Heme Oxygenase-1 Protein Expression Research Articles

Exploration about the protection mechanism of 5-hydroxy-1-methylhydantoin on paraquat poisoning model

To investigate the effects of 5-hydroxy-1-methylhydantoin (HMH) on kidney injury induced by paraquat (PQ). Fifteen SPF healthy Kunming mice were randomly divided into normal saline (NS) control group, PQ poisoning model group and HMH intervention group, with 5 mice in each group. PQ poisoning model was challenged by one-time gavage of 30 mg/kg PQ solution. The NS group received the same amount of NS by gavage. The HMH group was given 100 mg/kg of HMH immediately after the model was made and continued to be gavaged. Mice in each group were sacrificed 1 day after HMH gavage and heart blood and renal tissue were harvested for examination. The morphological changes of renal tissue were observed under light microscope by hematoxylin-eosin (HE) staining. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in renal tissue were detected according to the instructions of the kit. The expression of heme oxygenase-1 (HO-1) and interleukin-1β (IL-1β) in renal tissues were detected by Western Blot. The serum metabolites were detected by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), the overall distribution of each sample was observed by principal component analysis (PCA), the accuracy of the model was evaluated by multidimensional analysis orthogonal partial least squares-discriminant analysis (OPLS-DA), and the difference metabolites were screened by variable importance in the projection (VIP) value > 1. Light microscopic observation showed that: glomerular structure in NS group was clear, there was no hyperemia and inflammatory cell infiltration in renal interstitium and blood vessels. In PQ group, some glomeruli atrophy and necrosis, capillary congestion in glomeruli, infiltration of inflammatory cells around glomeruli, swelling of renal tubular epithelial cells, slight stenosis of lumen, and occasional necrosis and exfoliation of epithelial cells occurred. The degree of kidney injury in HMH group was significantly less than that in PQ group. Compared with the NS group, the content of MDA in the PQ group was significantly increased (nmol/g: 6.70±0.84 vs. 2.70±0.43, P < 0.01) and the activity of SOD was significantly decreased (kU/L: 33.30±4.66 vs. 50.20±3.23, P < 0.05), the protein expression of HO-1 and IL-1β were significantly increased (HO-1/β-actin: 1.11±0.12 vs. 0.61±0.13, IL-1β/β-actin: 0.93±0.13 vs. 0.32±0.06, both P < 0.05). Compared with the PQ group, the content of MDA in the HMH group was significantly decreased (nmol/g: 5.10±0.93 vs. 6.70±0.84, P < 0.05) and the activity of SOD was significantly increased (kU/L: 61.00±9.02 vs. 33.30±4.66, P < 0.05), the protein expression of HO-1 was significantly decreased (HO-1/β-actin: 0.77±0.07 vs. 1.11±0.12, P < 0.05), however, there was no significant difference in the protein expression of IL-1β (IL-1β/β-actin: 0.87±0.13 vs. 0.93±0.13, P > 0.05). Metabolite detection results showed that: compared with NS group, the levels of creatinine, glycine, succinic acid, fumaric acid and citric acid were significantly increased in the PQ group (VIP value was 1.50, 1.58, 1.64, 1.74 and 1.95 respectively, all P < 0.05), while the levels of palmitic acid,α-tocopherol and 6-phosphogluconic acid were significantly decreased (VIP value was 1.10, 1.55 and 1.56 respectively, all P < 0.05). Compared with the PQ group, the levels of creatinine and citric acid were significantly decreased in the HMH group (VIP value was 1.50 and 1.86, both P < 0.05), while trans-4-hydroxy-proline, D-glyceric acid, 2, 6-fructose phosphate, 6-phosphate gluconic acid and aminomalonic acid were significantly increased (VIP value was 1.36, 1.55, 1.63, 1.68 and 1.76 respectively, all P < 0.05). HMH protects kidney injury caused by PQ poisoning by correcting tricarboxylic acids cycle disturbance, lipid peroxidation and energy metabolism disturbance, and its mechanism is related to the regulation of HO-1 protein expression through Nrf2 pathway.

A SIRT1 agonist reduces cognitive decline in type 2 diabetic rats through antioxidative and anti‑inflammatory mechanisms.

Sirtuin 1 (SIRT1) is an NAD+-dependent protein deacetylase that is involved in cell differentiation, aging, apoptosis, physiological rhythms, metabolic regulation, oxidative stress and numerous other important biological processes. In the present study, the ability of a sirtuin-1 (SIRT1) agonist, SRT1720, to reduce cognitive decline in type 2 diabetes mellitus (T2DM) was investigated. Streptozotocin-induced male Sprague-Dawley rats were used to establish a T2DM model and the protective effect of SRT1720 and its underlying mechanisms were investigated. Body weight and fasting blood glucose (FBG) were recorded and cognitive function was measured with the Morris water maze. Levels of oxidative stress, inflammation, caspase-3 activity and nuclear factor κB (NF-κB) mRNA expression were detected with a series of commercial assay kits and reverse transcription-quantitative polymerase chain reaction, respectively. Western blot analysis was performed to determine the protein expression of NF-κB, endothelial nitric oxide synthase (eNOS), peroxisome proliferator-activated receptor γ (PPARγ), AMP-activated protein kinase (AMPK), heat shock 70 kDa protein (HSP70), SIRT1, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). The results revealed that SRT1720 significantly increased body weight, decreased FBG, improved cognitive function and reduced the levels of proteins associated with oxidative stress and inflammation damage in T2DM rats. Additionally, SRT1720 significantly decreased NF-κB p65 mRNA expression and increased eNOS and PPARγ expression. SRT1720 significantly reduced caspase-3 activity and HSP70 protein expression, and increased p-AMPK, SIRT1, Nrf2 and HO-1 protein expression. Collectively, the results indicate that SRT1720 may reduce cognitive decline in T2DM rats through antioxidative and anti-inflammatory action via NF-κB and AMPK-dependent mechanisms.

Curcumin improves exercise performance of mice with coronary artery ligation-induced HFrEF: Nrf2 and antioxidant mechanisms in skeletal muscle.

A hallmark of chronic heart failure (HF) with low ejection fraction (HFrEF) is exercise intolerance. We hypothesized that reduced expression of nuclear factor E2-related factor 2 (Nrf2) in skeletal muscle contributes to impaired exercise performance. We further hypothesized that curcumin, a Nrf2 activator, would preserve or increase exercise capacity in HF. Experiments were carried out in mice with coronary artery ligation-induced HFrEF. Curcumin was deliveried by a subcutaneous osmotic minipump at a dose of 50 mg·kg-1·day-1 for 8 weeks. In vivo, in situ, and in vitro experiments were employed to evaluate exercise capacity, muscle function, and molecular mechanisms. We found that: 1) the maximal speed, running distance to exhaustion, and limb grip force were significantly lower in HFrEF mice compared with sham. Curcumin-treated HF mice displayed enhanced exercise performance compared with vehicle-treated HF mice; 2) both soleus (Sol) and extensor digitorum longus (EDL) muscles of HFrEF mice exhibited reduced force and rapid fatigue, which were ameliorated by curcumin; and 3) protein expression of Nrf2, hemeoxygenase-1, SOD2, myogenin, and MyoD were significantly lower, but total ubiquitinated proteins, MURF1, and atrogen-1 were higher in Sol and EDL of HFrEF compared with sham mice, whereas these alterations in Nrf2 signaling and antioxidant defenses in HFrEF were attenuated by curcumin, which had no effect on cardiac function per se in mice with severe HFrEF. These data suggest that impaired Nrf2 signaling intrinsic to skeletal muscle contributes to exercise intolerance in HFrEF. Skeletal muscle Nrf2 should be considered as a novel therapeutic target in severe HF. NEW & NOTEWORTHY These studies suggest that impaired nuclear factor E2-related factor 2 (Nrf2) signaling is a critical mechanism underlying the enhanced oxidative stress in skeletal muscle in heart failure with low ejection fraction (HFrEF). Curcumin prevents the decline in running performance in HFrEF mice by upregulating antioxidant defenses in skeletal muscle, likely mediated by activating Nrf2 signaling. These findings suggest a novel therapeutic target for the improvement of exercise capacity and quality of life in HFrEF patients.

The role of heme oxygenase-1 on oxidative stress injury induced by zinc oxide nanoparticles in human umbilical vein endothelial cells line EA.hy926 cells

Objective: To explore the effect of heme oxygenase-1 (HO-1) on level of reactive oxygen species (ROS) induced by zinc oxide nanoparticles (ZnO-NPs) in Human umbilical vein endothelial cells line EA.hy926. Methods: The EA.hy926 cells in logarithmic growth phase were incubated with 0.0, 2.5, 5.0, 10.0 and 15.0 mg/L ZnO-NPs respectively. The ROS level, reflected by mean fluorescence intensity (MFI), was examined by flow cytometer after 4 hours exposure, the protein expression of HO-1 which was determined by Western Blot after exposed to ZnO-NPs for 24 hours. Cells incubated with 15.0 mg/L were set as the ZnO-NPs group; a blank control group was set at the same time. Cells were pretreated with HO-1 inhibitor zinc protoporphyrin (ZnPPIx) and HO-1 activator cobalt protoporphyrin (CoPPIx), they were classified as ZnPPIx group and CoPPIx group. 15 mg/L ZnO-NPs was chosen to conduct the experiment of HO-1 activation and inhibition. Cells were classified as ZnPPIX+ ZnO-NPs group and CoPPIx+ ZnO-NPs group after pretreated with 10 μmol/L ZnPPIx or CoPPIx for 1 h, added 15 mg/L ZnO-NPs to cell culture medium. In all groups ROS levels were detected after exposed to ZnO-NPs for 4 hours, the protein expression of HO-1 was detected after exposed to ZnO-NPs for 24 hours. Results: With the increased dose of ZnO-NPs, levels of ROS and HO-1 in EA.hy926 cells were clearly elevated (the MFI of 0.0, 2.5, 5.0, 10.0 and 15.0 mg/L ZnO-NPs incubated groups was 22 627.22±718.27, 24 726.47±568.52, 31 141.75±1 312.24, 39 824.82±4 774.74, 50 569.03±1 497.63 respectively, and HO-1 relative expression were 0.16±0.01, 0.19±0.02, 0.16±0.01, 0.23±0.02, 0.92±0.06 respectively). HO-1 expression in ZnPPIx pretreatment group decreased compared with ZnO-NPs group (1.05±0.05 vs. 1.12±0.01, P<0.05), meanwhile ROS level enhanced (62 683.95±2 589.59 vs. 53 654.53±2 229.01, P<0.05). However, CoPPIx pretreatment had higher HO-1 level and lower level of ROS compared with ZnO-NPs group (HO-1: 1.74±0.11 vs. 0.22±0.03, P<0.05; ROS: 32 845.04±993.48 vs. 53 654.53±2 229.01, P<0.05). Conclusions: Exposure to ZnO-NPs significantly induced ROS generation in EA.hy926 cells in a dose-dependent manner. HO-1 regulated ZnO-NPs-induced oxidative stress.

5-Aminolevulinic acid with ferrous iron improves early renal damage and hepatic steatosis in high fat diet-induced obese mice.

5-Aminolevulinic acid, a natural amino acid, activates mitochondrial respiration and induces heme oxygenase-1 expression. Obesity and type 2 diabetes mellitus are associated with age-related mitochondrial respiration defect, oxidative stress and inflammation. The aim of this study is to investigate the effects of 5-aminolevulinic acid with sodium ferrous citrate on early renal damage and hepatic steatosis. 7-Month-old C57BL/6 mice were fed with a standard diet or high fat diet for 9 weeks, which were orally administered 300 mg/kg 5-aminolevulinic acid combined with 47 mg/kg sodium ferrous citrate (5-aminolevulinic acid/sodium ferrous citrate) or vehicle for the last 5 weeks. We observed that 5-aminolevulinic acid/sodium ferrous citrate significantly decreased body weight, fat weight, hepatic lipid deposits and improved levels of blood glucose and oral glucose tolerance test. In addition, 5-aminolevulinic acid/sodium ferrous citrate suppressed increased glomerular tuft area in high fat diet-fed mice, which was associated with increased heme oxygenase-1 protein expression. Our findings demonstrate additional evidence that 5-aminolevulinic acid/sodium ferrous citrate could improve glucose and lipid metabolism in diabetic mice. 5-Aminolevulinic acid/sodium ferrous citrate has potential application in obesity or type 2 diabetes mellitus-associated disease such as diabetic nephropathy and nonalcoholic fatty liver disease.

Inhibition of lipopolysaccharide-induced inflammation in RAW264.7 macrophages by sinomenine through regulating heme oxygenase-1 expression and autophagy

To investigate the effect of sinomenine on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophages and the underlying mechanisms. Methods: The mouse RAW264.7 macrophages were treated with sinomenine and/or LPS with or without heme oxygenase-1 (HO-1) inhibitor Znpp. Real-time PCR, ELISA, immunofluenscence, and Western blot were used to detect the mRNA expression of TNF-α and IL-6, the release of TNF-α and IL-6, the protein expression of HO-1 and autophagy, respectively. Results: Compared with the control group, the mRNA expression and release of inflammatory cytokines TNF-α and IL-6 were increased, the green fluorescence of autophagy-related protein LC3 was accumulated and the protein expression of HO-1 was increased in RAW264.7 cells after LPS treatment (P<0.05). Compared with the LPS group, sinomenine treatment could reduce the mRNA expression and release of TNF-α and IL-6, accompanied by increasess in green fluorescence aggregation of LC3 and HO-1 production (P<0.05). HO-1 inhibitor Znpp could weaken the ability of sinomenine through suppressing TNF-α and IL-6 expression and decreasing the aggregation of LC3 green fluorescence (P<0.05). Conclusion: Sinomenine could alleviate LPS-induced inflammation in RAW264.7 macrophages, which might be related to HO-1 mediated autophagy. This study provides an experimental and theoretical basis for the clinical application of sinomenine in prevention and treatment of inflammation.

Hepatoprotective Effect of Ugonin M, A Helminthostachyszeylanica Constituent, on Acetaminophen-Induced Acute Liver Injury in Mice.

The present study aimed to discover the possible effectiveness of Ugonin M, a unique flavonoid isolated from Helminthostachys zeylanica—a traditional Chinese medicine used as anti-inflammatory medicine—and to elucidate the potential mechanisms of Ugonin M in the acute liver injury induced by acetaminophen (APAP). In this study, Ugonin M significantly ameliorated APAP-induced histopathological changes and the typical liver function biomarkers (i.e., alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (T-Bil)). It also affected APAP-induced abnormal lipid metabolism including total cholesterol (TC) and triglyceride (TG) in the serum. In inflammatory pharmacological action, Ugonin M suppressed the pro-inflammatory mediators such as nitric oxide (NO) and the lipid peroxidation indicator malondialdehyde (MDA). In addition, Ugonin M reinforced hemeoxygenase-1 (HO-1) protein expression and the production of antioxidant enzymes viz superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). Furthermore, inflammation-associated cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β as well as proteins such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were decreased by the pretreatment of Ugonin M. Moreover, this study found that pretreatment of Ugonin M apparently decreased nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) activation via inhibition of the degradation of NF-κB, inhibitory κB-α (IκB-α), extracellular regulated kinase (ERK), c-Jun-N-terminal (JNK), and p38 active phosphorylation. In conclusion, Ugonin M significantly showed a protective effect against APAP-induced liver injury by reducing oxidative stress and inflammation. Thus, Ugonin M could be one of the effective components of H. zeylanica that plays a major role in the treatment of inflammatory disorders.

Study of antioxidant effect of cannabinoid receptor type 2 agonist on rat hepatic stellate cell line

Objective: To investigate the action and antioxidant effects of CB2 agonist AM-1241 on rat hepatic stellate cell line (HSC-T6). Methods: HSC-T6 was randomly divided into four groups: control group, oxidative stress group, AM-1241 intervention group and AM-1241+AM-630 antagonist group. Survival rate of HSC-T6 was detected by thiazolyl blue assay under 24 h interventions with 0, 20, 50, 80 μmol/L AM-1241 and 0, 10, 20, 30, 40 μmol/L AM-630, respectively. Besides control group, the remaining groups were well cultured in low-glucose DMEM containing 100 mU/L glucose oxidase (GO) for 12 h to prepare the oxidative stress model. Then, AM-1241 intervention group was treated with 50 μmol/L low-glucose DMEM medium. After incubation for 12 h, the AM-1241+AM-630 antagonist group was treated with CB2 antagonist AM-630 (20 μmol/L) for 2 h, and cultured with 50 μmol/L AM-1241 in complete low-glucose medium for 12 h. The optimal drug concentration was selected according to the cell viability considered by the experiment results. Type III collagen (C III) content in the HSC-T6 supernatant was detected by enzyme-linked immunosorbent assay. Glutathione (GSH) content in HSC-T6 was detected by spectrophotometry. CB2 and heme oxygenase-1(HO-1) in each group of HSC-T6 were detected by western blotting. Results: HSC-T6 proliferation was inhibited in each group of AM-1241 in a concentration-dependent manner (P < 0.05). The inhibition was highest at 80μmol/L, and the cell survival rate was (41.61% ± 3.13%) (P < 0.05). AM-630 concentration group had no significant inhibitory effect on the proliferation of HSC-T6 (P > 0.05). HSC-T6 expressed CB2 receptor in each group. The expression level of CB2 in the AM-1241 intervention group was higher compare with control group (P < 0.05).The expression of Col III were significantly higher in oxidative stress group (P < 0.05) than in control group, and the expression of Col III of AM-1241 intervention group was significantly lower than that in oxidative stress group (P < 0.05). Col III level in AM-1241+AM-630 antagonistic group was significantly higher than that in AM-1241 intervention group (P < 0.05). There was no significant difference between AM-1241+AM-630 antagonistic group and oxidative stress group (P > 0.05). The content of GSH and HO-1 in oxidative stress group was higher (P < 0.05) than control group. The content of GSH and HO-1 in the AM-1241 intervention group was higher compared with oxidative stress group, while content of AM-1241 + AM-630 antagonist group was lower compared to AM-1241 intervention group (P < 0.05), and the differences were not statistically significant for oxidative stress group. Conclusion: CB2 agonist AM-1241 can inhibit the proliferation and activation of HSC-T6 and its mechanism may activate the nuclear translocation of Nrf2 binding to HSC-T6, initiating the up-regulation of antioxidant enzymes HO-1 and GSH protein expression, and thus increase the antioxidant effect of HSC-T6.

Role of Oleanolic acid in maintaining BBB integrity by targeting p38MAPK/VEGF/Src signaling pathway in rat model of subarachnoid hemorrhage

Vasogenic brain edema after subarachnoid hemorrhage (SAH) is an independent risk factor for death and poor prognosis. Disruption of the blood-brain barrier (BBB) is the main cause of vasogenic brain edema induced by SAH. Oleanolic acid (OA) is a natural pentacyclic triterpenoid with various biological functions. Previous studies have shown that prophylactic administration of OA could prevent the BBB disruption in autoimmune encephalomyelitis mice. In this context, we speculate that OA may play a neuroprotective role by protecting the integrity of the BBB and reducing vasogenic cerebral edema after SAH. To validate this hypothesis, a SAH model was established on Sprague Dawley rats using a standard intravascular puncture model. The effects of OA on various physiological indexes were observed, including SAH grades, mortality, neurological function score, brain edema and BBB permeability. Related proteins of the brain endothelial cell junction complex were also detected, including tight junctions (TJs) and adherent junctions (AJs). Results showed that OA significantly reduced the permeability of BBB and relieved brain edema by increasing protein expression of TJs and AJs, and decreased the SAH grades by increasing the protein expression of heme oxygenase-1 (HO-1) in SAH rats. Additionally, we found OA could inhibit up-regulation of VEGF and the phosphorylation of p38 mitogen-activated protein kinase (MAPK), and suppress p38MAPK/VEGF/Src signaling pathway which involved in BBB disruption following SAH. From the experimental results, we speculate that OA effectively alleviated SAH-induced vasogenic edema by targeting p38 MAPK/VEGF/Src axis.

Desoxo-narchinol A and Narchinol B Isolated from Nardostachys jatamansi Exert Anti-neuroinflammatory Effects by Up-regulating of Nuclear Transcription Factor Erythroid-2-Related Factor 2/Heme Oxygenase-1 Signaling.

We previously reported that desoxo-narchinol A and narchinol B from Nardostachys jatamansi DC (Valerianaceae) inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2), and the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 protein in lipopolysaccharide (LPS)-stimulated BV2 and primary microglial cells. In this study, we aimed to elucidate the molecular mechanism underlying the anti-neuroinflammatory effects of desoxo-narchinol A and narchinol B. These two compounds inhibited the nuclear factor (NF)-κB pathway, by repressing the phosphorylation and degradation of inhibitor kappa B (IκB)-α, nuclear translocation of the p65/p50 heterodimer, and DNA-binding activity of the p65 subunit. Furthermore, both compounds induced heme oxygenase-1 (HO-1) protein expression, which was mediated by the activation of nuclear transcription factor erythroid-2-related factor 2 (Nrf2). Activation of the Nrf2/HO-1 pathway by desoxo-narchinol A was shown to be regulated by increased phosphorylation of p38 and extracellular signal-regulated kinase (ERK), whereas only p38 was involved in narchinol B-induced activation of the Nrf2/HO-1 pathway. In addition, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling was also involved in the activation of HO-1 by desoxo-narchinol A and narchinol B. These compounds also increased the phosphorylation of glycogen synthase kinase 3 beta (GSK3β) at serine-9 residue, following phosphorylation of Akt. The anti-neuroinflammatory effect of desoxo-narchinol A and narchinol B was partially blocked by a selective HO-1 inhibitor, suggesting that this effect is partly mediated by HO-1 induction. In addition, both compounds also induced HO-1 protein expression in rat-derived primary microglial cells, which was correlated with their anti-neuroinflammatory effects in LPS-stimulated primary microglial cells. In conclusion, desoxo-narchinol A and narchinol B are potential candidates for the development of preventive agents for the regulation of neuroinflammation in neurodegenerative diseases.

Chlorogenic acid ameliorates experimental colitis in mice by suppressing signaling pathways involved in inflammatory response and apoptosis

Chlorogenic acid (ChA) exhibits a multitude of positive health effects, however, the signaling mechanisms by which ChA could influence the inflammatory response in experimental colitis are unknown. To answer this question, we induced colitis in mice by administration of 2.5% dextran sulfate sodium (DSS) in drinking water for seven days. Oral administration of ChA significantly ameliorated clinical symptoms, improved disease activity index and colon shortening induced by DSS. Furthermore, ChA administration resulted in a suppression of phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases 1 and 2 (JNK1/2), Akt and signal transducer and activator of transcription 3 (STAT3) with concomitant upregulation of phosphatase and tensin homolog (PTEN) expression. Immunohistochemical analysis showed a dose-dependent decrease in expression and nuclear translocation of nuclear factor-kappa B (NF-κB) p65 subunit, which was accompanied by suppression of pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) expression. Induction of apoptosis and oxidative stress was attenuated in a dose-dependent manner by suppressing Bax, caspase-8, caspase-9 and heme oxygenase-1 (HO-1) protein expression in mice administrated with ChA. The results of the current study suggest that ChA could be useful for the treatment of inflammation and attenuating colitis severity by suppressing activation of pro-inflammatory and apoptotic signaling pathways.

Ferulic acid protects against heat stress-induced intestinal epithelial barrier dysfunction in IEC-6 cells via the PI3K/Akt-mediated Nrf2/HO-1 signaling pathway

Purpose: Intestinal epithelial barrier dysfunction is crucial in the pathogenesis of intestinal and systemic diseases. Ferulic acid (FA) possesses promising antioxidant activities. In a previous study, we demonstrated potentially protective effects of FA against heat stress-induced intestinal epithelial barrier dysfunction in IEC-6 cells. However, the underlying mechanisms are unclear. The present study aimed to elucidate whether FA protects IEC-6 cells from heat stress-induced intestinal epithelial barrier dysfunction via antioxidative mechanisms.Materials and methods: IEC-6 cells were pretreated with FA prior to hyperthermia exposure at 42 °C for 6 h, and the levels of intracellular reactive oxygen species (ROS), malondialdehyde (MDA), nitrogen oxide (NO), and superoxide dismutase (SOD) activity were analyzed. The intestinal epithelial barrier function was determined by transepithelial electrical resistance (TER) values and 4-kDa fluorescein isothiocyanate-dextran (FD4) flux in IEC-6 cell monolayers. Expression of related proteins was detected by Western blotting.Results: FA suppressed heat stress-induced intestinal oxidative stress damage by reducing ROS, MDA and NO production, while enhancing SOD activity. Furthermore, FA treatment strengthened intestinal barrier function via increasing the phosphorylation levels of Akt, nuclear factor-erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1) protein expression, which was reversed by zinc protoporphyrin (an HO-1 inhibitor). Additionally, LY294002, a specific PI3K/Akt inhibitor, significantly suppressed FA-induced Nrf2 nuclear translocation and HO-1 protein expression and inhibited FA-induced occludin and ZO-1 protein expression.Conclusions: FA protected against heat stress-induced intestinal epithelial barrier dysfunction via activating the PI3K/Akt-mediated Nrf2/HO-1 signaling pathway in IEC-6 cells.

Orientin-mediated Nrf2/HO-1 signal alleviates H2O2-induced oxidative damage via induction of JNK and PI3K/AKT activation

Oxidative stress is closely associated with the pathogenesis of various diseases. Orientin (Ori), a flavonoid component isolated from natural plants, possesses antioxidant activity. Accordingly, we focused on exploring the potential therapeutic effects of Ori on hydrogen peroxide (H2O2)-induced oxidative impairment in RAW 264.7 cells and the underlying antioxidative mechanisms. Our findings suggested that Ori exposure effectively alleviated H2O2-stimulated cytotoxicity, inhibited reactive oxygen species (ROS) generation, and glutathione (GSH) depletion, which were involved in induction of heme oxygenase-1 (HO-1) by enhancing the nuclear factor-erythroid 2-related factor 2 (Nrf2) translocation, decreasing the Keap1 protein expression, and increasing the antioxidant response element (ARE) activity. However, knockdown of Nrf2 and HO-1 with siRNA mostly abolished the cytoprotective effects against H2O2-induced cell oxidative injury, reduced the expression of Nrf2 and HO-1, respectively. Moreover, Ori exposure significantly induced a c-Jun NH2-terminal kinase (JNK) and phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (AKT) phosphorylation, but JNK and PI3K/AKT inhibitors treatment effectively reduced levels of Ori-enhanced Nrf2 nuclear translocation and HO-1 protein expression, and blocked Ori-inhibited cytotoxicity and ROS accumulation triggered by H2O2 respectively. Taken together, Ori might exhibit a protective role against H2O2-stimulated oxidative damage by the induction of HO-1 expression through the activation of the JNK- and PI3K/AKT-Nrf2 signaling pathways.

Canagliflozin Inhibits Human Endothelial Cell Inflammation via the Induction of Heme Oxygenase-1

Vascular disease is the major cause of mortality in diabetes. Although many factors contribute to diabetes-associated vascular disease, inflammation of endothelial cells (ECs) plays a critical role. Sodium-glucose transporter 2 (SGLT2) inhibitors are recently approved anti-hyperglycemic drugs that cause a significant reduction in cardiovascular events in type 2 diabetic patients, but the mechanisms underlying this protection are not fully known. In the present study, we investigated the effect of SGLT2 inhibitors on the expression of the vasoprotective protein heme oxygenase-1 (HO-1) in human umbilical vein ECs. Treatment of ECs with canagliflozin, dapagliflozin, or empagliflozin stimulated the expression of HO-1 mRNA and protein; however, only clinically achievable concentrations of canagliflozin (3-10µM) increased HO-1 levels. The induction of HO-1 by canagliflozin was associated with an increase in Nrf2 activity and reactive oxygen species (ROS) formation. Canagliflozin also stimulated HO-1 promoter activity, and this was prevented by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. In addition, the canagliflozin-mediated induction of HO-1 was abolished by N-acetyl-L-cysteine or rotenone. Significantly, prior treatment of ECs with canagliflozin inhibited the induction of the adhesion molecules ICAM-1 and VCAM-1 in response to high concentrations of glucose (25mM) and oxidized low-density lipoprotein (50mg/L) that mimic the diabetic milieu. Significantly, the anti-inflammatory action of canagliflozin was abrogated by blocking HO-1 activity or expression. In conclusion, this study demonstrates that canagliflozin stimulates HO-1 gene expression in the vascular endothelium via the ROS-Nrf2 pathway, and that canagliflozin mitigates inflammation in human ECs exposed to a diabetic environment via the induction of HO-1. Thus, canagliflozin may exert some of it cardiovascular benefit in diabetes by targeting HO-1. Disclosure K.J. Peyton: None. G. Behnammanesh: None. W. Durante: None.

Effect of penehychdine hydrochloride pretreatment on Nrf2/ARE signaling pathway during myocardial ischemia-reperfusion in rats

Objective To evaluate the effect of penehychdine hydrochloride pretreatment on nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway during myocardial ischemia-reperfusion(I/R)in rats. Methods Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 2-3 months, weighing 220-240 g, were divided into 3 groups (n=12 each) using a random number table method: sham operation group (group S), myocardial I/R group and penehyelidine hydrochloride pretreatment group (group PHC). Myocardial I/R was induced by occlusion of the anterior descending branch of left coronary artery for 30 min followed by 120 min reperfusion.At 30 min before ischemia, penehyelidine hydrochloride 2 mg/kg was injected intraperitoneally in group PHC, and the anterior descending branch of left coronary artery was only exposed but not ligated in group S. Rats were sacrificed at the end of reperfusion, and hearts were removed for measurement of the myocardial infarct size(by 2, 3, 5-triphenyltetrazolium chloride staining), cell apoptosis (by TUNEL) and expression of Nrf2, heme oxygenase-1(HO-1), NQO1 and γ-glutamylcysteine synthetase(γ-GCS) protein and mRNA (by using Western blot or real-time fluorescence quantitative polymerase chain reaction). The percentage of myocardial infarct size and apoptosis index were calculated. Results Compared with group S, the percentage of myocardial infarct size and apoptosis index were significantly increased, and the expression of Nrf2, HO-1, NQO1 and γ-GCS protein and mRNA was down-regulated in I/R and PHC groups (P<0.05). Compared with group I/R, the percentage of myocardial infarct size and apoptosis index were significantly decreased, and the expression of Nrf2, HO-1, NQO1 and γ-GCS protein and mRNA was up-regulated in group PHC (P<0.05). Conclusion Penehyclidine hydrochloride pretreatment attenuates myocardial I/R injury through activating Nrf2-ARE signaling pathway in rats. Key words: Cholinergic antagonists; Myocardial reperfusion injury; Nuclear factor-E2 related factor 2; Response elements; Signal transduction

MIF protects against oxygen-glucose deprivation-induced ototoxicity in HEI-OC1 cochlear cells by enhancement of Akt-Nrf2-HO-1 pathway

Ischemia and oxidative stress play crucial roles in the pathophysiology of sudden sensorineural hearing loss (SSNHL). Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine and serves an important role in hearing function. The present study was designed to evaluate the effect of MIF on oxygen-glucose deprivation (OGD)-induced ototoxicity and to elucidate its molecular mechanism. In HEI-OC1 auditory cells, OGD reduced cell viability and increased supernatant lactate dehydrogenase (LDH) and MIF in a time-dependent manner. However, the reduced cell viability exerted by OGD was attenuated by antioxidant and MIF. Luciferase reporter assay demonstrated that MIF could activate NF-E2-related factor 2 (Nrf2), and real-time PCR showed increased mRNA expressions of Nrf2 and two Nrf2-responsive genes, including heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). MIF also suppressed oxidative stress induced by OGD, as demonstrated by decreased MDA and increased GSH in cellular supernatant. Inhibition of Nrf2 using siRNA suppressed HO-1 protein expression, the protective effect on OGD-induced injury and decrease in oxidative stress by MIF. Moreover, MIF prevented OGD-induced reduction of Akt1 phosphorylation at Ser473. LY294002, an inhibitor of PI3K/Akt signaling, attenuated the enhancement of Nrf2 protein and protective effect of MIF in OGD-treated cochlear cells. We demonstrate that MIF protects cochlear cells against OGD-induced injury through activation of Akt-Nrf2-HO-1 pathway.

Fibroblast growth factor 19 protects the heart from oxidative stress-induced diabetic cardiomyopathy via activation of AMPK/Nrf2/HO-1 pathway

Diabetes affects cardiac structure and function, where it leads to diabetic cardiomyopathy. Reactive oxygen species (ROS) produced by oxidative stress play an important role in the development of diabetic cardiomyopathy. Fibroblast growth factor (FGF) 19, an enterokine, is synthesized and released into the ileum. In the present study, we revealed that FGF19 induced an antioxidant response through stimulating the expression of nuclear erythroid factor 2 (NE-F2)-related factor 2 (Nrf2) and as well as reducing ROS production through the AMPK signaling pathway. Additionally, AMPK inhibition by the AMPK-specific inhibitor compound C decreased Nrf2 and heme oxygenase-1 (HO-1) protein expression. Taken together, these results suggested that FGF19, through the anti-oxidative defense system, attenuated the development of diabetic cardiomyopathy and restored cardiac function.

Heme oxygenase-1 exerts pro-apoptotic effects on hepatic stellate cells in vitro through regulation of nuclear factor-κB.

Heme oxygenase-1 (HO-1) is an antioxidant and cytoprotective protein, which has been proven to alleviate the proliferation of hepatic stellate cells (HSCs) and the development of liver fibrosis. However, the role of HO-1 in HSC apoptosis remains unclear. The aim of the present study was to investigate the effect of HO-1 on HSC apoptosis and its possible underlying mechanisms. HSCs-T6 were incubated with different concentrations of hemin (HO-1 chemical inducer) and Znpp-IX (HO-1 chemical inhibitor) for 12, 24 and 48 h. Cell viability was determined using an MTT assay. HSCs were classified into 4 groups as follows: Control, hemin, Znpp-IX and hemin+Znpp-IX co-treatment groups. Apoptosis was quantitatively measured by Annexin V/propidium iodide double staining and a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The mRNA and protein expression of HO-1, α-smooth muscle actin, B-cell lymphoma (Bcl)-2, caspase-3 and nuclear factor (NF)-κB p65 were measured using quantitative polymerase chain reaction and western blotting. The levels of tumor growth factor (TGF)-β and interleukin (IL)-6 in HSC supernatants were examined by ELISA. The results demonstrated that HO-1 exerted antiproliferative effects on HSCs in a time- and concentration-dependent manner. Increasing HO-1 expression induced HSC apoptosis in vitro as demonstrated by a significant decrease in Bcl-2 and an increase in caspase-3 expression. Additionally, the expression of NF-κB p65 and its downstream inflammatory factors TGF-β and IL-6 in the HO-1 overexpression group was significantly decreased compared with the control group. Therefore, the present study provided evidence that HO-1 serves an anti-fibrosis role in the liver by enhancing HSC apoptosis, which was partially associated with the regulation of NF-κB and its downstream effectors.

DOR agonist (SNC-80) exhibits anti-parkinsonian effect via downregulating UPR/oxidative stress signals and inflammatory response in vivo

The pathophysiology of Parkinson’s disease exhibit imperative roles in unfolded protein response stress-induced oxidative stress and inflammation in general. Although, delta opioid receptor (DOR), has been found to represent anti-parkinsonian effect at behavioral level, its underlying mechanism remains elusive till date. In the present study the role of DOR agonist, SNC-80 and the consorted molecular mechanisms, which translates to behavioral recuperation, has been delineated. In order to mimic PD, mice were intra-peritoneally injected with MPTP, following exposure to SNC-80 and L-DOPA to elucidate amelioration of the MPTP-induced behavioral impairments. The results obtained suggest that the severity of the compromised motor functions up-regulated the UPR stress sensors: IRE-1α/Bip/CHOP, oxidative stress along with the pro-inflammatory cytokines: IL1β/IFNγ/TNFα and IL-6. These inimical factors combined, aids the persistence of the disease in MPTP intoxicated mice. Supplementation with SNC-80 significantly improved motor functions via down-regulation of the UPR stress sensors and inflammatory cytokines. Additionally, SNC-80 could upregulate Nrf-2 and Heme oxygenase-1 (HO-1) protein expression indicating their involvement in SNC-80′s potential anti-oxidant function. There was also a significant reduction in protein carbonyl content indicating the positive role of SNC-80 in dampening MPTP induced oxidative stress. Concomitantly, L-DOPA also demonstrated an enhanced effect towards improvement of motor functions but did not suppress the UPR and inflammatory responses caused due to MPTP intoxication. Hence, these results suggest that SNC-80 could hold a pivotal role in replenishing motor functions essentially via regulating UPR and inflammation.

SB202190 inhibits endothelial cell apoptosis via induction of autophagy and heme oxygenase-1.

Activation of the p38 mitogen-activated protein kinase (MAPK) pathway has been implicated in various detrimental events finally leading to endothelial dysfunction. The present study therefore investigates the impact of the p38 MAPK inhibitor SB202190 on the expression of the cytoprotective enzyme heme oxygenase-1 (HO-1) as well as metabolic activity, apoptosis and autophagy of endothelial cells. Using human umbilical vein endothelial cells (HUVEC) SB202190 was found to cause a time- and concentration-dependent induction of HO-1 protein. Induction of HO-1 protein expression was mimicked by SB203580, another p38 MAPK inhibitor, but not by SB202474, an inactive structural analogue of p38 MAPK inhibitors. HO-1 induction by both SB202190 and SB203580 was also demonstrated by analysis of mRNA expression. On the functional level, SB202190 was shown to increase metabolic activity and autophagy of HUVEC along with diminishing basal apoptosis. Treatment of cells with tin protoporphyrin IX (SnPPIX), a well-characterised HO-1 enzymatic inhibitor, or HO-1 siRNA left SB202190-modulated metabolic activity and autophagy virtually unaltered but caused a significant reversal of the anti-apoptotic action of SB202190. Conversely, however, HO-1 expression by SB202190 became completely suppressed by the autophagy inhibitor bafilomycin A1. Bafilomycin A1 likewise fully reversed effects of SB202190 on metabolic activity and apoptosis, albeit significantly inducing apoptosis per se. Collectively, this work demonstrates SB202190 to confer upstream induction of autophagy followed by HO-1 induction resulting in potential protective effects against apoptosis. On the other hand, our data oppose HO-1 to contribute to SB202190-mediated increases in metabolic activity and autophagy, respectively.