation by increasing Smad1/5/9-mediated HO-1 expression in MC3T3-E1 cells and primary mouse calvarial cells.

Zingerone is a non-volatile compound found mainly in dried ginger. Zingerone increases the expression of osteogenic markers and has antioxidant effects. A previous study showed that zingerone accelerated osteoblast differentiation by suppressing the expression of Smad7, a member of the inhibitory Smad (I-Smad) family. However, it is not known if zingerone can induce osteoblast differentiation by regulating Smad1/5/9, a member of the receptor-regulated Smad (R-Smad) family. In addition, osteoblast differentiation induced by Smad1/5/9 mediated increases in the expression of heme oxygenase 1 (HO-1) has not been reported. This study investigated the effects of zingerone on osteoblast differentiation and confirmed the relationship between Smad1/5/9 and HO-1. Zingerone increased the expression of osteogenic genes including runt-related transcription factor 2 (Runx2), distal-less homeobox (Dlx5) and osteocalcin (OC) and also promoted Smad1/5/9 phosphorylation. Interestingly, HO-1 expression was also elevated by zingerone, and an inhibitor of HO-1 (Sn[IV] protoporphyrin IX dichloride [SnPP]) suppressed the zingerone-induced increase in HO-1 expression and expression of osteogenic marker genes such as Dlx5, Runx2 and OC. Protein phosphatase 2A Cα (PP2A Cα, an inhibitor of Smad1/5/9) suppressed the zingerone-induced increase in HO-1 expression and expression of osteogenic marker genes. The zingerone-induced increase in HO-1 luciferase activity was suppressed by PP2A Cα. Taken together; our data demonstrate that zingerone promotes osteoblast differentiation by increasing Smad1/5/9 mediated HO-1 expression.

Systemic Maternal Human sFLT1 Overexpression Leads to an Impaired Foetal Brain Development of Growth-Restricted Foetuses upon Experimental Preeclampsia.

The pregnancy disorder preeclampsia (PE) is characterized by maternal hypertension, increased level of circulating antiangiogenic soluble fms-like tyrosine kinase-1 (sFLT1), and reduced placental perfusion, leading to foetal growth restriction (FGR) and preterm birth. All these adverse effects are associated with neurocognitive disorders in the offspring. However, the direct interplay between increased antiangiogenesis during PE and disturbed foetal brain development independent of prematurity has not been investigated yet. To examine foetal brain development in sFLT1-related PE, hsFLT1/rtTA-transgenic mice with systemic (maternal or maternal/fetoplacental) human sFLT1 (hsFLT1) overexpression since 10.5 days postconception (dpc) were used, and histological and molecular analyses of foetal brains were performed at 18.5 dpc. Consequences of elevated hsFLT1 on placental/foetal vascularization and hypoxia of placentas and foetal brains were analysed using the hypoxia markers pimonidazole and hemeoxygenase-1 (HO-1). Immunohistochemical analysis revealed increased hypoxia in placentas of PE-affected pregnancies. Moreover, an increase in HO-1 expression was observed upon elevated hsFLT1 in placentas and foetal brains. PE foetuses revealed asymmetrical FGR by increased brain/liver weight ratio. The brain volume was reduced combined with a reduction in the cortical/hippocampal area and an increase of the caudate putamen and its neuroepithelium, which was associated with a reduced cell density in the cortex and increased cell density in the caudate putamen upon hsFLT1 overexpression. Mild influences were observed on brain vasculature shown by free iron deposits and mRNA changes in Vegf signalling. Of note, both types of systemic hsFLT1 overexpression (indirect: maternal or direct: maternal/fetoplacental) revealed similar changes with increasing severity of impaired foetal brain development. Overall, circulating hsFLT1 in PE pregnancies impaired uteroplacental perfusion leading to disturbed foetal oxygenation and brain injury. This might be associated with a disturbed cell migration from the caudate putamen neuroepithelium to the cortex which could be due to disturbed cerebrovascular adaption.

Abstract 299: Microvessel Oxidative Stress Predicts Changes In Leg Function Of Patients With Peripheral Arterial Disease After Supervised Exercise Therapy

Our objective was to evaluate baseline oxidative stress in the microvessels of patients with Peripheral Arterial Disease (PAD) 1) in association with Heme Oxygenase-1 (HO-1), a critical cytoprotective molecule for oxidative stress 2) as a predictor of the functional and pathophysiologic changes that occur in response to six months of Supervised Exercise Therapy (SET). We hypothesized that accumulated oxidative stress in the microvessels of PAD at baseline would predict changes in walking performance, myofiber pathology, and limb function at the end of a six-month program of SET. Methods: Twelve claudicating patients received six months of SET per ACC/AHA guidelines. Before and after SET, patients were evaluated for leg biomechanics, overground walking capacity (six-minute walking distance, SMWD), and maximum walking time on a treadmill. Subsequently, their more affected calf muscle was biopsied for quantification of HO-1 and oxidative stress (carbonyl content) in both myofibers and microvessels. We evaluated the association between HO-1 expression and carbonyl content with correlation and multiple regression. Results: HO-1 expression and Carbonyl content were strongly associated in the microvessels (n=1400, r=0.98, p&lt;0.001). Increasing Carbonyl content in the microvessels of each subject was associated with increasing HO-1 expression. In a subset of patients, HO-1 expression increased more slowly with increasing carbonyl content. Pre-SET Oxidative Stress in microvessels was a significant predictor of SET mediated change in SMWD (r=-0.75, p=0.012) and plantarflexion torque (r=0.8571, p=0.0065). Conclusion: Carbonyl content (oxidative stress) in microvessels of each subject was positively associated with HO-1 expression. However, in a subset of patients HO-1 expression increased more slowly with increasing carbonyl content. Baseline oxidative stress in the microvessels, which may be a function of the quality of HO-1 expression, was a significant predictor of SET mediated change in SMWD and plantarflexion torque. The data suggest that microvessel oxidative damage may contribute uniquely to calf muscle pathology and leg dysfunction in PAD patients.

Brazilin From Caesalpinia sappan L. Induced Apoptosis via mTOR and HO-1 Pathway in SW480 Human Colon Cancer Cells

The mTOR pathway is a crucial biological regulatory mechanism of cell growth, proliferation and cell death, and its inhibitors were new candidates of anticancer drugs through regulation of energy balance and metabolism. In the present study, whether brazilin and mTOR inhibitor (Torin1) exerts anti-cancer effects was evaluated and the mechanism of its regulation in colorectal cancer cells investigated. Brazilin showed dose- and time-dependent cytotoxicity of colorectal cancer cells (SW480 cells) through apoptosis pathways such as Bcl-2, Bax, as well as cleavage of caspase 3, caspase 9, and PARP1. In addition, brazilin reduced mammalian target of rapamycin (mTOR) phosphorylation in a dose- and time-dependent manner, and the mTOR inhibitor torin 1 blocked this phosphorylation. Brazilin also decreased heme oxygenase-1 (HO-1) expression in a dose- and time-dependent manner; however, hemin, a specific HO-1 substrate, markedly increased HO-1 expression. Torin 1 reduced hemin-induced HO-1 expression and increased colorectal cell death in a dose-dependent manner in the presence and absence of hemin. Moreover, nuclear factor erythroid 2–related factor 2 (Nrf2) translocation into nucleus fraction was crucial role in brazilin-mediated apoptosis of colorectal cancer cells. These results showed that brazilin and torin1 might regulate the mTOR signaling pathway by decreasing mTOR phosphorylation. Furthermore, mTOR signaling was associated with brazilin-regulated HO-1 expression, which induced apoptosis in colorectal cancer cells. These results suggest that synthetic and/or natural mTOR inhibitors were useful candidate for treatment of colorectal cancer cells.

Eicosapentaenoic Acid Increases Vascular and Pulmonary Endothelial Ferritin Levels with Enhanced Heme Oxygenase-1 Expression during Inflammation

Lead Author's Financial Disclosures Nothing to disclose. Study Funding Amarin Pharma Inc. and Elucida Research LLC. Background/Synopsis Inducible heme oxygenase-1 (HO-1) catalyzes the degradation of heme into biliverdin, carbon monoxide, and ferrous iron. Increased free iron triggers production of intracellular ferritin. Ferritin as well as the other HO-1 products have potent antioxidant, vasodilatory and anti-inflammatory actions. Unlike trials using mixed omega-3 formulations, eicospentaenoic acid (EPA) only treatment (icosapent ethyl) was associated with reduced cardiovascular (CV) events in high-risk patients with elevated triglycerides (REDUCE-IT) but the mechanism is not fully understood. Objective/Purpose This study compared the treatment effects of EPA on protein expression in endothelial cells (ECs) isolated from vein and pulmonary tissue under conditions of inflammation using the cytokine IL-6. Methods Human umbilical vein endothelial cells (HUVECs) and pulmonary microvascular ECs were first challenged with IL-6 (12 ng/mL) for 2 hours, and then treated with EPA (40 micromolar) for 24 hours. Proteomic analysis was performed using LC/MS to measure relative expression levels of >1,000 proteins simultaneously. Only significant (p<0.05) changes in expression between treatment groups >1-fold were included in the analysis. Results Cells treated with EPA significantly down/up-regulated expression of 310/276 and 212/229 proteins in vascular and pulmonary ECs, respectively, compared with IL-6 alone. In particular, EPA increased levels of HO-1 by 220% (p = 7.9E-50) and 190% (p = 2.8E-32) in vascular and pulmonary ECs, respectively. Increased HO-1 expression resulted in increased levels of ferritin light chain by 120% (p = 4.0E10-5) and 110% (p = 0.049) in vascular and pulmonary ECs, respectively, and ferritin heavy chain by 110% (p = 0.0002) and 160% (p = 1.4E10-6), respectively. Conclusions EPA increased expression of HO-1, a protein that has broad atheroprotective benefits, in both vascular and pulmonary ECs that correlated with increased ferritin levels during inflammation. The ability of EPA to enhance cytoprotective proteins has therapeutic implications for patients at risk for CV disease. Nothing to disclose.

Anti-Inflammatory and Anti-oxidant Functions of Cornuside by Regulating NF-[Formula: see text]B, STAT-1, and Nrf2-HO-1 Pathways.

Cornuside (CNS), found in the fruit of Cornus officinalis Seib, is a natural bisiridoid glucoside that possesses therapeutic effects by suppressing inflammation. This study aimed to determine whether CNS could inhibit the inflammatory response induced by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVECs) and mice, as well as to decipher the mechanisms. After activating HUVECs with LPS, the cells were treated with CNS. Cells were then isolated for protein or mRNA assays to analyze signaling and inflammatory molecules. In addition, mice received an intraperitoneal injection of LPS, followed by an intravenously administered dose of CNS. CNS inhibited cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) expressions induced by LPS. CNS decreased phosphorylated signal transducer and activator of transcription 1 (STAT1)-1 by promoting HO-1 expression, inhibiting nuclear factor (NF)-[Formula: see text]B-luciferase activity, and decreasing COX-2/prostaglandin E2 (PGE2) and iNOS/NO. Furthermore, CNS treatment in LPS-activated HUVECs increased the nuclear translocation of nuclear factor erythrocyte 2-related factor 2 (Nrf2) and combined Nrf2 to anti-oxidant response elements and decreased IL-1[Formula: see text] production. Reduced iNOS/NO expression by CNS was restored when HO-1 RNAi inhibited heme oxygenase-1 (HO-1). After CNS treatment in vivo, iNOS levels in lung tissue and tumor necrosis factor (TNF)-[Formula: see text] expression in the bronchoalveolar lavage fluid were significantly decreased. The results indicated that CNS increased HO-1 expression, reduced LPS-activated NF-[Formula: see text]B-luciferase activity, and inhibited iNOS/NO and COX-2/PGE2, all of which contributed to the inhibition of STAT-1 phosphorylation. Thus, CNS can be a potential new substance for treating inflammatory disorders.

Anti-Inflammatory and Antioxidant Effects of Soroseris hirsuta Extract by Regulating iNOS/NF-κB and NRF2/HO-1 Pathways in Murine Macrophage RAW 264.7 Cells

Until now, the physiological effects of Soroseris hirsuta were primarily unknown. Here we have evaluated the anti-inflammatory and antioxidant effects of Soroseris hirsuta extract (SHE) on lipopolysaccharide (LPS)-activated murine macrophages RAW 264.7 cells. SHE inhibited nitric oxide expression and inducible nitric oxide synthase expression in RAW 264.7 cells treated with LPS. Moreover, SHE suppressed LPS-induced phosphorylation of IκB kinase, inhibitor of kappa B, p65, p38, and c-JUN N-terminal kinase. Western blot and immunofluorescence analyses showed that SHE suppressed p65 nuclear translocation induced by LPS. Furthermore, SHE inhibited the reactive oxygen species in LPS-treated RAW 264.7 cells. SHE significantly increased heme oxygenase-1 expression and the nuclear translocation of nuclear factor erythroid 2-related factor 2. SHE suppressed LPS-induced interleukin-1β mRNA expression in RAW 264.7 cells. Thus, SHE is a promising nutraceutical as it displays anti-inflammatory and antioxidant properties.

Protective effects of selenium in tacrolimus-induced lung toxicity: potential role of heme oxygenase 1.

The present study aimed to evaluate the protective effects of selenium (Sel) administration against tacrolimus (Tac) - induced lung toxicity and to assess the relation between heme oxygenase 1 (HO-1) and these effects. The study was conducted on 36 Wistar male albino rats equally divided into four groups: (i) normal control; (ii) Sel (0.1 mg/kg per day p.o. for four weeks); (iii) TAC 3 mg/mL as single oral dose on 27th day; and (iv) Tac + Sel. Lung tissues, lung homogenate, and bronchoalveolar lavage of the sacrificed animals were investigated biochemically and histopathologically, by immunohistochemistry or by PCR. The Tac group showed significantly lower expression of HO-1. Administration of Sel was associated with increased HO-1 expression. Oxidative (malondialdehyde, reduced glutathione, superoxide dismutase, myeloperoxidase, and glutathione peroxidase activity) and nitrosative stress (nitric oxide) markers and markers of inflammation (interleukin 1β (IL-1β), IL-6, and IL-10) showed changes corresponding to HO-1 levels in rat groups. Tac group showed the highest expression of caspase-3. Sel exerted a protective role against Tac-induced lung toxicity.