Heme Oxygenase-1 mRNA Expression Levels Research Articles

Protective effects of chlorogenic acid against cyclophosphamide induced liver injury in mice

ABSTRACT We investigated possible protective effects of chlorogenic acid (CGA) against cyclophosphamide (CP) induced hepatic injury in mice. We measured aminotransferase alanine transaminase (ALT) and aspartate transaminase (AST) levels in the serum. We assayed catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in hepatic tissue. We assessed expression of nuclear transcription factor 2 (Nrf2) and Kelch sample related protein-1 (keap1) proteins in hepatic tissues using immunohistochemistry. The relative mRNA expression levels of heme oxygenase-1 (HO-1), NADH quinone oxidoreductase 1 (NQO1), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Hematoxylin & eosin staining was used to assess liver histopathology. We found that administration of CGA prior to induction of injury by CP decreased serum ALT, AST and MDA expressions in hepatic tissue, while CAT, SOD, GSH and GSH-Px concentrations were increased. We found that hepatocytes of animals administered CGA gradually returned to normal morphology. CGA increased the protein expression of Nrf2 in murine hepatic tissue. Administration of CGA up-regulated mRNA expression levels of HO-1, NQO1, TNF-α and IL-6 in hepatic tissue. CGA exhibited a marked protective effect on CP induced liver injury in mice.

Effects of Dietary Supplementation of Chinese Yam Polysaccharide on Carcass Composition, Meat Quality, and Antioxidant Capacity in Broilers.

The study aimed to evaluate the influences of the dietary supplementation of Chinese yam polysaccharide (CYP) on the carcass performance, antioxidant capacity, and meat quality of broilers. Three hundred and sixty healthy 1-day-old broilers with similar body weight (39 ± 1 g, gender balanced) were randomly divided into four groups (control, CYP1, CYP2, and CYP3 groups). In the control group, broilers were fed a basal diet with CYP, and the CYP1, CYP2, and CYP3 groups were fed diets supplemented with 250, 500, and 1000 mg/kg CYP, respectively. There were three replicates in each group, 30 birds in each replicate, and the feeding trial lasted for 48 days. Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) by one-way analysis of variance. The results showed that compared with the control group, dietary supplementation with 500 mg/kg CYP can improve live weight, half-eviscerated carcass percentage, eviscerated carcass percentage, and thigh muscle percentage. Moreover, dietary supplementation with 500 mg/kg CYP can improve the contents of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GPX), and glutathione s-transferase (GST) in serum (p < 0.05). Meanwhile, the mRNA expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), quinone oxidoreductase (NQO1), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), and catalase (CAT) in the liver; the mRNA expression levels of HO-1, NQO1, GPX1, and CAT in the breast muscle; and the mRNA expression levels of NQO1, SOD1, and CAT in the thigh muscle of broilers in the CYP2 group were significantly increased (p < 0.05). In addition, the yellowness and shear force of the thigh and breast muscles and the content of malondialdehyde (MDA) in the serum of broilers in the control group were higher than that in the CYP2 groups (p < 0.05). The results demonstrated that the CYP2 group had the best effect on improving meat quality. In conclusion, dietary supplementation with 500 mg/kg CYP can improve the meat quality of broilers by improving carcass quality, meat color, shear force, and antioxidant capacity.

9‑Hydroxy‑isoegomaketone inhibits LPS‑induced NO and inflammatory cytokine production in RAW264.7 cells.

Isoegomaketone (IK) is a known component of Perilla frutescens that reportedly exhibits anti-inflammatory, anti-cancer and anti-allergic properties. A novel compound known as 9-HIK has been isolated from the extract of a radiation mutant P. frutescens var. crispa using supercritical carbon dioxide. In the present study, 9-HIK induced heme oxygenase-1 (HO-1) mRNA expression in RAW264.7 cells, with maximal levels observed 4 h after 9-HIK treatment. In addition, 9-HIK inhibited the mRNA and protein expression of pro-inflammatory mediators, such as IL-6 and interferon-β, as well as the production of nitric oxide (NO) in lipopolysaccharide-stimulated RAW264.7 cells. Furthermore, N-acetyl-L-cysteine, a reactive oxygen species scavenger, inhibited NO production and HO-1 mRNA expression levels through the nuclear factor erythroid 2-related factor 2 pathway. Overall, 9-HIK displayed anti-inflammatory properties in LPS-induced RAW264.7 cells via direct suppression of inflammatory mediators and HO-1 induction.

Imatinib inhibits oxidative stress response in spinal cord injury rats by activating Nrf2/HO-1 signaling pathway.

Effect of imatinib on rats with spinal cord injury (SCI) was investigated through the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. Forty-eight Sprague-Dawley rats were randomly divided into sham operation group (n=12), model group (n=12), imatinib group (n=12) and inhibitor group (n=12). The results of immunohistochemistry showed that in comparison with sham operation group, the other three groups had overtly increased positive expression level of Bax and evidently reduced positive expression level of Bcl-2 (P<0.05). Compared with those in model group and inhibitor group, the positive expression level of Bax was distinctly lower, while that of Bcl-2 was notably increased in imatinib group (P<0.05). According to western blot analysis, the protein expression levels of Nrf2 and HO-1 were obviously higher in the other three groups than those in sham operation group (P<0.05), and they were remarkably higher in imatinib group than those in model group and inhibitor group (P<0.05). The results of qPCR assay revealed that the Nrf2 and HO-1 mRNA expression levels were markedly elevated in the other three groups compared with those in sham operation group (P<0.05). Based on ELISA, the other three groups exhibited notably raised content of IL-6, TNF-α, ROS and SOD compared with sham operation group (P<0.05), and imatinib group displayed remarkably decreased content of IL-6, TNF-α and ROS and markedly elevated SOD content in comparison with model group and inhibitor group (P<0.05). The results of TUNEL assay demonstrated that the rate of apoptosis was significantly raised in the other three groups compared with that in sham operation group (P<0.05), and it declined obviously in imatinib group compared with that in model group and inhibitor group (P<0.05). Imatinib inhibits oxidative stress response in SCI rats by activating the Nrf2/HO-1 signaling pathway, thus repressing apoptosis and inflammation.

Effects of heme oxygenase-1 knockdown on proliferation, invasion and metastasis of lung adenocarcinoma A549 cells and its mechanism

Objective: To investigate the effects of heme oxygenase-1 (HO-1) knockdown on proliferation, invasion and migration of lung adenocarcinoma A549 cells and explore the mechanism. Methods: The expression levels of HO-1 mRNA in human bronchial epithelial cells (HBECs) and human lung cancer cell lines (A549, H1299, H358 and H1993) were detected by real-time quantitative polymerase chain reaction (RT-qPCR), and immunohistochemistry (IHC) was used to detect the expression level of HO-1 in human lung adenocarcinoma specimens. The HO-1 short hairpin RNA (shRNA) was transfected into A549 cells by RNA interference technique. HO-1 stably deleted A549 cells were selected (HO-1 shRNA group) and verified by RT-qPCR and western blot. HO-1 shRNA A549 cells and control shRNA A549 cells were treated with the inducer of autophagy Torin1 or its inhibitor Bafilomycin A1 (Baf A1), respectively. The expressions of autophagic markers LC3B and p62 were determined by western blot. The proliferation, invasion and migration abilities of each group of A549 cells were assessed by cell counting, Transwell and wound healing assays, respectively. Results: The expressions of HO-1 mRNA in lung cancer cell lines (A549, H1299, H358 and H1993) were significantly higher than that of HBECs, and HO-1 upregulated in human lung adenocarcinoma. The expression of p62 protein and the ratio of LC3B-Ⅱ/ LC3B-Ⅰ in no treatment group, Torin1 treatment group and Baf A1 treatment group were significantly higher than those of the corresponding control group (P<0.05). After 11 days of culture, the number of cells in HO-1 shRNA group were 41.8%, 30.4% and 14.0% of the corresponding control group, respectively. The number of lower chamber cells in HO-1 shRNA group were (35.7±2.1), (27.0±1.0) and (38.0±1.0)/field, respectively, which were lower than (66.0±9.2), (39.3±1.2) and (43.0±2.6)/field of the corresponding control group, respectively (P<0.05). The migration distances of HO-1 shRNA group were (7.47±0.91) mm, (4.23±0.82) mm and (5.42±0.24) mm, which were lower than (10.07±1.26) mm, (7.14±0.07) mm and (12.04±0.80) mm of the corresponding control groups, respectively (P<0.05). Conclusion: Knockdown of HO-1 inhibits the proliferation, invasion and migration of A549 cells by impeding autophagy.

Propofol vs desflurane on the cytokine, matrix metalloproteinase-9, and heme oxygenase-1 response during living donor liver transplantation: A pilot study.

Background:We investigated the effects of propofol vs desflurane on ischemia and reperfusion injury (IRI)-induced inflammatory responses, especially in matrix metalloproteinase-9 (MMP-9) downregulation and heme oxygenase-1 (HO-1) upregulation, which may result in different clinical outcomes in liver transplant recipients.Methods:Fifty liver transplant recipients were randomized to receive propofol-based total intravenous anesthesia (TIVA group, n = 25) or desflurane anesthesia (DES group, n = 25). We then measured the following: perioperative serum cytokine concentrations (interleukin 1 receptor antagonist [IL-1RA], IL-6, IL-8, and IL-10); MMP-9 and HO-1 mRNA expression levels at predefined intervals. Further, postoperative outcomes were compared between the 2 groups.Results:The TIVA group showed a significant HO-1 level increase following the anhepatic phase and a significant MMP-9 reduction after reperfusion, in addition to a significant increase in IL-10 levels after the anhepatic phase and IL-1RA levels after reperfusion. Compared to DES patients, TIVA patients showed a faster return of the international normalized ratio to normal values, lower plasma alanine aminotransferase concentrations 24 hours after transplantation, and fewer patients developing acute lung injury. Moreover, compared with DES patients, TIVA patients showed a significant reduction in serum blood lactate levels. However, there were no differences in postoperative outcomes between the two groups.Conclusion:Propofol-based TIVA attenuated inflammatory response (elevated IL-1RA and IL-10 levels), downregulated MMP-9 response, and increased HO-1 expression with improved recovery of graft function and better microcirculation compared with desflurane anesthesia in liver transplant recipients.

High-Pressure Carbon Monoxide and Oxygen Mixture is Effective for Lung Preservation.

(1) Background: Heme oxygenase-1 (HO-1) degrades heme and generates carbon monoxide (CO), producing various anti-inflammatory, anti-oxidative, and anti-apoptotic effects. This study aimed to confirm the effects of CO on the ischemia–reperfusion injury (IRI) of donor lungs using a high-pressure gas (HPG) preservation method. (2) Methods: Donor rat and canine lungs were preserved in a chamber filled with CO (1.5 atm) and oxygen (O2; 2 atm) and were ventilated with either CO and O2 mixture (CO/O2 group) or air (air group) immediately before storage. Rat lungs were subjected to heterotopic cervical transplantation and evaluated after reperfusion, whereas canine lungs were subjected to allogeneic transplantation and evaluated. (3) Results: Alveolar hemorrhage in the CO/O2 group was significantly milder than that in the air group. mRNA expression levels of HO-1 remained unchanged in both the groups; however, inflammatory mediator levels were significantly lower in the CO/O2 group than in the air group. The oxygenation of graft lungs was comparable between the two groups, but lactic acid level tended to be higher in the air group. (4) Conclusions: The HO-1/CO system in the HPG preservation method is effective in suppressing IRI and preserving donor lungs.

Resveratrol reduces brain injury after subarachnoid hemorrhage by inhibiting oxidative stress and endoplasmic reticulum stress.

Previous studies have shown that resveratrol, a bioactive substance found in many plants, can reduce early brain injury after subarachnoid hemorrhage, but how it acts is still unclear. This study explored the mechanism using the experimental subarachnoid hemorrhage rat model established by injecting autologous blood into the cerebellomedullary cistern. Rat models were treated with an intraperitoneal injection of 60 mg/kg resveratrol 2, 6, 24 and 46 hours after injury. At 48 hours after injury, their neurological function was assessed using a modified Garcia score. Brain edema was measured by the wet-dry method. Neuronal apoptosis in the prefrontal cortex was detected by terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick-end labeling assay. Levels of reactive oxygen species and malondialdehyde in the prefrontal cortex were determined by colorimetry. CHOP, glucose-regulated protein 78, nuclear factor-erythroid 2-related factor 2 and heme oxygenase-1 mRNA expression levels in the prefrontal cortex were measured by reverse transcription polymerase chain reaction. Tumor necrosis factor-alpha content in the prefrontal cortex was detected by enzyme linked immunosorbent assay. Immunohistochemical staining was used to detect the number of positive cells of nuclear factor-erythroid 2-related factor 2, heme oxygenase 1, glucose-regulated protein 78, CHOP and glial fibrillary acidic protein. Western blot assay was utilized to analyze the expression levels of nuclear factor-erythroid 2-related factor 2, heme oxygenase 1, glucose-regulated protein 78 and CHOP protein expression levels in the prefrontal cortex. The results showed that resveratrol treatment markedly alleviated neurological deficits and brain edema in experimental subarachnoid hemorrhage rats, and reduced neuronal apoptosis in the prefrontal cortex. Resveratrol reduced the levels of reactive oxygen species and malondialdehyde, and increased the expression of nuclear factor-erythroid 2-related factor 2, heme oxygenase-1 mRNA and protein in the prefrontal cortex. Resveratrol decreased glucose-regulated protein 78, CHOP mRNA and protein expression and tumor necrosis factor-alpha level. It also activated astrocytes. The results suggest that resveratrol exerted neuroprotective effect on subarachnoid hemorrhage by reducing oxidative damage, endoplasmic reticulum stress and neuroinflammation. The study was approved by the Animals Ethics Committee of Shandong University, China on February 22, 2016 (approval No. LL-201602022).

Microsatellite polymorphism in the Heme oxygenase-1 gene promoter is associated with dermal collagen density in Japanese obese male subjects.

We previously reported elevated oxidative stress-related mechanical vulnerabilities of the skin as sparse distributions of hyperechoic areas. Although this helped establish a personalized skin care system to prevent skin disorders related to mechanical stress, obesity-related skin vulnerability involves individual differences. Here, we hypothesized that individual differences are caused by polymorphisms of GT repetitive sequences in the heme oxygenase1 (HMOX1) promoter region, which encodes an antioxidant enzyme. This cross-sectional study enrolled healthy male volunteers in a walking classroom aimed at weight control. Subjects with a body mass index <25 kg/m2 were classified as non-obese and those with body mass index ≥25 kg/m2 were classified as obese. Subject skin was categorized into sparse dermis or normal groups according to the distribution of hyperechoic areas by high-resolution skin ultrasonography (20 MHz). Genomic DNA and mRNA extracted from three body hairs with attached follicle cells were used to analyze GT repetitive sequences of the HMOX1 promoter, HMOX1 mRNA expression levels, and oxidative stress levels (8-hydroxy-2’-deoxyguanosine). Classifications of GT repetitive sequence of HMOX1 promoter were Short (<27 times) and Long (≥27 times). Higher numbers of subjects with sparse dermis were in the obese group compared with the non-obese group. In obese subjects, the number of subjects that had the Long allele of the HMOX1 promoter with sparse dermis was significantly higher compared with the normal group, whereas no association was observed between the polymorphism and ultrasonographic features in non-obese subjects. Thus, HMOX1 polymorphisms detected a risk of low collagen density in Japanese obese male subjects.

Cyclic helix B peptide protects HK‑2 cells from oxidative stress by inhibiting ER stress and activating Nrf2 signalling and autophagy.

Renal ischemia-reperfusion injury (IRI) is present in numerous diseases and is observed following certain treatments, including renal transplantation. Preventing tubular epithelial cells (TECs) from undergoing apoptosis is vital for treatment of renal IRI. Cyclic helix B peptide (CHBP) is a novel agent that has a protective effect on renal IRI in vivo. In the present study, the effect and underlying mechanism of CHBP on TECs was investigated. The HK-2 human renal proximal tubular epithelial cell line was treated with 500 µmol/l H2O2 for 4 h prior to determining the effect of CHBP pretreatment for 1 h on cell viability, caspase 3 activity and expression levels, expression levels of oxidative stress markers, endoplasmic reticulum (ER) stress markers, NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and autophagy markers. This was investigated using a Cell Counting kit 8, a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, western blotting, reverse transcription-quantitative polymerase chain reaction and immunocytochemistry. Results revealed that pretreatment with CHBP enhanced HK-2 cell viability, the glutathione/glutathione disulphide ratio, activation of Nrf2 and mRNA expression levels of HO-1 and the expression levels of beclin-1 and light chain 3 A/B-II/I. Conversely, CHBP pretreatment reduced the expression levels of reactive oxygen species, the activity and protein expression levels of capase-3, the mRNA and protein expression levels of C/EBP homologous protein and binding immunoglobulin protein, and the expression levels of phosphorylated (p)-mechanistic target of rapamycin (mTOR) Ser2448 and p62 during oxidative stress. However, the expression of p-mTOR Ser2481 was enhanced after CHBP pretreatment. CHBP pretreatment reduced the expression levels of reactive oxygen species, the activity and protein expression levels of capase-3, the mRNA and protein expression levels of C/EBP homologous protein and binding immunoglobulin protein, and the expression levels of phosphorylated (p)-mechanistic target of rapamycin (mTOR) Ser2481, p62 and p-mTOR Ser 2448 during oxidative stress. In conclusion, CHBP pretreatment protected HK-2 cells from H2O2-induced injury, inhibited ER stress and pro-apoptotic pathways, and activated the Nrf2 signalling pathway and autophagy. These results provide a potential mechanism of how CHBP protects against renal IRI.

Hemeoxygenase-1 as a Novel Driver in Ritonavir-Induced Insulin Resistance in HIV-1-Infected Patients.

Hemeoxygenase-1 (HO-1) has recently been identified as a major driver of metaflammation and obesity-related insulin resistance (IR). Drug-induced IR increases cardiovascular risk within the HIV-1-infected population receiving antiretroviral therapy (ART). We therefore investigated a possible role of HO-1 in ART-induced IR. Effects of HIV-1 protease inhibitor ritonavir and integrase inhibitor raltegravir (RAL) on expression levels of HO-1 and proinflammatory cytokines, including interleukin 1β (IL-1β), IL-6, IL-8, tumor necrosis factor-α (TNFα), chemokine (C-C motif) ligand 5 (CCL5), and monocyte chemotactic protein 1 (MCP-1), were studied in monocyte and hepatocyte cell lines. Plasma levels of HO-1 and inflammatory markers were measured in insulin-resistant and insulin-sensitive HIV-1-infected patients under ART and seronegative controls. We show that, in contrast to RAL, ritonavir treatment significantly increases mRNA expression levels of HO-1, IL-8, TNFα, CCL5, and MCP-1 in vitro in a dose-dependent manner. HO-1 plasma levels were significantly higher in insulin-resistant compared to insulin-sensitive patients on ritonavir-boosted ART (lopinavir/ritonavir group: 3.90 ± 1.15 vs 2.56 ± 1.07 ng/mL, P < 0.005 and darunavir/ritonavir group: 3.16 ± 1.37 vs 2.28 ± 1.23 U/mL, P < 0.05) and were correlated with expression levels of TNFα in individuals on ritonavir-boosted ART (lopinavir/ritonavir group: r = 0.108, P < 0.05 and darunavir/ritonavir group: r = 0.221, P < 0.05) but not in HIV-1-infected individuals receiving RAL or in seronegative controls. HIV-1-infected patients on stable ART are often faced with non-AIDS-related metabolic comorbidities, increasing their individual cardiovascular risk. Here, we provide insight into a novel mechanism of ritonavir-induced IR involving proinflammatory properties of HO-1. Our initial observations might also provide prognostic value in the future to identify patients at risk for the development type 2 diabetes mellitus.

Overexpression of heme oxygenase-1 induced by constitutively activated NF-κB as a potential therapeutic target for activated B-cell-like diffuse large B-cell lymphoma.

There is an urgent requirement for a new therapeutic target for activated B-cell-like lymphoma (ABC-DLBCL), which is known to have dismal outcome and constitutive activation of NF-κB. Heme oxygenase-1 (HO-1) can inhibit apoptosis and promote proliferation in many cancers. To our knowledge, no studies have been performed on the correlation between HO-1 and DLBCL. In this study, immunohistochemical analysis of 31tumor tissues from DLBCL patients [20 of ABC subtype and 11 of germinal center B-cell-like (GCB) subtype] and 11normal lymph nodes revealed that HO-1 overexpression was characteristic of ABC-DLBCL. In addition, HO-1 mRNA expression levels were consistent with the immunohistochemistry results. High levels of HO-1 expression were significantly correlated with the involvement of more than 1 extranodal site (p=0.025), with a high positivity rate of Ki-67 (p<0.01). Similar to its anti-apoptotic role in other malignancies, HO-1 upregulation suppressed apoptosis of the ABC-DLBCL cell line OCI-ly10, whereas its downregulation sensitized the tumor cells to chemotherapeutic drugs. Further study demonstrated that the HO-1 overexpression was mediated by constitutively activated NF-κB which together played an anti-apoptotic role in ABC-DLBCL. Combination of the NF-κB inhibitor Bay11‑7082 and the lentivirus vector Lenti-siHO-1 significantly decreased HO-1 protein expression and increased apoptosis in OCI-ly10 cells. However, in GCB-DLBCL cells with low levels of NF-κB expression, the TNF-α-mediated activation of NF-κB leading to HO-1 upregulation rescued the cells from apoptosis caused by HO-1 silencing. These results indicated that HO-1 can be a potential target for the treatment of ABC-DLBCL.

Biliary tract external drainage increases the expression levels of heme oxygenase-1 in rat livers.

BackgroundHeme oxygenase-1 (HO-1) protects cells by anti-oxidation, maintaining normal microcirculation and anti-inflammatory under stress. This study investigated the effects of biliary tract external drainage (BTED) on the expression levels of HO-1 in rat livers.MethodsBiliary tract external drainage was performed by inserting a cannula into the bile duct. Sixty Sprague–Dawley rats were randomized to the following groups: sham 1 h group; BTED 1 h group; bile duct ligation (BDL) 1 h group; sham 6 h group and BTED 6 h group. The expression levels of HO-1 mRNA were analyzed using real-time RT-PCR. The expression levels of HO-1 were analyzed using immunohistochemistry.ResultsThe expression levels of HO-1 mRNA in the liver of the BTED group increased significantly compared with the sham group 1 and 6 h after surgery (p < 0.05).The expression levels of HO-1 in the BTED group increased significantly compared with the sham group 1 and 6 h after surgery. The expression levels of HO-1 mRNA in the liver in the BDL group decreased significantly compared with the sham group 1 h after surgery (p < 0.05).The expression levels of HO-1 in the BDL group decreased significantly compared with the sham group at this time.ConclusionBiliary tract external drainages increase the expression levels of HO-1 in the liver.

Upregulated heme oxygenase-1 expression of mouse mesenchymal stem cells resists to chemotherapy-induced bone marrow suppression

Background Bone marrow hematopoietic function suppression is one of the most common side effects of chemotherapy. After chemotherapy, the bone marrow structure gets destroyed and the cells died, which might cause the hematopoietic function suppression. Heme oxygenase-1 (HO-1) is a key enzyme of antioxidative metabolism that associates with cell proliferation and resistance to apoptosis. The aim of this study was to restore or resist the bone marrow from the damage of chemotherapy by the HO-1 expression of mouse mesenchymal stem cells (mMSCs) homing to the mice which had the chemotherapy-induced bone marrow suppression. Methods One hundred and sixty female Balb/c mice (6–8-weeks old) were randomly divided into four groups. Each group was performed in 40 mice. The control group was intraperitoneally injected for 5 days and tail intravenously injected on the 6th day with normal saline. The chemotherapy-induced bone marrow suppression was established by intraperitoneally injecting cyclophosphamide (CTX) into the mice which performed as the chemotherapy group. The mMSCs were tail intravenously injected into 40 chemotherapically damaged mice which served as the mMSCs group. The difference between the HO-1 group and the mMSCs group was the injected cells. The HO-1 group was tail intravenously injected into the mMSCs that highly expressed HO-1 which was stimulated by hemin. The expression of HO-1 was analyzed by Western blotting and RT-PCR. Cell proliferation was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Histopathologic examinations were performed 1 week after injection. Results Compared with the control group, the expression levels of HO-1 mRNA and protein were significantly higher in the HO-1 group (all P &lt;0.05), even obviously than the mMSCs group. CTX treatment induced apoptosis and inhibited proliferation. After injected, the white blood cell (WBC), red blood cell (RBC) and platelet (PLT) declined fast and down to the bottom at the 7th day. The bone marrow structure was destroyed incomplete. In vitro, the survival rate of cells in chemotherapy group was less than 50% after 24 hours. In contrast, mMSCs could do a favor to the cellular cleavage and proliferation. They slowed down the cell mortality and more than 50% cells survived after 24 hours. The effects of blocking apoptosis and bone marrow recovery could be more effective in the HO-1 group. In the HO-1 group, it had observed that the bone marrow structure became complete and the hemogram closed to normal at 7th day. Conclusions HO-1 played an important role in promoting the recovery of CTX-induced hematopoietic damage. We suggest that HO-1 is able to restore the functions of chemotherapy-induced hematopoietic damage.

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.

Neuroprotective effects of valproic acid against hemin toxicity: Possible involvement of the down-regulation of heme oxygenase-1 by regulating ubiquitin–proteasomal pathway

During hemorrhagic stroke induced by intracerebral hemorrhage (ICH), brain injury occurs from the deleterious actions of hemoglobin byproducts; induction of heme oxygenase-1 (HO-1) also plays a critical role in the neurotoxicity in ICH. Valproic acid (VPA), which is a commonly used drug in the treatment of epilepsy, has been reported to have neuroprotective effects against various neuronal insults including ischemic stroke. We investigated the effect of VPA on HO-1-mediated neurotoxicity in an experimental model of ICH. We investigated the effects of VPA on HO-1 protein in primary cortical neurons: (1) the expression levels of HO-1 mRNA and protein measured by RT-PCR and Western blotting; (2) the cell viability and ROS generation by MTT reduction assay and ROS measurement; (3) the signal pathway regulated by VPA using IP-Western blotting; (4) the effects of VPA on hemin-induced cell death by hemin microinjection and immunohistochemistry in vivo. VPA treatment partially blocked cell death induced by hemin, which is released from hemoglobin during ICH, both in rat primary cortical neurons and rat brain. Treatment of VPA significantly decreased the expression of HO-1 protein both in vitro and in vivo. Hemin treatment induced HO-1 protein expression and this was partially blocked by pretreatment with VPA, which might be mediated by increased ubiquitination and degradation of HO-1 via ERK1/2 and JNK activation in primary cortical neurons. Our results indicate that VPA inhibits hemin toxicity by downregulating HO-1 protein expression, and provide a therapeutic strategy to attenuate intracerebral hemorrhagic injury.

Possible Role of Heme Oxygenase-1 and Prostaglandins in the Pathogenesis of Cerebral Malaria: Heme Oxygenase-1 Induction by Prostaglandin D2 and Metabolite by a Human Astrocyte Cell Line

Astrocytes are the most abundant cells in the central nervous system that play roles in maintaining the blood-brain-barrier and in neural injury, including cerebral malaria, a severe complication of Plasmodium falciparum infection. Prostaglandin (PG) D(2) is abundantly produced in the brain and regulates the sleep response. Moreover, PGD(2) is a potential factor derived from P. falciparum within erythrocytes. Heme oxygenase-1 (HO-1) is catalyzing enzyme in heme breakdown process to release iron, carbon monoxide, and biliverdin/bilirubin, and may influence iron supply to the P. falciparum parasites. Here, we showed that treatment of a human astrocyte cell line, CCF-STTG1, with PGD(2) significantly increased the expression levels of HO-1 mRNA by RT-PCR. Western blot analysis showed that PGD(2) treatment increased the level of HO-1 protein, in a dose- and time-dependent manner. Thus, PGD(2) may be involved in the pathogenesis of cerebral malaria by inducing HO-1 expression in malaria patients.

Prevention of cadmium accumulation in retinal pigment epithelium with manganese and zinc

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the elderly. Risk factors include old age, female gender, obesity, smoking, low dietary intakes of antioxidants and increased exposure to the toxic metal cadmium (Cd 2+). Supplementation with high-dose zinc (80 mg) provides some protection, but the mechanism(s) underlying such protection has not been fully elucidated. The present study had a focus on the human retinal pigment epithelial (RPE) cell line ARPE-19 in an attempt to demonstrate a reduction in intracellular Cd 2+ effect associated with heme oxygenase-1 (HO-1) expression by co-exposure with zinc (Zn 2+) or manganese (Mn 2+), which is known to be a more potent inhibitor of Cd 2+ uptake than Zn 2+. Our results indicated that co-exposure of 10 μM Cd 2+ with 5 μM Mn 2+ reduced the intracellular Cd 2+ effect by 50–60%, possibly by limiting the amounts of Cd 2+ entering cells through Mn 2+ transporter protein (ZIP8). A similar reduction in a Cd 2+ effect was achieved by co-exposure with 20 μM Zn 2+ while co-exposure with 5 and 10 μM Zn 2+ ions was ineffective. Mn 2+ ions as low as 2.5 μM were found to cause an increase in HO-1 mRNA expression levels in ARPE-19 cells, demonstrating for the first time that Mn 2+ is an inducer of HO-1. Mn 2+ ions at 1 μM induced HO-1 mRNA expression in the HEK293 human embryonic kidney cells. In contrast, Zn 2+ in 5, 10 or 20 μM concentrations did not induce expression of HO-1 in ARPE-19 cells or any other cells tested. These data suggest the superiority of Mn 2+ over Zn 2+ in preventing Cd 2+ uptake and accumulation in RPE to toxic levels. Further, induction of HO-1 by Mn 2+ could provide RPE with some resistance to enhanced oxidative stress arising from Cd 2+ accumulation in RPE as HO-1 is one of the frontline cellular antioxidant defense mechanisms.

Regulation of heme oxygenase-1 by transcription factor Bach1 in the mouse brain

Oxidative stress has been implicated in tissue damage from traumatic brain injury. Heme oxygenase-1 (HO-1) is an inducible enzyme that degrades prooxidant heme to radical-scavenging biliverdin/bilirubin in order to protect cells from oxidative stress. Although HO-1 is induced after induction of brain damage, the regulatory mechanism of HO-1 in the brain is still unclear. Bach1 is a transcriptional repressor of the HO-1 gene, and plays a critical role in tissue protection from oxidative stress by reperfusion injury of the myocardium. In this study, we examined the role of Bach1 in HO-1 regulation of the various brain sites by investigating the expression of Bach1 and HO-1 in brain tissues of mice bearing Bach1-deficient (Bach1(-/-)) or wild-type (Bach1(+/+)) genes. While the expression levels of Bach1 mRNA in the olfactory bulb were significantly higher than other brain areas, those at the cortex showed the lowest activity. Bach1(-/-) mice showed significantly higher HO-1 mRNA expression levels than Bach1(+/+) mice in all brain sites studied. Moreover, higher induction of HO-1 was observed around damaged tissues after cold injury in Bach1(-/-) than Bach1(+/+) mice. Thus, Bach1 plays an important role in regulating the constitutive and inducible expression levels of HO-1 in the brain. Although a significantly higher level of HO-1 was observed in Bach1(-/-) than Bach1(+/+) mice, genetic ablation of the Bach1 gene failed to show any tissue protective effect after cold injury was inflicted on the cortex.

Prostaglandin D2 induces heme oxygenase-1 in human retinal pigment epithelial cells

The retinal pigment epithelium (RPE) constitutes the blood–retinal barrier, whose function is impaired in various pathological conditions, including cerebral malaria, a lethal complication of Plasmodium falciparum infection. Prostaglandin (PG) D2 is abundantly produced in the brain to regulate sleep responses. Moreover, PGD2 is a potential factor derived from intra-erythrocyte falciparum parasites. Heme oxygenase-1 (HO-1) is important for iron homeostasis via catalysis of heme degradation to release iron, carbon monoxide and biliverdin/bilirubin, and may influence iron supply to the intra-erythrocyte falciparum parasites. Here, we showed that treatment of human RPE cell lines, ARPE-19 and D407, with PGD2 significantly increased the expression levels of HO-1 mRNA, in a dose- and time-dependent manner. Transient expression assays showed that PGD2 treatment increased the HO-1-gene promoter activity through the enhancer sequence, containing a Maf-recognition element. Thus, PGD2 may contribute to the maintenance of heme homeostasis in the brain by inducing HO-1 expression.