| With the improvement of people’s standard of living and eating habits changing, coronary atherosclerotic cardiopathy(coronary heart disease,CHD)has become a high incidence of chronic fatal diseases. Coronary atherosclerosis caused by coronary artery occlusion, leading to acute myocardial ischemia, triggering arrhythmia, myocardial infarction, cardiac shock and related disease. Acute myocardial infarction(AMI) is the most severe clinical manifestations of coronary heart disease(CHD). Thrombolytic drugs, coronary artery bypass, percutaneous coronary angioplasty are widely used in clinical. These treatments can cure acute myocardial infarction, restore blood supply. But, after the restoration of coronary artery blood supply, the myocardium injury is more serious, this is called myocardial Ischemia/Reperfusion(I/R) injury. How effective prevention and treatment of myocardial ischemia-reperfusion injury and study the mechanism of ischemia reperfusion injury is a hotspot of current research.In 1986, Murry first put forward a self-defensive protection mechanism of myocardial cell that myocardial ischemia preadaptation(isehemie preconditioning, IPC), namely transient ischemic preconditioning can improve myocardial ischemia and reperfusion of longer then damage tolerance ability.Ischemic preconditioning, however, the problem such as safety, operability and individual differences of clinical application of IPC is restricted. So people put forward the drug post-treatment(pharmacologic preconditioning,PPC), namely the simulation IPC through a variety of drugs less impatient ischemia-reperfusion injury.Vitamin C(VC) is a widely used clinical antioxidants. Studies have shown that the IPC for myocardial cell protective effect related to oxygen free radicals produce less. Myocardial ischemia reperfusion produces a large number of myocardial damage caused by oxygen free radicals. Vitamin C can reduce the production of oxygen free radicals, has certain protective effect on the cell membrane damage. Research has showed that vitamin C can reduce ischemia reperfusion injury of skeletal muscle, kidney and lung, but also can reduce the swelling and skeletal muscle skeletal muscle necrosis, but less on the study of myocardial protection effect and its mechanism.In this study, we established the SD rat in vitro of I/R and the original generation develop myocardial cell oxygen and glucose deprivation/reperfusion(OGD/R) model, and explore the vitamin C postconditioning on myocardial ischemia-reperfusion injury of protection, and further to study the protective mechanism. Vitamin C safety, economy, clear the heart protection is helpful to clinical applications, has very practical significance for the research.The present study included three parts as following:Part 1 The effects of vitamin C postconditioning on myocardialischemia/reperfusion injury in isolate rat heartsObjective: Vitamin C post-treatment with whole heart ischemia in rats in vitro whether plays a protective role, and the mitochondrial permeability transition pore(m PTP) and mitochondrial ATP sensitive potassium channel(mito KATP, channels).Methods: Using aortic retrograde perfusion(Langendorff) heart in vitro model, the whole heart ischemia 30 min, 120 min reperfusion, detect myocardial infarction area, hemodynamic, perfusion liquid, the content of lactose dehydrogenase(LDH) in myocardial tissue ATP and nicotinamide adenine dinucleotide(NAD+) content, and myocardial ultrastructure were observed. Use the m PTP opener lonidamine(LND) and the mito KATP channels inhibitor 5-hydroxydecanoate(5-HD) to explore m PTP and mito KATP channels in the role of VC protect myocardial ischemia-reperfusion injury.Rats were randomly divided into 7 groups:1 Control group: the hearts was perfused without I/R.2 I/R group: The whole heart ischemia 30 min and 120 min reperfusion;3 VC groups: the whole heart ischemia 30 min, then the heart were treated with 2 μM VC for 30 min and 120 min reperfusion;4 VC+5-HD group: the hearts were first treated for 20 min with 100 μM5-HD, and then treated with 2 μM VC for 30 min after global ischemia.5 VC+LND group: the hearts were treated with 2 μM VC for 30 min after global ischemia, and exposed to 30 μM LND for 20 min at the beginning of reperfusion.6 5-HD group: the hearts were first perfused for 20 min with 100 μM5-HD, and then perfused with K-H buffer for 30 min after global ischemia.7 LND group: the hearts were exposed to 30 μM LND for 20 min at the beginning of reperfusion.Results:1 The effects of VC on I/R-induced infarct area in isolated rat hearts.Compared with I/R group, VC post-treatment for 15 min, 30 min, 60 min significantly reduce the myocardial infarction area(P < 0.05).VC post-treatment 120 min compared with I/R group no statistical difference(P>0.05).VC post-treatment 30 min most reduce the myocardial infarction area(P<0.05), illustrate the medication time of VC best protection for 30 min.Compared with I/R group, four different concentrations of 0.5μM, 1μM,2μM and 10μM VC post-treatment were significantly reduce the myocardial infarction area(P < 0.05).Among them, 2 μM the best VC post-processing myocardial protection.VC with 5-HD or LND joint application has not been able to reduce the effect of myocardial infarction area(P<0.05), and give 5-HD or LND alone had no significant effect on the myocardial infarction area(P>0.05).2 The effects of VC on hemodynamics7 experimental groups in vitro cardiac hemodynamic parameters phase balance between no significant statistical difference(P > 0.05).Rats in vitro cardiac ischemia reperfusion, left ventricular systolic blood pressure(LVDP),heart rate(HR), left ventricular pressure change maximum rate(dp/dtmax) and left ventricular pressure change the minimum rate(- dp/dtmin) than the phase balance is reduced, and the left ventricular end-diastolic pressure(LVEDP)compared with the equilibrium phase was increased(P<0.05).VC(2μM) post-treatment group of hemodynamic indicators are better than I/R group(P < 0.05), showed that left ventricular systolic and diastolic function improved. However, VC and 5-HD or LND joint application does not have the protection of cardiac function(P<0.05).5-HD or LND used alone to ischemia reperfusion had no significant effect to improve cardiac function indexes.3 The effects of VC on the levels of LDH in the perfusateIn the I/R group, LDH activity increased compared with control group(P<0.05).VC post-treatment can significantly reduce the activity of LDH(P>0.05). VC with 5- HD or LND joint applications, have hindered the VC effect of the activity of LDH(P < 0.05). Compared with I/R group, give 5-HD or LND alone not significantly affect the activity of LDH(P>0.05).4 The effects of VC on the ATP content in myocardial tissueCompared with control group, the ATP levels of I/R group decreased significantly(P<0.05). Compared with I/R group, significantly increased the myocardial ATP level of VC post-treatment group, 5-HD or LND inhibited VC increases the effect of ATP. Give 5- HD alone or LND had no obvious effect on the ATP levels.5 The effects of VC on the m PTP openingIn I/R group and drug treatment of myocardial NAD + content is far lower than the control group(P < 0.05). Compared with I/R group, VC post-treatment group of NAD+ were significantly increased(P < 0.05),suggesting that the VC post-treatment can inhibit m PTP open to prevent NAD+decreased. The cooperative use of VC and 5- HD or LND hindered the inhibitory effect of VC is open to m PTP. Using 5- HD or LND alone does not affect myocardial NAD + levels after ischemia reperfusion.6 The comparison of the myocardial ultrastructure in the seven groupsMyocardial mitochondria structure of I/R group was greatly altered comparing with the control group. The myocardial fibers were in disorder,myocomma structure was unclear, and part of myofilament was broken or dissolved. Mitochondria were swollen, even ruptured, cristae were disorganized or had disappeared, and vacuolar degeneration and matrix loss could be seen in some mitochondria. The number of glycogen granules was significantly decrease. VC post-treatment significantly improve myocardial cell ultrastructure changes. But LND or 5- HD inhibits VC protection role.Conclusion:1 in vitro Langendorff heart ischemia reperfusion model, vitamin C post-processing to reduce myocardial infarction area, and in 30 min 2 microns concentration post-treatment conditions, making the most of vitamin C myocardial protection.2 vitamin C postconditioning improved the myocardial blood flow dynamics, and reduced the level of LDH.3 vitamin C postonditioning increased the amount of myocardial tissue ATP, improved myocardial energy metabolism.4 vitamin C prostconditioning increased myocardial tissue NAD+ content,inhibit m PTP open.5 vitamin C postconditioning obviously alleviated the pathological changes of the ultrastructure of mitochondria, reduced the damage degree of the myocardial cells.6 the effect of vitamin C can be canceled by mito KATP channel inhibitor5-HD and the m PTP opener LND. Myocardial protective effect of vitamin C is mediated by activation mito KATP channels and inhibition of m PTP open, and mito KATP channelsmay be located in the upstream of m PTP.Part 2 the effects of vitamin C postconditioning on oxygen-glucosedeprivation/reperfusion injury in primary neonatal ratcardiomyocytesObjective: To investigate whether the post-treatment of vitamin C could exert cardioprotective effect against oxygen-glucose deprivation/reperfusion(OGD/R) injury in primary neonatal rat cardiomyocytes.Methods:Primary cardiomyocytes were isolated from neonatal 1-3 d Sprague-Dawley rats. To establish an in vitro model of OGD/R, which resembles ischemia/reperfusion in vivo, primary cardiomyocytes were exposed to OGD for 10 h followed by a 3 h reperfusion.Neonatal rat cardiac myocytes in primary culture were randomly assigned to one of the following 7 groups:1 control group: cells were cultured in the DMEM medium containing10% FBS, under 37 ℃ 5% CO2 incubator.2 OGD/R group: cells were exposed to 10 h OGD treatment followed by a 3 h reperfusion only.3 VC group: time-related effects of VC on myocardial cell viability, VC(2 μM) was added to the medium for 1 h, 3 h, 6 h, 12 h, 24 h or 48 h after OGD treatment. The optimal duration of treatment was used in subsequent experiments. Concentration-related effects of VC on myocardial cell viability.VC was diluted for use with final concentrations 0.1 μM, 1 μM, 2 μM, and 10μM for 3 h(according to the optimal treatment time) after OGD treatment.The optimal concentration was used in subsequent experiments.4 VC + 5-HD group: first give cell 100 μM 5-HD for 20 min, before OGD, then cell medium change for the final concentration of 2 μM VC and cell incubation for 3 h, abandon the original medium, clean with sugar-free DMEM cell twice, then followed by a 3 h reperfusion.5 VC + LND groups: after undergoing OGD for 10 h, cells were treatmented with the final concentration of 2 μM VC and incubation for 3 h,clean with sugar-free DMEM cell twice. Then cells were incubated with normal culture medium containing 30 μM LND for 20 min, followed by a 3 h reperfusion.6 5-HD group: before OGD, cells were incubated with 100 μM 5-HD for20 min, abandon to medium, clean with DMEM medium twice. Then cells were to reperfusion for 3 h.7 LND group: cells were exposed to OGD for 10 h, followed by incubated with 30 μM LND for 20 min. Then cells were to reperfusion for 3 h.Results:1 The effects of VC on OGD/R-induced cell death in cultured ventricular myocytesDetermined by MTT experiment, results showed that the VC post-treatment for 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, myocardial cell vitality had significant difference compared with OGD/R group(P < 0.05). VC post-treatment for 3 h had the best effect of myocardial protection. 0.1 μM, 1μM, 2 μM and 10 μM of VC post-treatment compared with OGD/R group, the myocardial cell reply all have significant difference(P<0.05). Among them,2μM VC post-treatment is the best myocardial protection condition.5-HD or LND significantly canceled the effect of VC on the cell viability(P < 0.05). Compared with OGD/R group, 5-HD or LND alone had no obvious effect on the myocardial cell viability(P>0.05).2 The effects of VC on LDH release from cultured ventricular myocytesCompared with control group(10.25±1.84%), in the OGD/R group(46.71±4.51%) myocardial cell supernatant of LDH level significantly increased(P < 0.05). Compared with the OGD/R group, in the VC post-treatment group(26.90±3.36%), LDH were significantly reduced(P <0.05).5-HD or LND with VC application, are blocking the effect of VC to reduce the LDH level(P < 0.05). Compared with OGD/R group, treatment with 5-HD or LND alone had no obvious effect on levels of LDH(P>0.05).Conclusions:1 VC reduce OGD/R-induced cell death.2 VC post-treatment reduced OGD/R-induced LDH release.Part 3 the mechanism of vitamin C postconditioning on oxygen-glucosedeprivation/reperfusion injury in primary neonatal ratcardiomyocytesObjective: To explore the role of mitochondria in cardioprotection of VC postconditioning.Methods:Primary cardiomyocytes were isolated from neonatal 1-3 d Sprague-Dawley rats. To establish an in vitro model of OGD/R, which resembles ischemia/reperfusion in vivo, primary cardiomyocytes were exposed to OGD for 10 h followed by a 3 h reperfusion.Neonatal rat cardiac myocytes in primary culture were randomly assigned to one of the following 7 groups:1 control group: cells were cultured in the DMEM medium containing10% FBS, under 37 ℃ 5% CO2 incubator.2 OGD/R group: cells were exposed to 10 h OGD treatment followed by a 3 h reperfusion only.3 VC group: time-related effects of VC on myocardial cell viability, VC(2 μM) was added to the medium for 1 h, 3 h, 6 h, 12 h, 24 h or 48 h after OGD treatment. The optimal duration of treatment was used in subsequent experiments. Concentration-related effects of VC on myocardial cell viability.VC was diluted for use with final concentrations 0.1 μM, 1 μM, 2 μM, and 10μM for 3 h(according to the optimal treatment time) after OGD treatment.The optimal concentration was used in subsequent experiments.4 VC + 5-HD group: first give cell 100 μM 5-HD for 20 min, before OGD, then cell medium change for the final concentration of 2 μM VC and cell incubation for 3 h, abandon the original medium, clean with sugar-free DMEM cell twice, then followed by a 3 h reperfusion.5 VC + LND groups: after undergoing OGD for 10 h, cells were treatmented with the final concentration of 2 μM VC and incubation for 3 h,clean with sugar-free DMEM cell twice. Then cells were incubated with normal culture medium containing 30 μM LND for 20 min, followed by a 3 h reperfusion.6 5-HD group: before OGD, cells were incubated with 100 μM 5-HD for20 min, abandon to medium, clean with DMEM medium twice. Then cells were to reperfusion for 3 h.7 LND group: cells were exposed to OGD for 10 h, followed by incubated with 30 μM LND for 20 min. Then cells were to reperfusion for 3 h.Results:1 The effects of VC on OGD/R-induced m PTP openingCompared with control group, the myocardial cell fluorescence intensity of OGD/R group(26.89±1.62%) decreased significantly(P < 0.05).fluorescence intensity of VC post-treatment group(83.97±3.05%) was obviously higher than that of OGD/R group(P < 0.05). The fluorescence intensity of VC + 5-HD group(29.80±2.43%) and VC + LND(29.17±3.45%)were significantly lower than VC group, proved that 5- HD or LND canceled the effect of VC inhibited m PTP open. the fluorescence intensity of 5- HD group(28.07±2.90%) and LND(27.17±2.82%) was no significant difference compared with OGD/R group.2 The effects of VC on OGD/R-induced ROS levelsCompared with control group, myocardial intracellular ROS level of OGD/R group(557.93±47.44% of control) was significantly increased(P <0.05), VC post-treatment group(286.65±30.90% of control) reduced myocardial cells ROS levels compared with OGD/R group(P < 0.05). VC treatment with 5- HD or LND increased myocardial intracellular ROS level,which meant 5- HD and LND blocking the effect of VC to reduce ROS. Give5- HD and LND alone had no obvious effect on ROS levels in the cell.3 The effects of VC on OGD/R-induced calcium overloadThis experiment using calcium ion sensitive probe Fluo – 4 and Rhod-2respectively to observe the change of calcium concentration in the cytoplasm and mitochondria. The changes of fluorescence intensity and calcium concentration were positively. Compared with control group, the fluorescence intensity of OGD/R group in the cytoplasm and mitochondria(392.17±24.11%of control, 236.31±31.87 of control increased significantly(P < 0.05),suggesting that Ca2+ concentration increased significantly in the cytoplasm and mitochondria, and producing calcium overload. The fluorescence intensity of VC post-treatment group decreased significantly compared with the OGD/R group, showed that VC post-treatment inhibited calcium internal flow caused by OGD/R, reduced calcium overload. This effect of VC can be inhibited by 5- HD or LND. Give 5- HD or LND alone had no obvious effect to reduce calcium internal flow.4 The effects of VC on OGD/R-induced mitochondrial membrane potential dissipationUsing the confocal laser scanning microscopy detected TMRE fluorescence changes, and then evaluate the situation of the mitochondrial membrane potential. Compared with the control group, the fluorescence intensity of OGD/R group(34.39±3.71% of control) decreased significantly,showed that the depolarization of mitochondrial membrane potential. The TMRE fluorescence intensity decreased by post-treatment with VC(84.00±4.50% of control), suggesting that VC post-treatment reduced the depolarization of mitochondrial membrane potential. TMRE fluorescence intensity of VC + 5- HD group(35.60±3.46% of control) and VC + LND group(34.31±3.60% of control) decreased significantly compared with the VC group, suggesting that 5- HD or LND suppresses the role of VC to maintain the mitochondrial membrane potential. 5- HD group(32.49±2.02% of control)and LND group(30.81±2.46% of control) did not change significantly,showed that 5- HD and LND cannot reduce the change of mitochondrial membrane potential.Conclusions:1 VC prevented the m PTP opening.2 VC post-treatment reduced OGD/R-induced ROS generation.3 VC post-treatment reduced OGD/R-induced Ca2+ overload.4 VC alleviated the collapse of the mitochondrial membrane potential in cardiomyocytes.5 The effects of VC were reversed by the selective mito KATP channel inhibitor 5-HD and the m PTP opener LND.This effect may be mediated by the reduction in ROS and the activation of the mito KATP channels, a reduction in Ca2+overload, the inhibition of m PTP opening and the attenuation of mitochondrial membrane potential collapse. Furthermore, mito KATP channels may act upstream of m PTP. |
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