ObjectiveTo investigate the protective effect and the regulation mechanism of oxaloacetate (OAA) on myocardial ischemia reperfusion injury in rats. MethodsSixty rats, weight ranged from 200 to 250 grams, were randomly divided into 6 groups:a negative control group, a sham operation control group, a model control group, an OAA pretreatment myocardial ischemia-reperfusion model group (three subgroups:15 mg/kg, 60 mg/kg, 240 mg/kg). We established the model of myocardial ischemia reperfusion of rats and recorded the internal pressure of left ventricle (LVSP), the maximal rate of left ventricular pressure change (±dp/dtmax) and left ventricular end diastolic pressure (LVEDP). We restored reperfusion 180 minutes after ligating the left anterior descending coronary artery 30 minutes and determinated cardiac troponin Ⅰ (cTn-I), lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px). We took out heart tissues, stained it and calculated the infarcted size. We used the Western blot to detect the expression of NF-E2 related factor 2 (Nrf2), Kelch-like ECH-associated protein-1 (Keap1) and heme oxygenase-1 (HO-1). ResultsCompared with the sham operation group, heart function indexes in the negative control group had no significant difference (P>0.05). But in the model control group there was a decrease (P<0.05) And the serum levels of LDH, cTn-I, and myocardial infarcted size were significantly increased (P<0.01). Compared with the model control group, heart function indexes in the OAA pretreatment groups improved, the serum LDH, cTn-I activity, and infarct size decreased (P<0.05), SOD and GSH-Px activity increased (P<0.05). And these results were statistically different (P<0.01) in the high dose OAA pretreatment groups. Compared with the model control group, the expression of Keap1 in the OAA pretreatment group was down-regulated (P<0.001) while total Nrf2, nucleus Nrf2 and its downstream HO-1 was up-regulated (P<0.001), which suggested that OAA enhanced antioxidant capacity by (at least in part) Keap1-Nrf2 pathway, resulting in reducing myocardial damage and protecting myocardium after acute myocardial ischemia reperfusion injury. ConclusionOxaloacetate can provide protective effects on myocardial ischemia reperfusion injury through down-regulating the expression of Keap1 and up-regulating the expression of Nrf2 and its downstream peroxiredoxins to improve antioxidant capacity.
Objective To investigate the effects of simvastatin on monocrotaline-induced pulmonary hypertension in rats, and explore the potential mechanism of simvastatin by blocking heme oxygenase-1( HO-1) expression. Methods 52 male Sprague-Dawley rats were randomly divided into five groups, ie. a control group, a simvastatin control group, a pulmonary hypertension model group, a simvastatin treatment group, a ZnPP ( chemical inhibitor of HO) group. Mean pulmonary arterial pressure ( mPAP) and right ventricular systolic pressure ( RVSP) were detected by right heart catheter at 5th week. Right ventricular hypertrophy index ( RVHI) was calculated as the right ventricle to the left ventricle plus septum weight. Histopathology changes of small intrapulmonary arteries were evaluated via image analysis system.Immunohistochemical analysis was used to investigate the expression and location of HO-1. HO-1 protein level in lung tissue were determined by western blot. Results Compared with the model group, simvastatin treatment decreased mPAP and RVHI significantly [ ( 35. 63 ±5. 10) mm Hg vs. ( 65. 78 ±15. 51) mm Hg,0. 33 ±0. 05 vs. 0. 53 ±0. 06, both P lt; 0. 05 ] . Moreover, simvastatin treatment partially reversed the increase of arterial wall area and arterial wall diameter [ ( 50. 78 ±9. 03 ) % vs. ( 65. 92 ±7. 19) % ,( 43. 75 ±4. 23) % vs. ( 52. 00 ±5. 35) % , both P lt; 0. 01) . In the model group, HO-1 staining was primarily detected in alveolar macrophages. Simvastatin treatment increased HO-1 protein expression significantly, especially in the thickened smooth muscle layer and alveolar macrophages. Inhibiting HO-1 expression using ZnPP resulted in a loss of the effects of simvastatin. mPAP in the ZnPP group was ( 52. 88±17. 45) mm Hg, while arterial wall area and arterial wall diameter were ( 50. 78 ±9. 03) % and ( 52. 00 ±5. 35) % , respectively. Conclusions Simvastatin attenuates established pulmonary arterial hypertension andpulmonary artery remodeling in monocrotaline-induced pulmonary hypertension rats. The effect of simvastatin is associated with HO-1.
Objective To explore the expression and effect of heme oxygenase-1 ( HO-1) in ventilator-induced lung injury. Methods Twenty-four New Zealand rabbits were randomly assigned to three groups, ie. a conventional ventilation + PEEP group( C group) , a ventilator-induced lung injury group( VILI group) , and a VILI + HO-1 inducer hemin group( Hm group) .Western blot and immunohistochemistry assay were used to investigate the expression of HO-1 protein. Blood gas analysis, lung wet /dry ratio, lunghistopathology and lung injury score were used to evaluate lung injury. Results HO-1 protein expression significantly increased in the VILI group compared with the C group. HO-1 was found mainly in alveolar epithelial cells and vascular endothelial cells, as well as in alveolar macrophages and neutrophils. Compared with the VILI group, HO-1 protein and PaO2 /FiO2 increased, while lung wet/dry ratio and lung injury score decreased in the Hmgroup significantly( P lt;0. 05) . Conclusion High HO-1 expression can alleviate lung injury from large tidal volume ventilation, implying its protective role in lung pathogenesis.
ObjectiveTo evaluate the effect of ZnPP Ⅸ on the expressions of heme oxygenase-1 (HO-1) and glutathione-Stransferase-π （GST-π） and the chemosensitivity of drug-resistant hepatic carcinoma cell line Bel/Fu, and explore it’s possibility to reverse drug-resistance and the relevant regulating mechanism. Methods①MTT assay was adopted to detect the drug sensitivity for adriamycin, mitomycin, and 5-fluorouracil of Bel/Fu cell after ZnPP Ⅸ being induced for 24 h. ②RTPCR was carried out to detect the expressions of HO-1 and GST-π mRNA after Bel/FU cells being treated with different concentrations ZnPP Ⅸ for 24 h. ResultsAfter Bel/Fu cells being treated with ZnPP Ⅸ for 24 h, the 50% inhibiting concentration (IC50) for drugs was decreased dramatically （Plt;0.05）. Meanwhile, the expressions of HO-1 and GST-π mRNA in the treated cells also decreased dose-dependently (Plt;0.01). ConclusionsZnPP Ⅸ can increase the chemosensitivity of Bel/FU cells by down-regulation of HO-1 and GST-π expression. ZnPP Ⅸ is a potential agent to reverse multidrug resistance of hepatic carcinoma cells.
Objective To investigate the effect of heme oxygenase-1 (HO-1) activity on rat liver transplantation model. Methods One hundred and thirty-seven rats were divided into 4 groups. Study control group (n＝44)： 24 h before operation, saline 5 ml/kg was infused into peritoneal cavity of donor rats; Hemin group (n＝44)： hemin 100 mg/kg was infused into peritoneal cavity of donor rats 24 h before operation, and hemin 100 mg/kg was infused into portal vein during the preserve time in 4 ℃ saline； ZnPP group (n＝44)： ZnPP 5 mg/kg was infused into peritoneal cavity of donor rats 24 h before operation, and ZnPP 5 mg/kg was infused into portal vein during the preserve time in 4 ℃ saline； Normal control group (n＝5)： normal rats as normal control group. Expressions of HO-1 protein and mRNA were detected with immunohistochemistry and RT-PCR technique respectively. The apoptosis rate of hepatocytes was analyzed by flow cytometry. Results Expression of HO-1 mRNA in the liver of hemin group after transplantation was higher than that in study control group obviously, serum ALT and AST levels were lower than those in study control group (P＜0.05); HO-1 mRNA expression in ZnPP group liver was lower than that in study control group, serum ALT and AST levels were higher than those in study control group (P＜0.05). About liver cell apoptosis rate 48 h after liver transplantation, ZnPP group was the highest, hemin group was the minimum, and there had a significant difference between two groups (P＜0.05). Seven days after transplantation, the survival ratios of control study group, hemin group and ZnPP group were 7/12, 9/12 and 4/12 in turn, the inter-group differences had statistical significance (P＜0.05). Conclusion Activity of HO-1 could be induced by the transplant operation. HO-1 increases the survival rate after liver transplantation which was related with reducing apoptotic ratio of hepatocyte and improve hepatic function.
【Abstract】ObjectiveTo investigate whether heme oxygenase-1 can alleviate the ischemiareperfusion injury of the aged donor liver. MethodsThe activity of superoxide dismutase (SOD) and catalase (CAT), and the contents of tocopherol (Vit E), ascorbic acid (Vit C) and malondialdehyde (MDA) were measured in the livers of adult SD rats (n=5) and aged SD rats (n=5). The experimental aged donor group (n=30) received intraperitoneal injection of Hemin 24 hours before operation, the control aged donor group(n=30) received saline. The histologic changes and apoptosis in the donor liver were observed. ResultsThe activity of SOD and the contents of Vit E and Vit C decreased significantly in 5 aged rats（P＜0.05）, but the content of MDA increased（P＜0.05）. Before the harvesting of the grafts, the activity of SOD and the contents of Vit E and Vit C increased significantly in rats pretreated with Hemin （P＜0.05） and the content of MDA decreased（P＜0.05）. The apoptotic cells in the livers pretreated with Hemin also decreased significantly after reperfusion（P＜0.05）. ConclusionThe liver of aged rat presents oxidative stress and peroxidative state. Ischemia-reperfusion injury can be alleviated by the induction of HO-1.
Objective To explore the role of sulforaphane (SFN) on the regulation of airway mucin 5AC mucin expression in the airway epithelial cell line (A549), and the underlying mechanism. Methods The experiments were performed in culture of PMA and H2O2 induced A549 in vitro. A549 cells divided into three groups: the control group, the model group (PMA and H2O2) and the intervention groups (SFN+PMA; SFN+H2O2). The intervention group's cells were pretreated with SFN for 30 mins before exposure to stimuli (PMA or H2O2). The MUC5AC, Nrf2 and the antioxidant gene HO-1, NQO1, GCLC mRNA levels were analyzed by real time-PCR, and protein production was assayed by western blot. Results Compared with the control group, expression of MUC5AC mucin was increased after being stimulated by PMA or H2O2 (P<0.05), but it was significantly inhibited by SFN (P<0.05). SFN induced the expression of Nrf2 gene and the antioxidant gene HO-1 (compared with the control and model groups,P<0.05). Conclusion Sulforaphane involves the airway mucous hypersecretion induced by PMA and H2O2, and Nrf2/HO-1 signaling pathway may play an important role in mucin MUC5AC regulation.
Objective To investigate the expression of heme oxygenase-1 (HO-1) by high glucose treatment in human lung epithelial A549 cells. Methods The effect of high glucose on the expression of HO-1 in A549 cells was investigated with Western blot and reverse transcription PCR. HO-1 enzymic activity and reactive oxygen species (ROS) production were investigated with enzyme-linked immunosorbent test. Results Treatment with 25 mmol/L high glucose for 0, 24 h, 48 h, 72 h and in concentrations of 5 mmol/L, 10 mmol/L, 25 mmol/L, 40 mmol/L for 48 h induced increased expression on protein and mRNA level of HO-1 in a concentration- and time-dependent manner in A549 cells. High glucose treatment increased production of ROS and transforming growth factor-β1 (TGF-β1) in A549 cells, which thus mediated HO-1 expression. Following the increase in HO-1 expression, the enzymatic activity of HO-1 also increased in high glucose-treated cells. Pretreatment with N-acetyl-L-cysteine (NAC) and PI3K/Akt inhibitors attenuated the high glucose-induced increased HO-1 expression. Conclusions High glucose increases ROS and TGF-β1 production in A549 cells, which mediates HO-1 expression and increases HO-1 enzymic activity.
Objective To determine the anti-apoptosis effects of heme oxygenase-1 (HO-1) on lung injury after cardiopulmonary bypass (CPB), and to investigate its probable mechanisms. Methods A total of 144 male Wistar rats with wight of 250-350 g were divided into 3 groups: group A (control group), group B (cobalt protoporphyrin, CoPP), and group C [CoPP and zinc protoporphyrin (ZnPP)] randomly. A modified rat model of CPB-induced lung injury was established. And then the lung tissues were taken at different times for the relevant indicators test: before CPB (T0), immediately after CPB (T1), 2 h after CPB (T2), 6 h after CPB (T3), 12 h after CPB (T4), and 24 h after CPB (T5). The expression of HO-1 and Bcl-2 protein in each group was tested by immunohistochemistry, and cell apoptosis by TUNEL. Results The HO-1 protein expression in group B was significantly higher than that in groups A and C at any given time point, so was the HO-1 activity (P<0.05). There was no significant difference in Bcl-2 expression of lung tissue before CPB among each group (P>0.05). The Bcl-2 protein reduced gradually after CPB. The expressions of Bcl-2 protein in group B at all time points after bypass were significantly higher than that in groups A and C (P<0.05). The apoptosis index (AI) showed no significant difference before CPB in each group (P>0.05), and increased gradually after CPB. AI in group B at any time point after bypass was lower than that in groups A and C (P<0.05). The HE staining results showed that the damage of lung tissue in group B obviously reduced compared with groups A and C. Conclusion CoPP can induce a large amount of HO-1 expression in the lung tissue, and it is still highly expressed after CPB. So it plays an important role in anti-apoptosis through the up-regulation of Bcl-2 protein expression.
Objective To observe the effect of melatonin (MT) on retinal apoptosis in rats with ischemia-reperfusion injury (RIRI). Methods A total of 54 male healthy Sprague-Dawley adult rats were randomly divided into the normal control (CON) group (6 rats), RIRI group (24 rats) and MT group (24 rats). The rats of RIRI and MT group were induced using suture-occluded methods to establish RIRI model. The rats of MT group were injected with MT in the left carotid artery 30 minutes after RIRI, and RIRI group was injected with the same amount of saline. On 6, 24 hours and 3, 7 days after RIRI, the morphological changes of retina were evaluated by hematoxylin and eosin (HE) staining; the effects of MT on retinal cell apoptosis and Nrf2, HO-1 proteins were examined by immunohistochemistry staining. The correlation between active Caspase-3 and Nrf2 protein, active Caspase-3 and HO-1 protein in MT group were analyzed by linear regression analysis. Results HE staining results showed that the morphology of retinal cells was regular and retinal cells were well arranged in the CON and MT group. In the RIRI group, both the thickness of inner retinal layer and the number of retinal ganglion cells (RGC) were decreased. On 6, 24 hours and 3, 7 days after RIRI, the thickness of inner retinal layer (F=16.710, 62.303, 68.389, 57.132; P＜0.01) and RGC number (F=24.250, 11.624, 14.155, 32.442; P＜0.05) in MT group were more than those in RIRI group. Immunohistochemistry staining results showed that less active Caspase-3+ cells were observed in MT group as compared with those in RIRI group at each time points (F=49.118, 134.173, 76.225, 18.385; P＜0.01). There were more Nrf2+ (F=11.041, 31.480, 59.246, 6.740; P＜0.05) and HO-1+ cells (F=128.993, 21.606, 51.349, 8.244; P＜0.05) in MT group as compared with those in RIRI group at each time points. Linear regression analysis results showed that the difference of active Caspase-3+ cells were all linearly correlated with the Nrf2+ cells and HO-1+cells in the MT group (r2=0.810, 0.730; P＜0.01). Conclusion MT could reduce retinal cell apoptosis in RIRI rats, and its mechanism may be associated with increased Nrf2 and HO-1 expression, reduced active Caspase-3 expression.