Role of heme oxygenase-1 in cardioprotection against anoxia/re oxygenation induced injury and itsmechanisms

AIM:To investigate the role of HO-1 in pro tection of rat hearts against anoxia/reoxygenation-induced injury and its under lying mechanism.METHODS:Cardiac contractility,lactate dehydrogenase (LDH) and infarct area were analyzed by the Langendorff method in isolated rat hearts.RESULTS:After intraperitoneal injection of HO-1 inducer hemin,CO concentration in rat blood enhanced (P<0.01 vs control group).Pretreatm ent with hemin prevented the increase in LVEDP and decrease in LVDP,±dp/d tmax during the anoxia and reoxygenation period in hearts.Hemin had n o effect on changes of coronary flow,but it really inhibited the release of LDH from anoxia/reoxygenation hearts.Hemin also reduced the infarct area in anoxia heart after 2 h reoxygenation (P<0.01).CO concentration in rat blood redu ced after intraperitoneal injection of HO-1 inhibitor ZnPP (P<0.01 vs contr ol group).ZnPP aggravated the decrease in LVDP and ±dp/dtmax.Co mpared with anoxia/reoxygenation heart,pretreatment of ZnPP enhanced the LDH re lease and enlarged the infarct area (P<0.05).GC inhibitor methylene blue a nd cyclooxygenase-2 (COX-2) inhibitor celecoxib both partly abolished the protec tion effect of hemin on LVEDP,LVDP and ±dp/dtmax.Pretreatment o f methylene blue or celecoxib also cancelled the inhibition of LDH release and r eduction of infarct area caused by hemin (P<0.05).CONCLUSION:HO-1 inducer hemin protects heart from anoxia/reoxy genation-induced injury.The cardiac protection of HO/CO might be through GC pathway,and the activation of COX-2 might be also involved in this process.     

CCK-8 up-regulats signal pathway of LPS-induced HO-1 expression in rat lungs

AIM: To study the signal pathway involved in up-regulation of LPS-induced HO-1 expression by CCK-8. METHODS: Forty-two SD rats were divided into 7 groups (six rats each) randomly as follows: control group, LPS group, LPS+SP600125 (JNK-specific inhibitor) group, CCK-8+LPS group, CCK-8+LPS+SP600125 group, CCK-8 group and CCK-8 +SP600125 group. Lungs from the rats in these 7 groups were excised 6 h after the agents were administered. HO-1 mRNA expression was examined by RT-PCR. The protein expression of HO-1 was detected by Western blotting and immunofluorescence flow cytometry (FCM). RESULTS: There were significant positive expression of HO-1 mRNA in LPS group compared to control group. CCK-8 enhanced LPS-induced HO-1 mRNA expression and CCK-8 alone induced HO-1 mRNA expression as well. The mRNA expressions of HO-1 in LPS group, CCK-8+LPS group and CCK-8 group were 3.01 (P<0.01), 5.88 (P<0.01) and 3.45 (P<0.01) times as many as that in control group, respectively. SP600125 inhibited the mRNA expression of HO-1 induced by CCK-8 and (or) LPS. The change of HO-1 protein expression was in accordance with that of HO-1 mRNA expression by Western blotting and immunofluorescence FCM. CONCLUSION: These results suggest that JNK/c-Jun signal pathway plays an important role in the up-regulation of LPS-induced HO-1 expression by CCK-8.     

Up-regulation of HO-1 protects ADSCs under serum-free and hypoxic conditions

AIM:To investigate the protective effects of endogenous heme oxygenase 1 (HO-1) induced by cobalt protoporphyrin (Copp, a HO-1 inducer) on adipose tissue-derived stromal cells (ADSCs) under the condition of serum-free and hypoxia. METHODS:The ADSCs were isolated from SD rat and cultured. The cell apoptotic rate was detected by DAPI staining. The protein expression of HO-1, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) and cleaved caspase-1 in ADSCs was messured by Western blotting. IL-1β level in supernatant was determined by ELISA. The level of intracellular reactive oxygen species (ROS) was detected using DCFH-DA. RESULTS:The up-regulation of HO -1 was induced by CoPP in a dose dependent manner and was most significant at 20 μmol/L. The increased expression of HO-1 induced by CoPP significantly reduced the apoptotic rate of ADSCs, intracellular ROS level and IL-1β secretion, and inhibited the overexpression of NLRP3, ASC and cleaved caspase-1 under serum and oxygen deprivation. These protective effects were reversed by zinc protoporphyrin (ZnPP, an HO-1 inhibitor) given simultaneously. CONCLUSION: The up-regulation of HO -1 expression induced by CoPP plays protective effect on ADSCs under the condition of serum and oxygen deprivation via inhibiting the activation of NLRP3 inflammasome and reducing IL-1β secretion.     

Role of heme oxygenase in cholecystokinin octopeptide ameliorating acute lung injury induced by lipopolysaccharide

AIM: To study the role of heme oxygenase (HO)-1 in the mechanism of cholecystokinin-octapeptide (CCK-8) for attenuation of acute lung injury (ALI) induced by lipopolysaccharide (LPS).METHODS: Adult male rats were randomly divided into five groups: control group,LPS group,CCK-8+LPS group,LPS+ Hm (hemin,HO-1 donor) group and LPS+ZnPP (zinc protoporphyrin,specific inhibitor of HO-1) group.PMN number in bronchoalveolar lavage fluid (BALF),the structure of the lung,MDA content,HO-1 activity,the expressions of HO-1 mRNA and protein in the lung were detected respectively.RESULTS: The lung injury in LPS group was observed,at the same time the numbers of PMN,the content of MDA,the activity and the expression of HO-1 were all higher than those in control group (all P<0.05).The degree of lung injury,PMN numbers and MDA content were lower,while the activity and the expression of HO-1 in CCK-8+LPS and LPS+Hm group were higher than those in LPS group (all P<0.05).However,the degree of lung injury,PMN numbers and MDA content were higher,the activity and the expression of HO-1 were lower in LPS+ZnPP than those in LPS group respectively (all P<0.05).CONCLUSION: CCK-8 attenuates the LPS-induced ALI by means of anti-oxidation and inhibits PMN aggregation,which are both mediated by HO-1 partly.     

Extract of Ginkgo Biloba decreased expression of lectin-like oxidized low density lipoprotein receptor-1 induced by TNF-α in bovine aortic endothelial cells

AIM: To investigate the effects of extract of Ginkgo biloba (EGB) on the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) induced by tumor necrosis factor a(TNF-α) in bovine aortic endothelial cells(BAECs). METHODS: The BAECs were incubated with TNF-α, followed by EGB treatment. The effect of EGB on LOX-1 expression was investigated by RT-PCR and Western blotting. The content of endothelial nitric oxide synthase (eNOS) was determined by Western blotting. Potential involvement of signaling pathways of the effects was explored by using related inhibitors of the signal molecules. The concentration of NO^-₂/NO^-₃ was also tested. RESULTS: Increased LOX-1 expression was induced by TNF-α. EGB markedly prevented the increase of LOX-1 expression induced by TNF-α (P<0.05), and this effect was inhibited by inhibitor of NOS (P<0.05). EGB significantly prevented the decrease of eNOS expression induced by TNF-α (P<0.05). EGB also significantly prevented the decrease of NO^-₂/NO^-₃ production induced by TNF-α (P<0.05). CONCLUSION: EGB markedly prevents the increase of LOX-1 expression induced by TNF-α and the effect is mediated by eNOS.     

Abnormal T-cell receptor signal pathway in murine autoimmue cardiom yophy induced with adenine nucleotide translocase

AIM: To explore the molecular mechanism in the pathogenesis of dilated cardiomyopathy (DCM) by analyzing the expression of T cell signaling molecules in mice with autoimmune DCM. METHODS: Mouse DCM model was induced by immunizing the animals with adenine nucleotide translocase (ANT) synthetic peptides. P56lck in T cells was detected with real-time fluorescent quantitative PCR in both DCM-group and the sham-immunized controls. At the same time, flow cytometry was used for quantity of Th cell intracellular cytokine IFN-γ and IL-4, ELISA for examining the level of serum anti-ANT antibody, immune histochemistry for investigating the expression of CD45 in Th cells. RESULTS: The mRNA expression of P56lck (1 369.51±874.05 vs 47.93±10.21, P<0.01), the percentage of IFN-γ and IL-4 (especially IL-4) (8.27±1.29 vs 5.58±0.59, P<0.01; 9.93±1.53 vs 2.05±0.21, P<0.01), the level of anti-ANT autoantibody (0.105±0.015 vs 0.006±0.002, P<0.01) and expression of CD45 (0.154±0.021 vs 0.026±0.008, P<0.01) were all elevated significantly in DCM-group compared with the controls. CONCLUSION: Impairment of T cell receptor (TCR) signal transduction pathway plays an important role in the development of DCM induced with ANT synthetic peptides in mice.     

Probucol inhibits proliferation of rat aortic smooth muscle cells stimulated with oxidized low-density lipoprotein

AIM: To investigate the relationships between antiproliferative mechanisms of probucol and protein expressions of signaling molecules ERK1/2, MKP-1, HO-1 and Trx-1 in rat aortic smooth muscle cells (RASMCs) stimulated with ox-LDL. METHODS: The effects of probucol on cell cycle, cell proliferation and the expressions of ERK1/2, MKP-1, HO-1 and Trx-1 in the presence of ox-LDL were observed by means of MTT test, FCM and Western blotting. RESULTS: (1) Probucol significantly inhibited the proliferation of RASMCs stimulated with ox-LDL. A value in 100 μmol/L probucol+35 mg/L ox-LDL group was reduced by 34.9% as compared to ox-LDL group (P<0.01). (2) Probucol protected against ox-LDL-induced RASMCs proliferation through inducing cell growth arrest at G0/G1 phase and cell apoptosis. (3) ox-LDL increased the expression of p-ERK1/2 by 34.7% (P<0.01) and decreased MKP-1 by 60.0% (P<0.01), respectively, as compared to control. Probucol attenuated the increase in ox-LDL-stimulated p-ERK1/2 level by 15.7%, but increased MKP-1 expression by 2 times (P<0.01). (4)ox-LDL at concentration of 35 mg/L decreased the intracellular Trx-1 expression by 28.9% (P<0.05), and slightly increased the level of HO-1 expression as compared to control (P<0.05). Probucol enhanced the expression of Trx-1 by 91.6% (P<0.01) and HO-1 by 31.9% (P<0.01), respectively as compared to ox-LDL group. CONCLUSION: Probucol inhibits ox-LDL-stimulated the proliferation of RASMCs through increases in MKP-1/HO-1 expression, suppression of cell cycle progression and induction of cell apoptosis.     

Effect of probucol and simvastatin on reactive oxygen species production and HO-1 expression induced by advanced glycation end products in rat renal microvascular endothelial cells

AIM: To investigate the effect of probucol and simvastatin on the production of reactive oxygen species (ROS) and heme oxygenase-1 (HO-1) expression induced by advanced glycation end products (AGEs) in rat renal microvascular endothelial cells (RMECs). METHODS: RMECs isolated and cultured from rat kidney were divided into 4 groups: normal control group, AGEs group, probucol group and simvastatin group. The levels of ROS were determined by the molecular probes of DCFH-DA. The expression of HO-1 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. RESULTS: (1) AGEs up-regulated ROS production and HO-1 expression in RMECs. (2) Probucol up-regulated HO-1 expression in RMECs, and inhibited the increasing level of ROS and expression of HO-1 in RMECs induced by AGEs. (3) Simvastatin also inhibited the increasing level of ROS in RMECs induced by AGEs, but it had no effect on HO-1 expression in RMECs with or without AGEs.CONCLUSION: Protective effect of probucol on the dysfunction of RMECs induced by AGEs may be related with its effect on the expression of HO-1 at mRNA and protein levels. Simvastatin also plays roles in antioxidation and renal protection, but is ineffective in the modulation of HO-1 expression.     

Effect of curcumin on ox-LDL-induced HAEC injury

AIM: To investigate the effect of curcumin on oxidized low-density lipoprotein (ox-LDL)-induced injury of human aortic endothelial cells (HAECs). METHODS: HAECs were pre-treated with curcumin at different concentrations and then treated with ox-LDL. The cell viability was assessed by MTT assay. The cell proliferation ability was analyzed by EdU assay. ELISA was used to determine the concentrations of interleukin-6 (IL-6), transforming growth factor β1 (TGFβ1), high mobility group box-1 protein (HMGB1) and secretory receptor for advanced glycation end products (sRAGE) in the HAEC culture medium. The binding activity of peroxisome proliferator-activated receptor γ (PPARγ) was evaluated by electrophoretic mobility shift assay. The protein levels of HO-1, HMGB1, RAGE,IL-6,TGFβ1 and phosphorylated PPARγ in the HAECs were determined by Western blot. RESULTS: The viability and the proliferation ability decreased significantly in the HAECs treated with ox-LDL. The PPARγ/HO-1 signaling pathway was inhibited while its down-stream HMGB1/RAGE signaling pathway was activated by ox-LDL. The levels of IL-6, TGFβ1, HMGB1 and sRAGE were increased. Pre-treatment with curcumin activated PPARγ/HO-1 signaling pathway and inhibited HMGB1/RAGE signaling pathway in ox-LDL treated HAECs in a concentration-dependent manner. The levels of IL-6, TGFβ1, HMGB1 and sRAGE were also decreased dramatically by pre-treatment of curcumin in a concentration-dependent manner. CONCLUSION: ox-LDL induces HAEC damage by inhibiting PPARγ/HO-1 to activate HMGB1/RAGE inflammatory signaling. Curcumin exerts protective effect on ox-LDL treated HAECs via activating PPARγ/HO-1 signaling pathway.     

Expression of oxygenase-1 protein and mRNA on mouse acute viral myocarditis caused by coxsackie viruses B3

AIM: To observe the dynamic variation of oxygenase-1 protein and mRNA on mouse acute viral myocarditis caused by coxsackie viruses B3.METHODS: A total 72 inbred male BALB/c mice of 4-6 weeks were divided randomly into 2 groups as follows: 32 mice were inoculated intraperitoneally (ip) with virus free 1640 culture solution 0.1 mL on day 0 as blank group (C); 40 mice were ip 0.1 mL tissue culture infectious dose 50 (TCID50 is 10-4.36/mL) coxsackie viruses B3 (CVB3) on day 0 as VMC group (V), then each mouse in both groups was ip 0.1 mL NS every day. 8 mice in each of C group and V group were sacrificed on 4, 8, 15, and 21 d respectively after infections. The blood specimens gathered by taking out the eyeballs of mice were tested for the content of carboxyhemoglobin (COHb) using spectrophotometer method.The heart tissue slides were also stained by immunohistochemistry (IHC) for HO-1 and in situ hybridization (ISH) for HO-1 mRNA. The histological and ultrastructure changes were observed under light microscope and electron microscope.RESULTS: (1)The histopathological changes of myocardial cells: many inflammatory cells were found in the heart and large area myocardial cells necrosis was observed under light microscope. The inflammatory area was reduced at late stage in the heart of mouse in group V, while the myocardium in group C was normal. (2) The myocardial observation by electron microscopy: the myofibril in group V was dissolved and mitochondrial membrane disappeared, mononuclear cell infiltration was also observed under electron microscopy, which contained many lysosomes. The myocardial cells in group C were normal. (3) The changes of blood COHb level: compared with group C, the group V COHb level showed significantly higher on the day 8 and day 15 after CVB3 innoculation (0.047±0.005 vs 0.031±0.004; 0.076±0.006 vs 0.030±0.005, P<0.01). No obvious change in group C was observed. (4) The result of HO-1 IHC staining: myocardial cells had positive expression in group V, group C was negative. The absorbance (A) values in group V was significantly higher than that in group C (P<0.01) at different time points. (5) The result of HO-1 ISH was similar to HO-1 IHC. The A values in group V was all higher than that in group C (P<0.01).CONCLUSION: Viral myocarditis caused by coxsackie viruses B3 induces the expression of HO-1 mRNA and protein, and these expressions may play self-protection in inflammation injury in myocardial cells.     

Effect of rhSOD on pulmonary nuclear factor-kappa B and MIP-1α expression in meconium-induced acute lung injury

AIM: To evaluate the role and mechanisms of recombinant human superoxide dismutase (rhSOD) in meconium-induced acute lung injury (ALI) by evaluating pulmonary MIP-1α and NF-κB expression. METHODS: 24 health male Sprage-Dawley rats were randomized to 3 groups (8, each group), followed by intratracheal (IT) administration with (1) saline at 1 mL/kg (control group); (2) 20% human newborn meconium suspension at 1 mL/kg, followed by saline at 1 mL/kg (Mec/saline group); (3) 20% human newborn meconium suspension at 1mL/kg, followed by rhSOD at 20 mg/kg (Mec/rhSOD group). The animal was killed 24 h after treatment. The measurements included the bronchoalveolar lavage (BAL) cell count, RT-PCR analysis of pulmonary MIP-1α mRNA expression, Western blotting analysis of pulmonary NF-κB expression. RESULTS: Meconium-induced ALI was characterized by increased BAL cell count, increased expressions of pulmonary MIP-1α mRNA and NF-κB protein ［(4.68±1.40)×109 cells/L vs (0.53±0.19)×109 cells/L, 3.60±0.75 vs 1.56±0.33, 0.72±0.31 vs 0.23±0.12, respectively in control rats, all P<0.01］. IT administration of rhSOD early in the ALI rat significantly decreased meconium-induced BAL cell count ［(3.13±0.77)×109 cells/L vs (4.68±1.40)×109 cells/L in Mec/saline rats, P<0.01］, inhibited the expression of pulmonary MIP-1α mRNA (2.20±0.39 vs 3.60±0.75, in Mec/saline rats, P<0.01) and NF-κB protein (0.44±0.21 vs 0.72±0.31 in Mec/saline rats, P<0.05). CONCLUSION: The early IT administration of rhSOD in ALI rat following meconium aspiration protects lung from inflammatory injury through inhibiting meconium-induced pulmonary MIP-1α mRNA and NF-κB protein expression.     

Increased central reactive oxygen species mediate the attenuated baroreflex function in heart failure

AIM: To determine the effect of reactive oxygen species on the baroreflex and to investigate the intracellular mechanism responsible for baroreflex dysfunction in the heart failure state.METHODS: In the rat model of cardiomyocytes infarct induced heart failure, baroreflex function was evaluated by measuring the relationship between renal sympathetic nerve activity(RSNA)responses and change of blood pressure by intravenous injection of nitroglycerin and phenylephrine. Alteration in baroreflex function was measured under the different reactive oxygen species(ROS)level induced by intracerebroventricular administration of several chemicals. RESULTS: (1)The range of RSNA response, average slope and maximum gain of baroreflex function curve were(92.2±9.9) mmHg,(0.07%±0.01%)/mmHg and(1.20%±0.10%)/mmHg, respectively, in CHF rats, which were significantly lower than those in sham rats(65.6±7.4) mmHg,(0.13%±0.02%)/mmHg and(3.00%± 0.20%)/mmHg(P<0.01).The minimum RSNA of baroreflex curve was higher in CHF rats than that in sham rat［(21.6%±4.8%)vs(7.5%±2.1%), P<0.01］.(2)Intracerebroventricular(icv)infusion of superoxide scavenger tempol and NADPH oxidase inhibitor apocynin significantly improved the blunted baroreflex in CHF rats. On contrast, icv administration of superoxide dismutase inhibitor diethyldithiocarbamate(DETC)decreased baroreflex function in sham rats.(3)The superoxide production in the hypothalamus of CHF rats was higher than that in sham rats［(73.9±9.8)RLU·5min-1·mg-1vs(40.6±7.1)RLU·5min-1·mg-1, P<0.01］.(4)Protein expression of NADPH oxidase subunits gp91phox in the paraventricular nucleus of the hypothalamus were increased by 1.3 fold in CHF rats than that in sham rats. CONCLUSION: Elevated intracellular ROS in the hypothalamus plays an important role in the attenuation of baroreflex function in the heart failure state and results from upregulation of NADPH oxidase protein expression.     

Effect of mycophenolic acid on T lymphocyte proliferation and cytokine expression in vitro

AIM: To investigate the effect of mycophenolic acid (MPA) alone or in combination with anti B7-1 mAb on proliferation of T lymphocytes and the possible mechanisms. METHODS: The proliferation of T lymphocytes was detected by BrdU incorporation method. The expressions of IL-2, IFN-γ and IL-10 in mRNA and protein levels were detected by RT-PCR and ELISA, respectively. RESULTS: (1) MPA markedly inhibited the T lymphocyte proliferation as compared with control (P<0.01). (2) MPA significantly inhibited the levels of IL-2 and IFN-γ (42.73 ng/L±14.64 ng/L vs 99.70 ng/L±9.15 ng/L, P<0.01; 7.87 ng/L±4.22 ng/L vs 82.42 ng/L±25.55 ng/L, P<0.05), and significantly increased content of IL-10 compared with control (770.95 ng/L±126.85 ng/L vs 545.71 ng/L±22.45 ng/L, P<0.05). MPA in combination with anti B7-1 mAb obviously enhanced the content of IL-10 compared with MPA alone (941.90 ng/L±56.61 ng/L vs 770.95 ng/L±126.85 ng/L, P<0.05). (3) The expression levels of IL-2 and IFN-γ mRNA in the MPA group were obviously lower than those in control (0.74±0.10 vs 1.17±0.15, 0.52±0.05 vs 0.75±0.12, P<0.01). MPA in combination with anti-B7-1 mAb showed a statistically significant increase in IL-10 mRNA expression (1.28±0.06 vs 0.84±0.09, P<0.01) as compared with MPA alone. CONCLUSION: MPA induces the changes of cytokine expressive spectrum and the Th1 and Th2 shift might be involved in the immunosuppressive effect. The combination of MPA with anti B7-1 mAb might have a synergic effect.