| The incidence of ischemic heart diseases is high in recent years,including angina pectoris and acute myocardium infarction.With the wide application of thrombolytic therapy,early primary percutaneous coronary intervention?PCI?and extracorporeal circulation,reperfusion therapies can regain blood perfusion and restore oxygen supply in a short period of time,accordingly playing effective roles for ischemic heart disease.Despite this improvement,reperfusion sometimes can also lead to the destruction of cardiac structure or function,which is generally referred to as myocardial ischemia reperfusion injury?MIRI?.Clinically,it can result in malignant arrhythmia,cardiac insufficiency and sudden death,which are seriously harmful to health.Therefore,MIRI has always been one of the focus topics for cardiovascular physicians.How to reduce ischemia reperfusion injury when restoring coronary blood perfusion as early as possible has become a major problem in urgent need of a solution for the treatment of coronary heart disease,especially for acute myocardial infarction.The mechanisms leading to the pathogenesis of MIRI are complex and multifactorial.Changes in mitochondrial membrane permeability,calcium overload or calcium redistribution,oxygen radical generation and complement system activation are important in above process.The mitochondrial permeability transition pore?mPTP?and ATP-sensitive potassium(KATP)channel are the two pivotal action sites in ischemia reperfusion injury.KATP channel is mainly divided into sarcolemmal KATP and mitochondrial KATP channel(mitoKATP).The opening of mPTP leads to the loss of inner membrane potential,respiratory chain uncoupling,mitochondrial ATP synthesis retardation,and ultimately mitochondrial swelling and cell apoptosis.The mitoKATP channel is widely believed to play an important role in cardioprotection by preconditioning and postconditioning,consequently reducing MIRI.Furthermore,the opening of mitoKATP channel inhibits mPTP,accordingly blocking the cascade reaction of downstream apoptotic protease activation of mPTP and inhibiting myocardial apoptosis.C-reactive protein?CRP?is an acute-phase plasma protein synthesized by the liver.CRP can react with pneumococcal C polysaccharide in the presence of calcium.In physiological conditions,the CRP levels are very low,and rising obviously when acute inflammation or tissue damage occurs.Therefore,CRP was mainly used for test and evaluation of acute injury and inflammation at the earliest moment.Then,CRP was found to be a powerful independent risk indicator that predicted future cardiovascular events in the 1980s,which had stimulated a strong interest among doctors,especially cardiologists.Subsequently,many large epidemiological investigations and experimental studies support CRP as a strong predictor of the development of cardiovascular and cerebrovascular diseases such as atherosclerosis,coronary heart disease and ischemic stroke.Furthermore,recent research has implicated CRP as a stimulating factor involved in atherosclerosis progression and ischemia reperfusion injury.However,whether CRP plays a direct role in aggravating ischemia reperfusion injury remains a question,and the underlying mechanisms are not well understood.In addition,as mentioned above,the mPTP and mitoKATP channel are the two pivotal action sites in ischemia reperfusion injury.Diazoxide,a selective mitoKATPTP channel agonist,and cyclosporin A,an inhibitor of mPTP,can both reduce ischemia reperfusion injury.However,the relationships between CRP and these two channels are uncertain.Discussing these problems can help us provide new therapeutic targets to reduce reperfusion injury,which can allow more patients to benefit from reperfusion therapies.Furthermore,a number of signaling pathways,including Jun N-terminal kinase/stress activated protein kinase?JNK/SAPK?,extracellular signal-regulated kinase?ERK?and phosphatidyl inositol 3-kinase/serine-threonine kinase?PI3K/Akt?play pivotal roles in ischemia reperfusion.Information is scarce regarding the connection between CRP and these signaling pathways.Investigating these problems can help us thoroughly understand the mechanisms of CRP-mediated myocardial ischemia reperfusion injury,thereby providing sufficient theoretical basis to reduce reperfusion injury.In addition,statins have a wide range of biological effects except reducing lipid levels,including inhibition of inflammatory response,improvement of endothelial function and plaque stabilization.Both ischemia reperfusion injury and statin effects are our research fields in recent years.It has been confirmed that pathological levels of CRP can directly participate in the process of atherosclerosis.We hypothesized that CRP,as a stimulating factor,could aggravate myocardial ischemia reperfusion injury.Thus,CRP could be a new therapeutic target,and ischemia reperfusion injury could be reduced by decreasing CRP levels in clinical application.Although no CRP specific blocker has been widely applied to clinical practice,some related basic and clinical studies have confirmed that aspirin,statins and other drugs can reduce CRP levels.The Justification for Use of statins in Prevention:an Intervention Trial Evaluating Rosuvastatin?JUPITER?study placed emphasis on ostensibly healthy people with elevated hsCRP and decreased LDL cholesterol levels who benefited from statin therapy.The results showed that statin treatment was associated with a reduction of high-sensitivity CRP?hsCRP?and decreased incidence of cardiovascular events and all-cause mortality.Previous studies in our research group have found that statins can activate mitoKATP channels and reduce reactive oxygen species and calcium overload,thereby inhibiting mPTP opening and further reducing myocardial ischemia reperfusion injury.This study will also discuss the protective roles of statin on CRP-induced myocardial ischemia reperfusion injury.Therefore,we simulated ischemia reperfusion using oxygen-glucose deprivation/reoxygenation?OGD/R?in isolated Sprague-Dawley rat cardiomyocyte primary cultures and compared the changes of myocardial cell viability,activity levels of lactate dehydrogenase?LDH?and other indicator,to definite the effects of CRP on myocardial ischemia reperfusion injury.At the same time,the opening degree of mPTP and mitochondrial membrane potential were measured after adding diazoxide or cyclosporin A to OGD/R,to understand the relationship between CRP and mitoKATP,mPTP.The mechanisms of CRP on the correlation signal pathways were also discussed to provide a research basis for early detection of specific CRP blockers.We also aimed to investigate the possible signaling pathways involved in the protective effects of atorvastatin?Ator?against CRP-induced myocardial ischemia reperfusion injury,by observing the changes of the above indexes after the combined treatment with Ator based on CRP intervention.Thus,these findings can provide a more sufficient theoretical basis for the treatment of patients with high CRP levels in clinical work to reduce ischemia reperfusion injury.Part one The effects of C-reactive protein on myocardial oxygen-glucose deprivation/reoxygenation in SD ratsObjective:To investigate the effects of C-reactive protein intervention on myocardial oxygen-glucose deprivation/reoxygenation?OGD/R?injury in rats,to simulate the process of myocardial ischemia reperfusion injury in vivo.Methods:We simulated ischemia reperfusion using oxygen-glucose deprivation/reoxygenation?OGD/R?in neonatal Sprague-Dawley rat cardiomyocytes,which were prepared from 1 to 2 days-old neonatal SD rat;reperfusion injury was induced by 3 hours of hypoxia with glucose and serum deprivation followed by 1 hour of reperfusion.The determination of CRP concentrationThe CRP concentrations used in previous studies were commonly 5?g/mL to 50?g/mL.Therefore,we tested different final concentrations of CRP starting at 5?g/mL.Our pre-experiments showed that incubating cardiac myocytes with a final concentration of 10?g/mL of CRP before OGD has significant effects on ischemia reperfusion injury,as established by MTS assays.Primary cultured neonatal rat myocardial cells were randomLy divided into the following groups:1.Control group:cardiomyocytes were cultured under normal conditions in high-glucose DMEM,in room air at 37°C.2.OGD/R group:the cell samples were subjected to 3 hours OGD and 1hour of reoxygenation.3.CRP+OGD/R group:before OGD/R,cardiomyocytes were incubated with 10?g/mL of CRP for 24 hours.After OGD/R,cell viability was tested with MTS assay,and cardiomyocyte damage was evaluated by lactate dehydrogenase?LDH?leakage,to evaluate the extent of myocardial damage.Mitochondrial membrane potential was measured using tetramethylrhodamine ethyl ester?TMRE?and mitochondrial permeability transition pore?mPTP?opening was measured using calcein/AM;both TMRE and caocein/AM were visualized with laser scanning confocal microscopy?LSCM?.Results:1.The effect of CRP intervention on myocardial cell viability after OGD/RCell viability was measured with the MTS assay,and calculated as the percentage of the control group.OGD/R-induced damage significantly decreased cell viability to 82.36%±6.18%of the control?P=0.0031?.Intervention with 10?g/mL CRP further decreased the cell viability to 64.84%±4.06%of the control?P<0.0001?,and the cell viability in the CRP+OGD/R group was significantly different from the value in the OGD/R group?P=0.0007?.2.The effect of CRP intervention on LDH levels of myocardial cells after OGD/RCompared with control group,the LDH levels in the OGD/R group were significantly increased?95.10U/L±21.57U/L versus 145.30 U/L±16.06 U/L,P=0.0319?.In addition,a significant difference was observed in LDH leakage in the CRP pretreatment group?208.20 U/L±19.23 U/L,P=0.0122?compared with the OGD/R group lacking CRP.3.The effect of CRP intervention on mPTP opening after OGD/RFluorescent intensities were expressed as percent changes compared with the values in control group.Compared with the control group,the green fluorescence intensity of myocardial cells in OGD/R group decreased significantly?60.93%±2.82%,P<0.0001?.After treatment with 10?g/mL CRP,the average fluorescence intensity values of calcein/AM significantly decreased compared with the OGD/R group?33.08%±3.48%,P<0.0001?.4.The effect of CRP intervention on mitochondrial membrane potential after OGD/RFluorescent intensities were expressed as percent changes compared with the values in control group.The red fluorescence intensity of the OGD/R group was significantly lower than the level in the control group?57.26%±2.95%,P<0.0001?.After the CRP intervention,the fluorescence intensity was further decreased markedly?33.31%±2.03%,P<0.0001?.Conclusions:1.The intervention of CRP significantly reduced the vitality of myoca-rdial cells after OGD/R.2.The CRP intervention increased the LDH levels in cell culture medium after OGD/R.3.The intervention of CRP increased the degree of mPTP opening after OGD/R.4.The CRP intervention significantly reduced the levels of mitochondrial membrane potential after OGD/R.5.CRP aggravated the oxygen-glucose deprivation/reoxygenation injury in cardiomyocytes.Part two The relationships between CRP and mitoKATP,CRP and mPTP during oxygen-glucose deprivation/reoxygenation,and the effects of CRP on signaling pathways.Objective:To explore the relationships between CRP and mitoKATP,CRP and mPTP during oxygen-glucose deprivation/reoxygenation,and to discuss the effects of CRP on the signaling pathways involved in oxygen-glucose deprivation/reoxygenation injury in cardiomyocytes.These findings can help us to understand the possible mechanisms of CRP-induced oxygen-glucose deprivation/reoxygenation injury.Methods:We simulated ischemia reperfusion using oxygen-glucose deprivation/reoxygenation?OGD/R?in neonatal Sprague-Dawley rat cardiomyocytes;reperfusion injury was induced by 3 hours of hypoxia with glucose and serum deprivation followed by 1 hour of reperfusion.Primary cultured neonatal rat myocardial cells were randomLy divided into the following groups:1.Control group:cardiomyocytes were cultured under normal conditions in high-glucose DMEM,in room air at 37°C.2.OGD/R group:the cell samples were subjected to 3 hours OGD and 1hour of reoxygenation.3.CRP+OGD/R group:before OGD/R,cardiomyocytes were incubated with 10?g/mL of CRP for 24 hours.4.CRP+CsA+OGD/R group:cardiomyocytes were treated with 10?g/mL of CRP for 24 hours and then subjected to 3 hours OGD.After the onset of reoxygenation,the cells were exposed to 10?M CsA for 20 minutes,continuous reperfusion of a total of 1h.5.CRP+Diazoxide+OGD/R group:cells were incubated with 10?g/mL of CRP for 24 hours,washed with medium to remove CRP,and then exposed to 100?M diazoxide for 90 minutes before OGD/R.After OGD/R,cell viability was tested with MTS assay,and cardiomyocyte damage was evaluated by LDH leakage,to evaluate the extent of myocardial damage.Mitochondrial membrane potential was measured using TMRE and mPTP opening was measured using calcein/AM;both TMRE and caocein/AM were visualized with LSCM.In the first three groups,the expression levels of ERK,JNK and others were detected by Western blot,to explore the signaling pathways associated with CRP-mediated oxygen-glucose deprivation/reoxygenation injury in cardiomyocytes.Results:1.The effects of cyclosporine A and diazoxide on the decreased cell viability induced by CRPCell viability was measured with the MTS assay,and calculated as the percentage of the control group.The cell viability of OGD/R group after CRP intervention decreased further than OGD/R group?64.84%±4.06%?.However,after pretreatment with cyclosporin A or diazoxide,the cell viability increased significantly?91.30%±5.18%,92.63%±5.74%?.Compared with CRP+OGD/R group,the differences were both statistically significant?P<0.0001?.2.The effects of cyclosporine A and diazoxide on the increased LDH leakage induced by CRPThe LDH level of CRP+OGD/R group was significantly higher than the level in OGD/R group?208.20U/L±19.23U/L versus 145.30 U/L±16.06 U/L,P=0.0122?,and it can be reduced in advance with the treatment of cyclosporine A or diazoxide?92.72U/L±17.56U/L,93.37U/L±23.99U/L,P=0.0015,0.0029?.3.The effects of cyclosporine A and diazoxide on the mPTP opening induced by CRPFluorescent intensities were expressed as percent changes compared with the control group values.In OGD/R group,the fluorescence intensity was weaker than the level in control group,and the fluorescence intensity after CRP intervention was further decreased?33.08%±3.48%?.However,additional intervention with diazoxide or CsA enhanced the fluorescence intensities,compared with CRP+OGD/R group?85.09%±4.11%,81.17%±4.13%,P<0.0001?.4.The effects of cyclosporine A and diazoxide on the decreased mitochondrial membrane potential induced by CRPFluorescent intensities were expressed as percent changes compared with the value in control group.The fluorescence intensity in CRP+OGD/R group was significantly decreased?33.31%±2.03%?.Similarly,the fluorescence intensity was significantly increased after pretreatment with cyclosporine A or diazoxide?77.35%±2.38%,72.26%±2.45%,P<0.0001?.5.The effects of CRP on signaling pathways during oxygen-glucose deprivation/reoxygenation.The expression levels of ERK,JNK and others were detected by Western blot.The levels of phosphorylated ERK1/2?phospho-ERK1/2?and total ERK1/2 were both markedly increased after treatment with CRP.The difference was especially significant for phospho-ERK1/2?93.53%±1.94%versus 170.4%±3.00%,P<0.0001?.However,no remarkable changes of the total levels of p38MAPK and JNK were observed after CRP treatment followed by OGD/R compared with the OGD/R group.Conclusions:1.Cyclosporine A and diazoxide can offset the above changes caused by CRP,demonstrating that both mPTP and mitoKATP were involved in CRP-mediated myocardial ischemia reperfusion injury2.CRP aggravated oxygen-glucose deprivation/reoxygenation injury by activation of the ERK signaling pathwayPart three The cardioprotective effects and mechanisms of atorvastatin on CRP-induced oxygen-glucose deprivation/reoxygenation injuryObjective:To investigate the protective effects of atorvastatin pretreatment on oxygen-glucose deprivation/reoxygenation injury induced by CRP and to explore the possible mechanisms.Methods:We simulated ischemia reperfusion using oxygen-glucose deprivation/reoxygenation?OGD/R?in neonatal Sprague-Dawley rat cardiomyocytes;reperfusion injury was induced by 3 hours of hypoxia with glucose and serum deprivation followed by 1 hour of reperfusion.Primary cultured neonatal rat myocardial cells were randomLy divided into the following groups:1.Control group:cardiomyocytes were cultured under normal conditions in high-glucose DMEM,in room air at 37°C.2.OGD/R group:the cell samples were subjected to 3 hours OGD and 1hour of reoxygenation.3.CRP+OGD/R group:before OGD/R,cardiomyocytes were incubated with 10?g/mL of CRP for 24 hours.4.Ator+CRP+OGD/R group:cells were pre-conditioned with 1?M Ator for 3 hours,10?g/mL of CRP for 24 hours,and then subjected to OGD/R.After OGD/R,cell viability was tested with MTS assay,and cardiomyocyte damage was evaluated by LDH leakage,to evaluate the extent of myocardial damage.Mitochondrial membrane potential was measured using TMRE and mPTP opening was measured using calcein/AM;both TMRE and caocein/AM were visualized with LSCM.The expression levels of ERK,Akt and JNK were detected by Western blot,to explore the signaling pathways associated with the protective effects of Ator involved in CRP-mediated oxygen-glucose deprivation/reoxygenation injury in cardiomyocytes.Results:1.The effects of Ator pretreatment on cell viability induced by CRP after OGD/RCell viability was measured with the MTS assay,and calculated as the percentage of the control group.Compared with CRP+OGD/R group,the cell viability was significantly increased after pretreatment with the Ator?64.84%±4.06%versus 98.61%±5.01%,P<0.0001?.2.The effects of Ator pretreatment on LDH leakage induced by CRP after OGD/RThe LDH level in CRP+OGD/R group?208.20U/L±19.23U/L?was significantly higher than the values in control group?95.10U/L±21.57U/L?and OGD/R group?145.30U/L±16.06U/L?.After pretreatment with the Ator,the LDH level was significantly decreased?94.35U/L±14.83U/L?compared with the CRP+OGD/R group?P=0.0013?.3.The effects of Ator pretreatment on mPTP opening induced by CRP after OGD/RFluorescent intensities were expressed as percent changes compared with the values in control group.The fluorescence intensity in OGD/R group was lower than the intensity in control group,and it further decreased in CRP+OGD/R group.However,after Ator intervention,the fluorescence intensity was significantly higher than that in CRP+OGD/R group?92.74%+2.47%versus 33.08%versus 3.48%,P<0.0001?.4.The effects of Ator pretreatment on mitochondrial membrane potential induced by CRP after OGD/RFluorescent intensities were expressed as percent changes compared with the value in control group.Compared with OGD/R group,the fluorescence intensity in CRP intervention group further decreased?33.31%±2.03%?.The effects of CRP on mitochondrial membrane potential were offset after treatment with Ator.The fluorescence intensity increased significantly?84.00%±2.31%,P<0.0001?.Ator pretreatment inhibited the decline of mitochondrial membrane potential induced by CRP.5.The effects of Ator pretreatment on signaling pathways during CRP-mediated oxygen-glucose deprivation/reoxygenationThe expression levels of ERK,Akt and JNK were detected by Western blot.Compared with the level after simple CRP pretreatment,Akt expression was markedly activated after co-treatment with Ator and CRP?122.7%±5.30%versus 184.2%±6.96%,P=0.0003?.In addition,the levels of both ERK and phospho-ERK were lower in the Ator and CRP co-treatment group compared with the levels in CRP group after OGD/R.The difference was especially significant for phospho-ERK.In summary,we concluded that Ator protected cardiomyocytes from CRP-mediated ischemia reperfusion injury mainly by activating the Akt signaling pathway and decreasing phosphorylated ERK levels.Conclusions:1.The pretreatment of Ator increased the cell viability in CRP-mediated OGD/R injury.Ator has a cardioprotective effect.2.The pretreatment of Ator significantly reduced the LDH level in CRP-mediated OGD/R injury.3.Ator pretreatment reduced the degree of mPTP opening in CRP-mediated OGD/R injury.4.Ator pretreatment increased the mitochondrial membrane potential in CRP-mediated OGD/R injury.5.Ator protected cardiomyocytes from OGD/R injury mainly by activat-ing the Akt signaling pathway and decreasing phosphorylated ERK levels. |
PDF Full Text Request