ABSTRACT
OBJECTIVE: To investigate whether lipoxin A4 (LXA4) increases expression of heme oxygenase-1(HO-1) in cardiomyocytes, whether LXA4-induced HO-1 protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury, and what are the mechanisms involved in the LXA4-induced HO-1 induction. METHODS: Rat cardiomyocytes were exposed to H/R injury with or without preincubation with LXA4 or HO-1 inhibitor ZnPP-IX or various signal molecule inhibitors. Expressions of HO-1 protein and mRNA were analyzed by using Western blot and RT-PCR respectively. Activity of nuclear factor E2-related factor 2 (Nrf2) binding to the HO-1 E1 enhancer was assessed by chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) were measured by using electrophoretic mobility shift assay. RESULTS: Pretreatment of the cells undergoing H/R lesion with LXA4 significantly reduced the lactate dehydrogenase and creatine kinase productions, increased the cell viability, and increased the expressions of HO-1 protein and mRNA and HO-1 promoter activity. HO-1 inhibition abolished the protective role of LXA4 on the cells undergoing H/R lesion. LXA4 increased p38 mitogen-activated protein kinase (p38 MAPK) activation, nuclear translocation of Nrf2, Nrf2 binding to the HO-1 ARE and E1 enhancer in cardiomyocytes with or without H/R exposure. CONCLUSION: The protection role of LXA4 against H/R injury of cardiomyocytes is related to upregulation of HO-1, via activation of p38 MAPK pathway and nuclear translocation of Nrf2 and Nrf2 binding to the HO-1 ARE and E1 enhancer, but not via activation of phosphatidyinositol-3-kinase or extracellular signal-regulated kinase pathway.
Subject(s)
Antioxidant Response Elements/genetics , Heme Oxygenase-1/biosynthesis , Hypoxia/enzymology , Lipoxins/pharmacology , Myocytes, Cardiac/pathology , NF-E2-Related Factor 2/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cardiotonic Agents/pharmacology , Cell Shape/drug effects , Cell Survival/drug effects , Creatine Kinase/metabolism , Enzyme Induction/drug effects , Heme Oxygenase-1/genetics , Hypoxia/genetics , Hypoxia/pathology , L-Lactate Dehydrogenase/metabolism , Male , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/enzymology , Oxygen/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Transport/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Wistar , Transcription, Genetic/drug effects , Up-Regulation/drug effectsABSTRACT
Studies have shown that lipoxin A4 (LXA4) and activation of LXA4 receptor provided protection against myocardial ischemia/reperfusion injury in animal models. However, the mechanisms by which LXA4 induced protective role on myocardial ischemia/reperfusion injury remains unclear. In the present studies, we investigated the protective effects of LXA4 on H9c2 cardiomyocytes exposed to hypoxia/reoxygenation (H/R) injury and involvement of heme oxygenase-1 (HO-1)- and K(+) channel-dependant pathways in the LXA4 action. H9c2 cardiomyocytes were pretreated with or without LXA4 or HO-1 specific interfering RNA (siRNA) or various blockers and openers of K(+) channels before exposing to H/R injury. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) in cellular supernatants and necrosis factor-α (TNF-α) in cellular lysates were measured by using ELISA. Expressions of HO-1 mRNA and protein were analyzed by using RT-PCR and Western blot respectively. Pretreatment of the cells undergoing H/R injury with LXA4 significantly reduced the LDH and CK levels induced by H/R injury, and increased the expressions and activity of HO-1. However, the protective effects of LXA4 were completely blocked by transfection of the cells with HO-1 siRNA, and were partially but significantly blocked by pretreatment of the cells with various blockers of K(+) channels. The LXA4-induced expressions of HO-1 in the cells were also inhibited by HO-1 siRNA and various blockers of K(+) channels. The inhibitory effects of LXA4 on enhanced TNF-α levels induced by H/R injury were abolished by transfection of the cells with HO-1 siRNA. In conclusion, the protective role of LXA4 on cardiomyocytes against H/R injury is related to upregulation of HO-1 via reduced production of TNF-α and activation of ATP-sensitive K(+) channels and calcium-sensitive K(+) channel.
Subject(s)
Lipoxins/metabolism , Myocytes, Cardiac/metabolism , Potassium Channels/metabolism , Animals , Cell Hypoxia , Creatine Kinase/genetics , Creatine Kinase/metabolism , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Lipoxins/pharmacology , Myocardial Reperfusion Injury/metabolism , Myocytes, Cardiac/pathology , RNA, Messenger/metabolism , Rats , Up-RegulationABSTRACT
OBJECTIVE: To evaluate the effect of Xiangdan Injection on mRNA expression of the endothelial vaso-active factors of patients with coronary heart disease and blood stasis. METHODS: Fifty-six patients were randomly divided into two groups:twenty-eight patients were treated according to the therapeutic guide for coronary heart disease as the control group and 28 were given the same treatment plus Xiangdan Injection as the treated group. The expressions of ET-1 and eNOS mRNA were examined with RT-PCR before experiment and ten days later. RESULTS: The positive rate of eNOS mRNA of the treated group increased, while the positive rate of ET-1 mRNA of the treated group decreased after ten day's treatment, with significant differences as compared with that before the experiment. Xiangdan Injection up-regulated the eNOS mRNA expression and suppressed the ET-1 mRNA expression. Changes of expression were not observed in the control group. CONCLUSION: Xiangdan Injection improves the endothelial function of patients with coronary heart disease and blood stasis by regulating the expressions of ET-1 and eNOS mRNA.
