Protein Expression Levels Of Heme Oxygenase-1 Research Articles

Interventional effect of bone marrow mesenchymal stem cell transplantation with different doses of X-ray irradiation induced hepatic injury in mice

Objective: To investigate the interventional effect of bone marrow mesenchymal stem cell (BMMSC) transplantation with different doses of X-ray irradiation induced hepatic injury in mice. Methods: Eighteen female C57BL/6J mice were randomly divided into 0, 2, and 3 Gy irradiation groups and 0, 2, and 3 Gy transplantation groups. The irradiation group was used as the control and injected with an equal volume of culture medium. The mice in the transplantation group were irradiated with different doses of X-ray irradiation, and BMMSCs were intravenously infused into the bone marrow. The mice were sacrificed for sampling at the end of the 21st day. Mice body weight changes were recorded daily. The changes in the content of peripheral blood lymphocytes, red blood cells, platelets, and hemoglobin were detected by an automatic blood tester. The morphological changes in mice liver tissues were observed by hematoxylin-eosin staining. The serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by a biochemical analyzer. The reduced glutathione contents in liver tissue were detected by the microplate method. The malondialdehyde content in liver tissue was detected by thiobarbituric acid. The content of total superoxide dismutase (T-SOD) in liver tissue was detected by the hydroxylamine method. The expression of the F4/80 protein in liver tissue was detected by the immunohistochemistry method. The protein expression of nuclear transcription factor erythroid 2 related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in liver tissue was determined by the western blotting method. The mRNA expression of NLRP3, IL-6, and Nrf2 in liver tissue was detected by a real-time quantitative polymerase chain reaction. The multiple-group comparisons were analyzed by factorial analysis of variance. The inter-group comparisons were analyzed by the LSD method for statistical analysis. Results: The contents of peripheral blood lymphocytes, erythrocytes, platelets, and hemoglobin were significantly decreased in the 3 Gy irradiation group than the 0 Gy irradiation group (P<0.05), while the activities of serum ALT and AST were significantly increased (P<0.05). The malondialdehyde content, F4/80 protein expression level, nucleotide-binding domain and leucine-rich repeats, nucleotide oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), and interleukin 6 mRNA expression levels were significantly increased in liver tissue, while the contents of T-SOD and glutathione, Nrf2 and HO-1 protein expression levels, and Nrf2 mRNA expression level in liver tissue were significantly decreased (P<0.05). The contents of peripheral blood lymphocytes, red blood cells, platelets, and hemoglobin were significantly increased in the 3 Gy transplantation group than the 3 Gy irradiation group (P<0.05), while the activities of serum ALT and AST were significantly decreased (P<0.05). The malondialdehyde content, F4/80 protein expression level, NLRP3 and interleukin-6 mRNA expression levels in liver tissue were significantly decreased (P<0.05), while the content of T-SOD and glutathione, Nrf2 and HO-1 protein expression levels, and Nrf2 mRNA expression level in liver tissue were significantly increased (P<0.05). Conclusion: X-ray irradiation at a dose of 3 Gy can induce liver oxidative damage in mice. BMMSC transplantation can improve X-ray irradiation-induced liver oxidative damage in mice, and its mechanism of action may be related to the regulation of the Nrf2/HO-1 pathway.

Effect of "Tiaoshen Tongluo" acupuncture on nerve function injury, muscle tension and neurotransmitters through Nrf2/ROS pathway in spastic rats after stroke

To observe the effect of "Tiaoshen Tongluo" acupuncture (TTA) at "Dingzhongxian" (MS5) and right "Dingpangxian" (MS8) on neurological injury, muscle tension and neurotransmitters through nuclear transcription factor E2 related factor 2 (Nrf2)/reactive oxygen species (ROS) signaling pathway in spastic rats after stroke, so as to explore its mechanisms underlying relief of post-stroke spasm (PSS). A total of 90 male SD rats were randomly divided into 6 groups, i.e. sham operation, PSS model, medication, non-acupoint acupuncture, TTA, TTA+ML385 groups, with 15 rats in each group. The PSS model was established by middle cerebral artery occlusion. After modeling, rats of the medication group were treated by gavage of baclofen (0.4 mg/kg), once daily for 7 days. For rats of the non-acupoint acupuncture group, the spot about 10 mm above the iliac crest and below the armpit of the affected side was needled, and for those of the TTA group and TTA+ML385 group, EA stimulation (1 mA, 2 Hz/15 Hz) was applied to MS5 and right MS8 for 10 min, once daily for 7 consecutive days. Intraperitoneal injection of ML385 ［ a specific nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor, 30 mg/kg］ was given to rats of the TTA+ML385 group before TTA was performed. The rats' neurological deficit score (0-4 points) was evaluated by referring to Zea Longa's methods and the muscular spasm degree of the quadriceps femoris of the left hindlimb (0-4 points) assessed by using Ashworth scale (MAS). The muscular tension of the left quadriceps femoris was measured by using a tension sensor, and Hoffman (H)-reflex response and M and H waves of electromyogram of the muscle between the metatarsals of the left foot were measured using an electrophysiological recorder. The cerebral infarction volume was measured after 2,3,5-triphenyltetrazolium chloride (TTC) staining. The contents of γ-aminobutyric acid (GABA), glycine (Gly), glutamic acid (Glu) and aspartic acid (Asp) of the right cortical infarct area were detected by using high performance capillary electrophoresis, and the contents of 5-hydroxytryptamine (5-HT), dopamine (DA) and norepinephrine (NE) were detected by fluorescence spectrophoto-metry, as well as the level of ROS in the right cerebral cortical infarction tissues was detected by dihydroethidium staining. The expression levels of Nrf2 and heme oxygenase-1 (HO-1) proteins in the infarcted cerebral area were detected using Western blot. Compared with the sham operation group, the neurological deficit score, MAS score, percentage of cerebral infarction volume, Hmax/Mmax ratio, contents of Glu and Asp and ROS level were significantly increased (P<0.001), whereas the muscle tone, stimulation threshold for inducing H-reflex, GABA, Gly, 5-HT, DA and NE contents, cerebral Nrf2 and HO-1 protein expression levels were apparently decreased (P<0.001) in the model group. In comparison with the model group, the neurological deficit score, MAS score, percentage of cerebral infarction volume, Hmax/Mmax ratio, contents of Glu, Asp and ROS levels were decreased (P<0.001), and the muscle tone, stimulation threshold for inducing H-reflex, GABA, Gly, 5-HT, DA and NE contents, Nrf2 and HO-1 protein expressions were increased (P<0.001, P<0.01) in both the medication and TTA groups. No significant differences were found between the non-acupoint group and model group, and between the medication and TTA groups in all the indexes mentioned above (P>0.05). After administration of ML385, the effects of TTA in reducing neurological deficit score, MAS score, Hmax/Mmax, percentage of cerebral infarct volume, Glu, Asp, ROS, and up-regulating H-reflex threshold, GABA, Gly, 5-HT, DA, NE, Nrf2 and HO-1 levels were eliminated (P<0.001，P<0.05,P<0.01). TTA can improve neurological behavior and muscle spasm in rats with PSS, which may be associated with its functions in regulating the levels of neurotransmitters in the cortical infarcted area by activating the Nrf2/ROS signaling pathway.

Methylmercury-induced reactive oxygen species-dependent and independent dysregulation of MAP kinase-related signaling pathway in cultured normal rat cerebellar astrocytes

Methylmercury (MeHg), a global environmental pollutant, could seriously damage the central nervous system (CNS) and cause neurological disorders such as cerebellar symptoms. Although numerous studies have revealed detailed toxicity mechanisms of MeHg in neurons, toxicity in astrocytes is barely known. Here, we tried to shed light on the toxicity mechanisms of MeHg exposure in cultured normal rat cerebellar astrocytes (NRA), focusing on the involvement of reactive oxygen species (ROS) in MeHg toxicity by assessing the effects of major antioxidants Trolox, a free-radical scavenger, N-acetyl-L-cysteine (NAC), a potent thiol-containing antioxidant, and glutathione (GSH), an endogenous thiol-containing antioxidant. Exposure to MeHg at just approximately 2 µM for 96 h increased cell viability, which was accompanied by the increase in intracellular ROS level and at ≥ 5 µM induced significant cell death and lowered ROS level. Trolox and NAC suppressed 2 µM MeHg-induced increases in cell viability and ROS level corresponding to control, although GSH with 2 µM MeHg induced significant cell death and ROS increase. On the contrary, against 4 µM MeHg-induced cell loss and ROS decrease, NAC inhibited both cell loss and ROS decrease, Trolox inhibited cell loss and further enhanced ROS decrease, and GSH moderately inhibited cell loss and increased ROS level above the control level. MeHg-induced oxidative stress was suggested by increases in the protein expression levels of heme oxygenase-1 (HO-1), Hsp70, and Nrf2, except for the decrease in SOD-1 and no change in catalase. Furthermore, MeHg exposure dose-dependently induced increases in the phosphorylation of MAP kinases (ERK1/2, p38MAPK, and SAPK/JNK) and phosphorylation and/or expression levels of transcription factors (CREB, c-Jun, and c-Fos) in NRA. NAC successfully suppressed 2 µM MeHg-induced alterations in all of the above-mentioned MeHg-responsive factors, whereas Trolox suppressed some MeHg-responsive factors but failed to suppress MeHg-induced increases in the protein expression levels of HO-1 and Hsp70 and increase in p38MAPK phosphorylation. Protein expression analyses in NRA exposed to 2 µM MeHg and GSH were excluded because of devastating cell death. These results suggested that MeHg could induce aberrant NRA activation, and ROS must be substantially involved in the toxicity mechanism of MeHg in NRA; however, other factors should be assumed.

The relationship between nuclear factor-erythrocyte-related factor 2 and antioxidant enzymes in the placenta of patients with gestational diabetes and the metabolism of umbilical cord endothelial cells.

The paper aimed to explore the relationship between nuclear factor erythroid 2 related factor 2 (Nrf2), antioxidant enzymes and the metabolism of umbilical cord endothelial cells in the placenta of patients with gestational diabetes (GDM). A total of 200 pregnant women who underwent an obstetric examination at the Municipal Maternity and Child Health Hospital from March 2018 to December 2019 were selected, with an average age of 30.91±3.24. According to the plasma glucose level and oral glucose tolerance test (OGTT), pregnant women were divided into a control group and an observation group. Blood samples were collected from these pregnant women, serum was removed, put into a centrifuge tube and stored in the refrigerator of the laboratory at - 80 °C. Placental tissue was collected for biochemical analysis. GSH level was detected by absorbance kit, and serum MDA content was detected by spectrophotometry. The expression levels of Heme oxygenase-1(HO-1), Nrf2, and NQO1 protein in placental tissue were analyzed by gel electrophoresis. Western blot analysis was used to detect the protein expression levels of Bach1 and Keap1 in endothelial cells. PCR real-time analysis was used to detect the expression of GSH and NQO1 mRNA. Results showed that the SOD and GSH levels in the serum of the observation group were lower than those of the control group (P<0.05). The protein expression levels of HO-1, Nrf2 and NQO1 in the observation group were higher than those in the control group (P<0.05). The GSH level of the HNE+ observation group was lower than that of the HNE+ control group before stimulation (P<0.05). 1.5 hours after the stimulation, the GSH levels of the two groups of cells were decreased. After 6 hours, the GSH levels of the two groups began to increase. The GSH level of HUVEC in the HNE+ observation group was lower than that of the HNE+ control group after 48 hours. The expression level of Bach1 protein in the HNE+ observation group was lower than that in the HNE+ control group (P<0.05). The expression level of Keap1 protein in the HNE+ observation group and HNE+ control group did not change (P>0.05). The expression levels of GSH and NQO1 mRNA in the HNE+Nrf2 silence group were lower than that in the Nrf2 silence group (P<0.05). The expression levels of GSH and NQO1 mRNA in the HNE+Nrf2 overexpression observation group were higher than those of the HNE+Nrf2 silence group (P<0.05). The apoptosis of trophoblast in the placenta of GDM patients was significantly decreased. The continuous lack of redox signals in fetal endothelial cells in patients with gestational diabetes can destroy the defense ability of cells in the uterus against oxidative stress. Nrf2 antioxidant defense pathway can provide therapeutic targets for reducing oxidative stress associated with diabetes and aging.

Protective Effects of Scutellarin on Acute Alcohol Intestinal Injury.

The present study aims to investigate the roles of scutellarin (SCU) on acute alcohol intestinal injury. Mice were divided into six groups: alcohol, three administration, negative control and positive drug bifendate control. The administration group mice were intraperitoneally injected with SCU for 3 consecutive days followed by alcohol gavage at an interval of 1 h. After the mice were sacrificed, colon tissue damage was evaluated by histopathological examination; the activities of inducible nitric oxide synthase (iNOS) and catalase (CAT), as well as the content of malondialdehyde (MDA) were detected using biochemical kits; the levels of inflammatory cytokines mRNA were determined by real-time fluorescence quantitative PCR; the protein expression levels of hemeoxygenase-1 (HO-1) and phosphorylated nuclear factor-ĸB p65 were measured via western blotting. The results showed that alcohol induced severe colon morphological degradation, epithelia atrophy, and more inflammatory cells infiltration in the submucosa. SCU treatment prevented this process, especially in the middle and high dose groups. Alcohol treatment caused excessive lipid peroxidation product accumulation of MDA, restrained the activity of antioxidant enzyme CAT, induced HO-1 expression in the colon, whereas low dose SCU treatment significantly down-regulated the MDA level, enhanced the CAT level, and accelerated HO-1 signals. SCU prevented alcohol stimulation triggered inflammatory response in colon tissues through significantly downregulating the iNOS activity, transcript levels of Tnf-α, Il-1β and Il-6, and phosphorylation levels of NF-κB p65. These findings suggest that SCU protects the colon via antioxidant and anti-inflammatory mechanisms, making it a promising drug against alcohol-induced colon damage.

Layer-specific strain for assessing the effect of naringin on systolic myocardial dysfunction induced by sepsis and its underlying mechanisms.

ObjectiveThis study aimed to investigate the protective effects of naringin on myocardial deformation and oxidative responses in rats with sepsis-induced myocardial dysfunction (SIMD).MethodsGlobal and segmental layer-specific longitudinal strain (LS) was assessed by speckle tracking echocardiography. Serum levels of creatine kinase, lactate dehydrogenase, superoxide dismutase, and malondialdehyde were measured. The activity of cleaved caspase-3 was determined by immunohistochemistry. Protein expression levels of Kelch-like ECH-related protein 1 (Keap1), nuclear erythroid factor 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) were measured by western blotting.ResultsNaringin inhibited the lipopolysaccharide-induced decrease in global and layer-specific LS of the left ventricle. Naringin also increased superoxide dismutase expression and decreased malondialdehyde, creatine kinase, lactate dehydrogenase, and cleaved caspase-3 expression in rats with SIMD. Furthermore, naringin increased Nrf2 and HO-1 protein expression levels, and decreased Keap1 protein expression levels in rats with SIMD.ConclusionLayer-specific LS analysis of myocardial function by speckle tracking echocardiography can reflect early changes in myocardial systolic function. Naringin may possess a protective effect through moderating lipopolysaccharide-induced myocardial oxidative stress via the Keap1/Nrf2/HO-1 pathway in rats with SIMD.

Possible mechanism of heme oxygenase-1 expression in rat malignant meningioma KMY-J cells subjected to talaporfin sodium-mediated photodynamic therapy.

We previously demonstrated that heme oxygenase-1 (HO-1) induction may contribute to a protective response against photodynamic therapy (PDT) using talaporfin sodium (TS) in rat malignant meningioma KMY-J cells. In the present study, we examined the mechanism of HO-1 induction by PDT with TS (TS-PDT) in KMY-J cells. KMY-J cells were incubated with 25 μM TS for 2 h and then exposed to 664 nm diode laser irradiation at 1 J/cm2. The gene and protein expression levels of HO-1 and hypoxia-inducible factor-1α (HIF-1α) were determined by real-time RT-PCR and western blot analysis, respectively. Cell viability was measured using the cell counting kit-8 assay. mRNA and protein levels of HO-1 in KMY-J cells were increased significantly at 3, 6, and 9 h after laser irradiation and the increased mRNA level of HO-1 was decreased by antioxidant N-acetyl cysteine treatment. The protein level of HIF-1α, which mediates transcriptional activation of the HO-1 gene, was increased significantly at 1 h after laser irradiation. Additionally, induction of mRNA expression of HO-1 by TS-PDT was diminished by HIF-1α inhibitor echinomycin. We also demonstrated that echinomycin significantly augmented the cytotoxic effect of TS-PDT. Our findings indicate that TS-PDT may induce HO-1 expression via reactive oxygen species production and then HIF-1 pathway activation in KMY-J cells, and the HO-1 induction may cause attenuation of the therapeutic effect of TS-PDT.

TSG-6 Attenuates Oxidative Stress-Induced Early Brain Injury in Subarachnoid Hemorrhage Partly by the HO-1 and Nox2 Pathways

BackgroundEarly brain injury (EBI) refers to acute brain injury during the first 72 h after subarachnoid hemorrhage (SAH), which is one of the major causes of poor prognosis after SAH. Here, we investigated the effect and the related mechanism of TSG-6 on EBI after SAH. Materials and methodsThe Sprague-Dawley rat model of SAH was developed by the endovascular perforation method. TSG-6 (5μg) was administered by an intraventricular injection within 1.5 h after SAH. The effects of TSG-6 on EBI were assessed by neurological score, brain water content (BWC) and TUNEL staining. Immunofluorescence staining was used to assay NF-κB/p-NF-κB expression in microglia. Protein expression levels of heme oxygenase-1 (HO-1), NADPH oxidase 2 (Nox2), Bcl-2, Bax, and cleaved-caspase-3 were measured to investigate the potential mechanism. The enzyme activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the level of reactive oxygen species (ROS) were analyzed using commercially available kits. ResultsThe results showed that TSG-6 treatment alleviated the neurobehavioral dysfunction and reduced BWC and the number of TUNEL-positive neurons in EBI after SAH. TSG-6 decreased the ROS level and enhanced the enzyme activity of SOD and GSH-Px after SAH. Furthermore TSG-6 inhibited the NF-κB activation, increased the protein expression levels of HO-1 and Bcl-2 and decreased the expression levels of Nox2, Bax, and cleaved-caspase-3. The administration of TSG-6 siRNA abolished the protective effects of TSG-6 on EBI after SAH. ConclusionWe found that TSG-6 attenuated oxidative stress and apoptosis in EBI after SAH partly by inhibiting NF-κB and activating HO-1 pathway in brain tissue.

Otoprotective Effects of Zingerone on Cisplatin-Induced Ototoxicity.

Previous studies have described the effects of zingerone (ZO) on cisplatin (CXP)-induced injury to the kidneys, liver, and other organs but not to the cochlea. This study aimed to investigate the effects of ZO on CXP-induced ototoxicity. Eight-week-old Sprague–Dawley rats were used and divided into a control group, a CXP group, and a CXP + ZO group. Rats in the CXP group received 5 mg/kg/day CXP intraperitoneally for five days. Rats in the CXP + ZO group received 5 mg/kg/day CXP intraperitoneally for five days and 50 mg/kg/day ZO intraperitoneally for seven days. Auditory brainstem response thresholds (ABRTs) were measured before (day 0) and after (day 10) drug administration. Cochlear histology was examined using hematoxylin and eosin (H&E) staining and cochlear whole mounts. The expression levels of cytochrome P450 (CYP)1A1, CYP1B1, inducible nitric oxide synthase (iNOS), nuclear factor kappa B (NFκB), tumor necrosis factor alpha (TNFα), and interleukin 6 (IL6) were estimated using quantitative reverse transcription-polymerase chain reaction. The expression levels of heme oxygenase 1 (HO1) and caspase 3 were analyzed via Western blotting. The auditory thresholds at 4, 8, and 16 kHz were attenuated in the CXP + ZO group compared with the CXP group. The mRNA expression levels of CYP1A1, CYP1B1, iNOS, NFκB, TNFα, and IL6 were lower in the CXP + ZO group than in the CXP group. The protein expression levels of HO1 and caspase 3 were lower in the CXP + ZO group than in the CXP group. Cotreatment with ZO exerted otoprotective effects against CXP-induced cochlear injury via antioxidative and anti-inflammatory activities involving CYPs, iNOS, NFκB, and TNFα.

Achyranthes bidentata polypeptide k suppresses neuroinflammation in BV2 microglia through Nrf2-dependent mechanism.

Activated microglia play a critical role in regulating neuroinflammatory responses in central nervous system. Previous studies have shown that Achyranthes bidentata polypeptide k's (ABPPk's) neuroprotective effects are partly due to its anti-inflammatory effect, but the mechanism remains unknown. This study is aimed to investigate the anti-inflammatory effect of ABPPk on lipopolysaccharide (LPS)-activated neuroinflammation in BV2 microglia. We pretreated BV2 microglia with different concentrations of ABPPk (0.04-5 µg/mL) for 30 minutes, and then stimulated microglia with LPS for 24 hours. Pro-inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nitric oxide (NO) and prostaglandin E2 (PGE2) production were measured by enzyme-linked immunosorbent assay (ELISA) kits. Inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), phosphorylated nuclear factor kappa B (NF-κB), heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) expression levels were detected by western blot. Glutathione (GSH) level was measured by GSH-Glo™ Glutathione assay. Immunofluorescent staining was used to detect the nuclear translocation of NF-κB and Nrf2. BV2 microglia transfected with Nrf2 siRNA were used to investigate the effect of Nrf2 on the anti-inflammatory activity of ABPPk. ABPPk (0.2-5 µg/mL) reduced the iNOS mediated NO and COX-2 mediated PGE2 production significantly in LPS-activated BV2 microglia. ABPPk (1 and 5 µg/mL) also suppressed the production of TNF-α and IL-6 significantly. NF-κB is phosphorylated and translocated into nuclear in LPS-activated BV2 microglia, but ABPPk is shown to inhibit the phosphorylation and translocation of NF-κB in a concentration-dependent way. ABPPk increased the protein expression levels of HO-1 and Nrf2, as well as the GSH content in BV2 microglia. Immunofluorescent staining showed that ABPPk also promoted nuclear translocation of Nrf2. After knocking down Nrf2 in BV2 cells with siRNA interference, ABPPk's inhibitory effect on pro-inflammatory mediators also disappeared. The present study suggests that ABPPk inhibits neuroinflammation in BV2 microglia through Nrf2-dependent mechanism. This provides some strong evidence for the potential of this neuroprotective natural compound to treat neurodegenerative diseases such as ischemic stroke and Parkinson's disease.

Effect of estradiol on high glucose‑induced osteoblast injury.

Estradiol (E2) serves an important role in the changes of postmenopausal bone turnover rate and the development of osteoporosis. The present study aimed to investigate the effects of E2 on high glucose (HG)-induced osteoblast injury. Cell Counting Kit-8 was used to determine cell viability. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting was used to analyze the mRNA and protein expression levels of osteocalcin, Runt-related transcription factor 2 (Runx2), nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO1). Flow cytometry was performed to analyze apoptosis. The results revealed that cell viability was lower in cells treated with HG (100, 200 or 300 mg/dl) compared with the control group. Cell viability was decreased in cells treated with 200 mg/dl HG on days 3, 5 and 7. In addition, cell viability was increased by 0.1 µM E2. E2 with HG co-treatment increased cell viability, osteocalcin and Runx2 mRNA expression levels and nuclear Nrf2 and HO1 protein expression levels compared with the HG-only group. All these changes, with the exception of Runx2, were reversed by silencing Nrf2 expression using small interfering (si)RNA (siNrf2). Additionally, apoptosis was reduced by E2 in HG-treated cells, which was reversed by siNrf2 transfection. These results demonstrated that E2 may prevent HG-induced osteoblast injury by activating Nrf2/HO1 signaling pathways.

Quercus ilex Extract Ameliorates Acute TNBS-Induced Colitis in Rats.

Quercus ilex L. (Fagaceae) is one of the most commonly used plants in folk medicine in the Mediterranean region to treat gastrointestinal disorders. The aim of the present study was to evaluate the effects of a polyphenol extract from mature leaves of Q. ilex on acute 2,4,6-trinitrobenzene sulfonic acid-induced colitis in rats. A polyphenol extract from mature leaves of Q. ilex (250 and 500 mg/kg/day) was administered by gavage 48, 24, and 1 h prior to the induction of colitis with 2,4,6-trinitrobenzene sulfonic acid as well as 24 h later. The inflammation response was assessed by histology, myeloperoxidase activity, and Th1 proinflammatory cytokine production. The protein expression of cyclooxygenase-2 and inducible nitric oxide synthase, and signaling pathways were studied by Western blotting in the colon tissues. The polyphenol extract from mature leaves of Q. ilex decreased neutrophil infiltration, interleukin-1β and TNF-α production, and proinflammatory proteins cyclooxygenase-2 and inducible nitric oxide synthase overexpression. Also, the polyphenol extract from mature leaves of Q. ilex was capable of blocking the activation of mitogen-activated protein kinases and nuclear transcription factor-kappa B. Furthermore, the reduction of inflammation by polyphenol extract from mature leaves of Q. ilex treatment was accompanied by a recovery of Nrf2 and heme oxygenase-1 protein expression levels. In conclusion, this study demonstrates that a polyphenol extract from mature leaves of Q.ilex improves 2,4,6-trinitrobenzene sulfonic acid-induced colitis, probably through mitogen-activated protein kinase/nuclear transcription factor-kappa B inhibition and Nrf2/heme oxygenase-1 activation signaling pathways, thus reducing the production of Th1 proinflammatory cytokines and cyclooxygenase-2 and inducible nitric oxide synthase overexpression.

Effects of Four Compounds from Gentianella acuta (Michx.) Hulten on Hydrogen Peroxide-Induced Injury in H9c2 Cells.

In previous studies, Gentianella acuta (Michx.) Hulten was reported to contain xanthones, iridoids, terpenoids, and sterols and is mainly used to cure hepatitis, jaundice, fever, headache, and angina pectoris. In this study, we used bioassay guided fractionation to identify compounds from G. acuta and investigated their activity against hydrogen peroxide (H2O2)-induced apoptosis of H9c2 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutamate-cysteine ligase catalytic (GCLC) expression were assessed using quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was evaluated using western blot. The results showed that all four compounds had protective effects on H9c2 cells. The transcription levels of HO-1 and GCLC significantly increased in H9c2 cells pretreated with norswertianolin (1), swetrianolin (2), demethylbellidifolin (3), and bellidifolin (4). However, compared to the model group, the transcription levels of Nrf2 were not enhanced by pretreatment with compounds 1, 2, and 4. The protein expression levels of HO-1 and GCLC in H9c2 cells were greater than that in the H2O2-treated group, and the expression of Nrf2 was not significantly changed except by swetrianolin treatment; inhibitors can reverse the protective effect by ZnPP (15 μM), BSO (10 μM), and brusatol (10 μM). The results indicated that the four compounds isolated from G. acuta inhibited the oxidative injury induced by H2O2 by activating the Nrf2/ARE pathway in H9c2 cells and provide evidence that G. acuta may be a potential therapeutic agent for the treatment of cardiovascular diseases.

Biliverdin protects the isolated lungs from ischemia/reperfusion injury via anti-apoptosis

To observe the effect of biliverdin (BV) on the lung ischemia/reperfusion injury (LIRI), and to investigate the mechanism of BV in treatment of LIRI. Thirty-two male Sprague-Dawley (SD) rats were randomly divided into control group, LIRI group, glutathione (GSH) group and BV-treated group, with 8 rats in each group. The rat LIRI model was reproduced by isolated lung perfusion system. Fifteen minutes after perfusion balance, lungs of control group were perfused continuously for 90 minutes, and the lungs in other groups were reperfused for 90 minutes after 1-hour ischemia, while the perfusion was added 10 μmol/L BV in BV-treated group and 4 mmol/L GSH in GSH group respectively. During the perfusion, the respiratory related indicators, such as tidal volume (VT), static compliance (Cst), arterial partial pressure of oxygen (PaO2), airway resistance (Raw), were dynamically observed. After perfusion, the wet/dry weight ratio (W/D) of lungs was determined. Pathological changes in lung tissue were observed under light microscope. The cell apoptosis was observed by TdT-mediated dUTP nick end labeling (TUNEL) method, and the apoptosis index was calculated. The protein expression levels of heme oxygenase-1 (HO-1), phosphorylated c-Jun N-terminal kinase (p-JNK), and caspase-3 were determined by Western Blot. Compared with control group, VT, Cst and PaO2 in LIRI group were significantly decreased from reperfusion for 30 minutes, and Raw was significantly increased. The pathological results showed that there was different degree of hyperemia edema, inflammatory cells infiltration and bronchial endothelium injury in the lung tissue of LIRI group. The W/D ratio of LIRI group was significantly higher than that of control group (8.98±2.34 vs. 5.89±0.52, P < 0.05). TUNEL results showed that the tan apoptosis cells of LIRI group were more than control group, and the apoptosis index was significantly higher than that of the control group [(13.88±2.35)% vs. (2.26±0.60)%, P < 0.05]. Compared with control group, the protein expression levels of HO-1, p-JNK, and caspase-3 in LIRI group were significantly increased [HO-1 (gray value): 0.55±0.13 vs. 0.16±0.02, p-JNK (gray value): 0.46±0.08 vs. 0.16±0.05, caspase-3 (gray value): 0.65±0.13 vs. 0.26±0.03, all P < 0.05]. Compared with LIRI group, the respiratory indicators in BV-treated group were improved significantly, the lung tissue injury was significantly reduced, the W/D ratio was significantly decreased (6.39±0.45 vs. 8.98±2.34, P < 0.05), and the cell apoptosis index was significantly reduced [(4.49±1.10)% vs. (13.88±2.35)%, P < 0.05], as well as the protein expression levels of HO-1, p-JNK, and caspase-3 were significantly lowered [HO-1 (gray value): 0.19±0.03 vs. 0.55±0.13, p-JNK (gray value): 0.31±0.06 vs. 0.46±0.08, caspase-3 (gray value): 0.33±0.05 vs. 0.65±0.13, all P < 0.05], which indicating that BV could alleviate LIRI via anti-apoptosis. The improvement effect of BV on PaO2, apoptosis index, protein expressions of HO-1 and caspase-3, and JNK phosphorylation were better than positive drug GSH, indicating that BV could protect LIRI ideally. BV alleviates LIRI via its anti-JNK pathway and its anti-apoptosis property.

Induction of docosahexaenoic acid to expression of heme oxygenase-1 in retinal pigment epithelial cells

Background Oxydative stress is an important pathogenesis of age-related macular degeneration.Resent evidences indicate that docosahexaenoic acid (DHA) plays an important role during the development of retinal photoreceptor cells and protect the cells against oxydative stress by inducing the expression of heme oxygenase-1 (HO-1). However, whether DHA can induce the expression of HO-1 in human retinal pigment epithelium (RPE) cells is unelucidated. Objective This study was to investigate the effect of DHA on the expression of HO-1 in RPE cells and its molecular mechanism. Methods Human RPE cell line ARPE-19 was cultured in vitro and treated with 30, 50, 100 and 120 μmol/L DHA for 4 to 24 hours, respectively, and the cells were cultured without DHA as the control group.The cytotoxicity of DHA was detected by lactate dehydrogenase(LDH), and the expression of HO-1 mRNA and protein were detected by real-time PCR and Western blot assay, respectively.The enzymatic activity of HO-1 was detected by colorimetry.The reactive oxygen species (ROS) proportion in the cells was detected using fluorescence probe H2DCFDA, and immunofluorescence technology was adopted to detect the nuclear translocation of nuclear facotor-E2-related factor 2 (Nrf2). The expression of Nrf2 protein in the cells was detected by Western blot after intervention of ROS inhibitor N-acetylcysteine (NAC) and transfection of Nrf2 small interfering RNA (siRNA). Results The LDH leakage rate was significantly different after 0, 3, 50, 100 and 120 μmol/L DHA treated the cells for 24 hours (F=8.14, P<0.05), and the LDH leakage rate in the 120 μmol/L DHA group was significantly higher than that of 0, 30, 50 and 100 μmol/L DHA group (all at P<0.05). The relative expression levels of HO-1 mRNA and HO-1 protein or HO-1 enzymatic activity in the cells were significantly different among different concentrations of DHA group in 8 hours after treatment (F=16.24, P<0.05; F=11.34, P<0.05; F=11.81, P<0.05), and the expressions of these factors were considerably higher in the 30, 50 and 100 μmol/L DHA group than those in the 0 μmol/L DHA group (all at P<0.05). The ROS relative fluorescence intensity and nuclear Nrf2 positive cells proportion were statistically significant among different concentrations of DHA groups (F=11.08, P<0.05; F=16.42, P<0.05), and the ROS relative fluorescence intensity and nuclear Nrf2 positive cells proportion were evidently higher in the 30, 50 and 100 μmol/L DHA group than those in the 0 μmol/L DHA group (all at P<0.05). The relative expression levels of HO-1 protein and the proportion of nuclear Nrf2 positive cells were significantly lower in the NAC pretreated 100 μmol/L DHA group than those in the 100 μmol/L DHA group.In addition, the HO-1 relative expression level and the positive cells proportion of nuclear Nrf2 were significantly lower in the of Nrf2 siRNA transfection group than those in the blank siRNA transfection group (both at P<0.05). Conclusions DHA with concentration below 100 μmol/L can protect RPE cells from oxidative stress by inducting the expression of HO-1 in the cells via ROS/Nrf2 pathway. Key words: Antioxidants/pharmacology; Cytoprotection/drug effects; Oxidative stress; Pigment epithelium of eye/drug effects; Heme oxygenase-1; Nuclear factor-E2-related factor 2/metabolism; Reactive oxygen species/metabolism; Docosahexaenoic acid

Effect of oxidative stress on heme oxygenase-1 expression in patients with gestational diabetes mellitus

The anti-oxidative stress effect of heme oxygenase-1 (HO-1) expression is being increasingly studied. However, few studies regarding HO-1 have been conducted in patients with gestational diabetes mellitus (GDM). In the present study, HO-1 expression was compared in peripheral blood mononuclear cells at 24–28 weeks of pregnancy in patients with GDM and healthy females, to investigate the correlation between HO-1 and oxidative stress by calculation of MDA content in the peripheral blood serum (thiobarbituric acid method), tested ROS (flow cytometry method), HO-1mRNA (RT-PCR method), and HO-1 protein (western blotting method) of Mononuclear cells. The results show that the levels of serum malonaldehyde (MDA), reactive oxygen species (ROS), HO-1 mRNA and HO-1 protein expression in peripheral blood mononuclear cells were higher in the GDM group than in the control group. Correlation analysis showed that the expression levels of HO-1 protein were positively correlated with the HO-1 mRNA expression levels (r=0.680; P=0.000), and the levels of ROS (r=0.572; P=0.000) and MDA (r=0.614; P=0.000). HO-1 mRNA expression levels were found to positively correlate with the levels of MDA (r=0.451; P=0.010) and fasting plasma glucose (FPG; r=0.337; P=0.039). Partial correlation analysis demonstrated that, after removing the effects of body mass index, FPG and 2-h plasma glucose, HO-1 protein expression levels were positively correlated with the levels of HO-1 mRNA expression (r=0.611; P=0.005), ROS (r=0.526; P=0.021) and MDA (r=0.519; P=0.015). These findings indicate that pregnant females with GDM may be protected against oxidative injury due to the induction of adaptive and compensatory expression of HO-1 to guard against oxidative stress induced by high glucose levels.

Antioxidant N-Acetylcysteine Attenuates the Reduction of Brg1 Protein Expression in the Myocardium of Type 1 Diabetic Rats

Brahma-related gene 1 (Brg1) is a key gene in inducing the expression of important endogenous antioxidant enzymes, including heme oxygenase-1 (HO-1) which is central to cardioprotection, while cardiac HO-1 expression is reduced in diabetes. It is unknown whether or not cardiac Brg1 expression is reduced in diabetes. We hypothesize that cardiac Brg1 expression is reduced in diabetes which can be restored by antioxidant treatment with N-acetylcysteine (NAC). Control (C) and streptozotocin-induced diabetic (D) rats were treated with NAC in drinking water or placebo for 4 weeks. Plasma and cardiac free15-F2t-isoprostane in diabetic rats were increased, accompanied with increased plasma levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6), while cardiac Brg1, p-STAT3 and HO-1 protein expression levels were significantly decreased. Left ventricle weight/body weight ratio was higher, while the peak velocities of early (E) and late (A) flow ratio was lower in diabetic than in C rats. NAC normalized tissue and plasma levels of 15-F2t-isoprostane, significantly increased cardiac Brg1, HO-1 and p-STAT3 protein expression levels and reduced TNF-alpha and IL-6, resulting in improved cardiac function. In conclusion, myocardial Brg1 is reduced in diabetes and enhancement of cardiac Brg1 expression may represent a novel mechanism whereby NAC confers cardioprotection.

Melatonin synergistically increases resveratrol‐induced heme oxygenase‐1 expression through the inhibition of ubiquitin‐dependent proteasome pathway: a possible role in neuroprotection

Melatonin is an indoleamine secreted by the pineal gland as well as a plant-derived product, and resveratrol (RSV) is a naturally occurring polyphenol synthesized by a variety of plant species; both molecules act as a neuroprotector and antioxidant. Recent studies have demonstrated that RSV reduced the incidence of Alzheimer's disease and stroke, while melatonin supplementation was found to reduce the progression of the cognitive impairment in AD. The heme oxygenase-1 (HO-1) is an inducible and redox-regulated enzyme that provides tissue-specific antioxidant effects. We assessed whether the co-administration of melatonin and RSV shows synergistic effects in terms of their neuroprotective properties through HO-1. RSV significantly increased the expression levels of HO-1 protein in a concentration-dependent manner both in primary cortical neurons and in astrocytes, while melatonin per se did not. Melatonin + RSV showed a synergistic increase in the expression levels of HO-1 protein but not in the HO-1 mRNA level compared to either melatonin or RSV alone, which is mediated by the activation of PI3K-Akt pathway. Treatment of melatonin + RSV significantly attenuated the neurotoxicity induced by H(2) O(2) in primary cortical neurons and also in organotypic hippocampal slice culture. The blockade of HO-1 induction by shRNA attenuated HO-1 induction by melatonin + RSV and hindered the neuroprotective effects against oxidative stress induced by H(2) O(2) . The treatment of MG132 + RSV mimicked the effects of melatonin + RSV, and melatonin + RSV inhibited ubiquitination of HO-1. These data suggest that melatonin potentiates the neuroprotective effect of RSV against oxidative injury, by enhancing HO-1 induction through inhibiting ubiquitination-dependent proteasome pathway, which may provide an effective means to treat neurodegenerative disorders.

Vascular Heme Oxygenase-1 Induction Suppresses Tissue Factor and Plasminogen Activator Inhibitor-1 Expressions

Objective - Heme oxygenase-1(HO-1), the rate-limiting enzyme of heme degradation, has recently been considered to have protective roles against various pathological conditions. 10 years have passed since we lost the first and the only patient of HO-1 deficiency. Since the patient of HO-1 deficiency showed endothelial cell injury and extremely enhanced coagulation and fibrinolytic parameters, we examined the effect of HO-1 modulation on tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) expression on endothelial cells.Methods and Results -Human umbilical vein endothelial cell (HUVEC) was stimulated with hemin (100mM), HO-1 inducer, and mRNA and protein levels for HO-1, TF and PAI-1 were examined. Total RNA was extracted from HUVEC, and was analyzed by real time RT-PCR. Protein expression levels of HO-1, TF and PAI-1 were measured by ELISA. Hemin stimulation increased HO-1 mRNA levels by 20 times. On the other hand, TF mRNA and antigen levels were minimum even after 8 hours of stimulation. Importantly, hemin stimulation reduced PAI-1 mRNA more than half after 4 hours.After HO-1 induction by hemin (100 mM) for 6 hours, HUVEC cultures were exposed to 10 ng/ml tumor necrosis factor (TNF).Prior exposure to hemin significantly increased HO-1 mRNA by 60 times in 30 minutes after stimulation with TNF. However, TNF alone could not induce HO-1 mRNA and protein levels in HUVEC. Although stimulation with TNF enhanced expressions of both TF and PAI-1 mRNA, they were significantly inhibited more than half by prior treatment with hemin. TF antigen levels were similarly decreased (5.0 to 0.7 pg/ml). PAI-1 antigen levels were also inhibited by prior treatment with hemin (1.8 to 0.1 ng/ml)(3) To see if hemin effect on HUVEC is due to HO-1 production, HO-1 inhibitor tin-protoporphyrin IX (SnPP-IX) was added to the cultures. The inhibitor effect of hemin on TF and PAI-1 productions was cancelled when HUVEC was cocultured with SnPP-IX.Conclusions - These results indicate that hemin exert inhibitory effect on TF and PAI-1 expressions through HO-1 production. Induction of HO-1 may be beneficial in the prevention of thrombosis associated with inflammation.