| BackgroundEndogenous inflammatory response is a vital factor in formation and progression of myocardial ischemia reperflision injury (IRI). Regulation of inflammatory response is proved to afford cardioprotection. It is well established that the vagal nerve can modulate inflammatory responses, termed the "cholinergic anti-inflammatorypathway"(CAP). Our previous works have demonstrated that postconditioning with the vagal stimulation can significantly attenuate the local and systemic inflammatory responses by myocardial IRI, and provide an obvious cardioprotection. However, vagal stimulation is still a complex invasive process and needs the expensive equipment. Thus, the substitutional drugs to activate CAP are more practical.The α7nicotinic acetylcholiner receptor (a7nAChR) is a key component of CAP. It can recognize, accept and transfer the sign from the vagal nerve and afford an immunomodulatory effect. The a7nAChR agonists have widely applied to treat various diseases associated with inflammation. Thus, we chose a specific a7nAChR agonist as a substitute of vagal stimulation and investigated its cardioprotction. Our recent works have proved that the a7nAChR agonist PNU282987can produce cardioprotection. However, the dose-effect relationship and mechanisms of PNU282987for cardioprotction are not well clarified.Glycogen synthase kinase3β (GSK-3β) is a multifunctional serine threonine kinase. The actived GSK-3β can amplify the inflammatory response, induce the irreversibly high level opening of the mitochondrial permeability transition pore (mPTP) and activate the apoptotic pathways in turn that aggravates myocardial IRI. Depressing the activation of GSK-3β is a common mechanism of various endogenous and exogenous myocardial protection interventions. Therefore, it is very possible to be the main mechanism of the cardioprotection of the postconditioning with PNU282987.The extensive opening of mPTP is another mechanism leading to mitochondrial-dependent apoptosis pathway. It has been demonstrated that depressing mPTP opening can significantly attenuate myocardial IRI. Just the same as inflammatory response, mPTP is also controlled by the GSK-3β. Moreover, those clues suggest that there may be some relationship between the inflammatory response and mPTP through the IRI. In order to clarify this hypothesis, we designed this randomized, controlled in vitro experiment. The aims of the present study included:1) establishing the in vitro model neonatal rat cardiomyocyte anoxia/reoxygenation injury;2) evaluating the protection of PNU282987against cardiomyocyte anoxia/reoxygenation injury in vitro and it’s dose-effect relationship;3) assessing the role of glycogen synthase kinase-3β (GSK-3β) in the cardioprotection of PNU282987;4) clarifing the relationship between mPTP and inflammatory response in the pathological process of myocardial IRI, and how they work in the cardioprotection of PNU282987.Part1Primary cultures of neonatal rat cardiomyocytes and establishment of in vitro cardiomyocyte anoxia/reoxygenation injury modelTo explore a mature and stable method for isolation and culture of neonatal rat cardiomyocytes and establish in vitro rat cardiomyocyte anoxia/reoxygenation injury model with an anaero-pack system.Cardiac ventricular myocytes were isolated from1-to2-day-old Sprague-Dawley rats using0.125%trypsin and0.1%collagenase digestions and purified by differential velocity adherent technique for1h combined with5-BrdU treatment. The activity of cardiomyocytes was investigated through the phase-contrast micrscopy. The purity of cardiomyocytes was examined by cardiac troponia T (cTnT) immunofluorescence and2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining.The in vitro rat cardiomyocyte anoxia/reoxygenation injury model was established by an anaero-pack system. After cardiomyocytes were cultured for72h, they were randomly divided into7groups:Sham group, cultured as normal;3h group, only anoxia for3h;3h/6h group, anoxia for3h followed by6h reoxygenation;6h group, only anoxia for6h;6h/6h group, anoxia for6h followed by6h reoxygenation;9h group, only anoxia for9h;9h/6h group, anoxia for9h followed by6h reoxygenation. At the end of the experiment, lactate dehydrogenase (LDH) release rate was detected by the LDH Release Assay Kit and myocardial apoptosis was detected by the dead cell apoptosis Kit with Annexin V/propidine iodide (Annexin V/PI) for flow cytometry.Neonatal cardiomyocytes were active and contracted strongly. After cultured for48-72h, they contracted synchronously. The purity of cardiomyocytes was up to94.2±2.0%.The results showed that compared to Sham group, the rate of LDH release, early and late apoptosis were significantly higher, but the rate of normal cell was significantly lower in3h,3h/6h,6h,6h/6h,9h and9h/6h groups.As compared to3h group, the LDH release rate, early and late apoptosis were higher, but the rate of normal cell was lower in6h and9h groups. There were no significant differences in the LDH release rate, early and late apoptosis, and the rate of normal cell in the3h/6h group than in the3h group.As compared to6h group, the LDH release rate, early and late apoptosis were significantly increased, but the rate of normal cell was significantly decreased in9h and6h/6h groups.The LDH release rate, early and late apoptosis, and the rate of normal cell did not differ between9h/6h and9h groups.Part2Experimental study on cardioprotective and anti-inflammatory effects of postconditioning with different-dose PNU282987in rat cardiomyocyte anoxia/reoxygenation injury modelAims of this part experiment were to evaluate cardioprotective and anti-inflammatory effects of postconditioning with different-dose PNU282987in rat cardiomyocyte anoxia/reoxygenation injury model.Based on the different treatments, the cardiomyocytes were randomly divided into6groups:Sham group, the cardiomyocytes were cultured as normal but only adding the same concentration DMSO as the other groups6h prior to detection; anoxia/reoxygenation injury group (AR group), the reoxygenation medium contained DMSO with the same concentration as the other groups;3μM PNU282987 postconditioning group (3μM group), the reoxygenation medium contained3μM PNU282987;10μM PNU282987postconditioning group (10μM group), the reoxygenation medium contained10μM PNU282987;30uM PNU282987postconditioning group (30μM group), the reoxygenation medium contained30μM PNU282987;60μM PNU282987postconditioning group (60μM group), the reoxygenation medium contained60μM PNU282987. At the end of the experiment, the LDH release rate and apoptosis were detected, and the supernatant concentrations of interleukin-6(IL-6) and tumor necrosis factor-a (TNF-a) were assaryed with the Enzyme-linked immunosorbent assay (ELISA) method.The results showed that compared to the Sham group, the LDH release rate, early and late apoptosis, and the supernatant concentrations of TNF-a and IL-6were significantly higher, but the rate of normal cell was significantly lower in the AR,3μM,10μM,30μM and60μM groups.The LDH release rate and the supernatant concentrations of TNF-a and IL-6were significantly decreased, but the rate of normal cell was significantly increased in the3μM,10μM,30μM and60μM groups compared with those in the AR group. The early apoptosis was also reduced significantly in the10μM,30μM and60μM groups compared to that in the AR group, but there was no significantly difference in3μM group than in the AR group. The rate of late apoptosis did not differ among the AR,3μM,10μM,30μM and60μM groups.The LDH release rate, early apoptosis and the supernatant concentrations of TNF-a and IL-6were significantly lower, but the rate of normal cell was significantly higher in the10μM group compared with those in the3μM group.The LDH release rate, early apoptosis and the supernatant concentrations of TNF-a and IL-6were significantly lower, but the rate of normal cell was significantly higher in the30μM group compared with those in the10μM group.The LDH release rate, early apoptosis and the supernatant concentrations of TNF-a and IL-6were significantly increased, but the rate of normal cell was significantly decreased in the30μM group compared with those in the10μM group.Part3Experimental study on the role of GSK-3β in the cardioprotection of postconditioning with PNU282987The aim of this part experiment was to assess the role of GSK-3β in cardioprotection of postconditioning with PNU282987.Based on the different treatments, the cardiomyocytes were randomly divided into4groups:Sham group, the cardiomyocytes were cultured as normal but only adding the same concentration DMSO as the other groups6h prior to detection; anoxia/reoxygenation injury group (AR group), the reoxygenation medium contained DMSO with the same concentration as the other groups; GSK-3β inhibitor TDZD-8postconditioning group (TDZD-8group), the reoxygenation medium contained10μM TDZD-8); PNU282987postconditioning group (PNU282987group), the reoxygenation medium contained30μM PNU282987. At the end of the experiment, the LDH release rate, myocardial apoptosis and the supernatant concentrations of IL-6and TNF-a were measured. The GSK-3β, phosphorylated GSK-3βSer9, NF-κBp65and phosphorylated NF-κBp65Ser536in all samples were assessed by Western-blotting technique. The mitochondrial membrane potential was measured by Rhodamine123staining flow cytometry and confocal laser scanning microscope.The results showed that compared to the Sham group, the LDH release rate, early and late apoptosis and the supernatant concentrations of TNF-a and IL-6were significantly higher, but the rates of normal cell and mitochondrial membrane potential were significantly lower in the PNU282987, TDZD-8and PNU282987groups. There were no significant differences in the GSK-3β among all groups. The phosphorylated GSK-3βser9was significantly increased in the PNU282987, TDZD-8and PNU282987groups compared that in the Sham group. Both NF-icBp65and phosphorylated NF-KBp65Ser536were significantly increased in the PNU282987, TDZD-8and PNU282987groups than in the Sham group.As compared to the AR group, the LDH release rate and early apoptosis, and the supernatant concentrations of TNF-a and IL-6were significantly lower in TDZD-8and PNU282987groups. The rate of normal cell and mitochondrial membrane potential were significantly increased in the TDZD-8and PNU282987groups. The rate of late apoptosis did not differ among the AR, TDZD-8and PNU282987groups. Compared to the AR group, the phosphorylated GSK-3βSer9was significantly increased in the TDZD-8and PNU282987groups. There was no significant difference in the NF-κBp65among the AR, TDZD-8, PNU282987groups. The phosphorylated NF-κBp65Ser536was significantly decreased in the TDZD-8and PNU282987groups than in the AR group.The LDH release rate and early apoptosis, and the supernatant concentrations of TNF-a and IL-6were significantly higher in the PNU282987group than in the TDZD-8group. The rate of normal cell and mitochondrial membrane potential were significantly lower in the PNU282987group than in the TDZD-8group. The phosphorylated GSK-3(3Ser9was significantly lower, but the phosphorylated NF-KBp65Ser536was significantly higher in the PNU282987group compared with those in the TDZD-8group.Part4The roles of mitochondrial permeability transition pore and inflammatory response in the cardioprotection of pstconditioning with PNU282987and their interactionBased on the results from the above third part of this experiment, this part experiment was designed to evaluate the roles of mitochondrial permeability transition pore (mPTP) and inflammation response and their interaction in the cardiomyocyte anoxia/reoxygenation injury, and to clarify how they modulate the cardioprotection of postconditioning with PNU282987.According to the different treatments, the cardiomyocytes were randomly divided into6groups:Sham group, the cardiomyocytes were cultured as normal but only adding the same concentration DMSO as the other groups6h prior to detection; anoxia/reoxygenation injury group (AR group), the reoxygenation medium contained DMSO with the same concentration as the other groups; PNU282987postconditioning group (PNU282987group), the reoxygenation medium contained30μM PNU282987; mPTP opening inhibitor, Ciclosporin A(CSA) postconditioning group (CSA group), the reoxygenation medium contained1μM CSA; mPTP opening activator, atractyloside (ATR) postconditioning group (ATR group), the reoxygenation medium contained20μM ATR and DMSO with the same concentration as the other groups; PNU282987combined with ATR postconditioning group (P+A group), the reoxygenation medium contained30μM PNU282987and20μM ATR). At the end of the experiment, LDH release rate, myocardial apoptosis, the supernatant concentrations of IL-6and TNF-a, NF-κBp65, phosphorylated NF-κBp65Ser536and mitochondrial membrane potential were measured.The results showed that the LDH release rate, early and late apoptosis, NF-κBp65, phosphorylated NF-KBp65and the supernatant concentrations of TNF-a and IL-6were significantly higher, but the rates of normal cell and mitochondrial membrane potential were significantly lower in the AR, PNU282987, CSA, ATR and P+A groups compared with those in the Sham group.As compared to the AR group, the early apoptosis, phosphorylated NF-κBp65Ser536and the supernatant concentrations of TNF-a and IL-6were significantly decreased, but the rate of normal cell was significantly increased in the PNU282987, CSA and P+A groups. The LDH release rate were significantly decreased, but mitochondrial membrane potential was significantly increased in the PNU282987and CSA groups compared with those in the AR group.The LDH release rate, early apoptosis, phosphorylated NF-κBp65Ser536, and supernatant concentrations of TNF-a and IL-6were significantly increased in the ATR group than in AR group. Both the rate of normal cell and mitochondrial membrane potential were significantly decreased in the ATR group than in the AR group.There were no significant differences in the late apoptosis and NF-κBp65among the AR, PNU282987, CSA, ATR and P+A groups.The LDH release rate, early apoptosis and the rate of normal cell did differ significantly between the PNU282987and CSA groups. The phosphorylated NF-κBp65Ser536and supernatant concentrations of TNF-a and IL-6were significantly lower in the PNU282987group than in the CSA group. However, mitochondrial membrane potential was significantly higher in the CSA group than in the PNU282987group.As compared to the PNU282987group, the LDH release rate, early apoptosis, phosphorylated NF-κBp65Ser536and the supernatant concentrations of TNF-a and IL-6were significantly increased, but the rate of normal cell and mitochondrial membrane potential were significantly decreased in the P+A group.The LDH release rate, early apoptosis, phosphorylated NF-κBp65Ser536and the supernatant concentrations of TNF-a and IL-6were significantly decreased, but the rate of normal cell and mitochondrial membrane potential were significantly increased in the P+A group compared with those in the ATR group.The interaction between PNU282987postconditioning and ATR postconditioning was analyzed by factorial analysis of variance. The results revealed there were antagonistic actions between them on the LDH release rate, early and late apoptosis, rate of normal cell, and the supernatant concentrations of TNF-a and IL-6. However, there were additional interaction between them on the phosphorylated NF-κBp65Ser536and mitochondrial membrane potential.ConclusionsBased on the results of all experiments, the following conclusions can be drawn:1. An in vitro rat cardiomyocyte anoxia/reoxygenation injury model can be successfully establish by the means of anoxia for6h followed by6h reoxygenation with an anaero-pack system.2. The postconditioning with different-dose PNU282987can product anti-inflammatory effect and provide obvious cardioprotection. The anti-inflammatory effect of postconditioning with PNU282987is dose-dependent, the cardioprotection postconditioning with PNU282987has a "ceiling effect", and the maximum effective concentration is30μM.3. Both PNU282987postconditioning and TDZD-8postconditioning can attenuate cardiomyocyte anoxia/reoxygenation injury via depressing the activity of GSK-3β, a key regulator of inflammatory response and mPTP. However, the cardioprotection induced by TDZD-8postconditioning is stronger than that by PNU282987postconditioning.4. The degree of cardiomyocyte injury by anoxia/reoxygenation is dependent on the level of mPTP opening. Also, the mPTP can influence the inflammatory response by cardiomyocyte anoxia/reoxygenation injury. The CSA postconditioning can significantly depresse the inflammatory response and maintain the normal mitochondrial membrane potential via inhibiting the mPTP opening, which affords an obvious cardioprotection. Also, ATR postconditioning can significantly aggravate the cardiomyocytes anoxia/reoxygenation injury via increaseing inflammatory response and decreaseing the mitochondrial membrane potential.5. Extensive mPTP opening can antagonize the cardioprotection and the decrease effects of the PNU282987postconditioning on IL-6and TNF-a. At the same time, the PNU282987postconditioning can antagonize the myocardial injury and increase effect on IL-6and TNF-a due to the extensvive mPTP opening.6. Other than inhibition of apoptosis pathway induced by the mPTP opening, there are other mechanisms of PNU282987postconditioning against cardiomyocytes apoptosis... |
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