| BackgroundAcute myocardial infarction (AMI) is the most serious type of coronary heart disease seriously jeopardizing health and quality of life. The basic principle of treatment is to rapidly open criminal vessel, timely restore coronary blood flow,and rescue Impaired myocardium.In recent years, Reperfusion therapy such as Coronary artery bypass surgery, Venous thrombolytic therapy and percutaneous coronary angioplasty have been widely used in Clinical practice.However, the quick restoration of coronary blood flow can lead to myocardial reperfusion injury.Thus,to realize restoration of myocardial microcirculation reperfusion,rather than merely open criminal vessel,is the best way for the treatment of acute myocardial infarction. How to effectively mitigate such damage has been the greatest challenge in reperfusion therapy for maximum benefit in AMI treatment. The main pathogenesis of myocardial ischemia reperfusion injury includes:mitochondrial dysfunction, generation of reactive oxygen species, calcium ion overload.And intracellular calcium overload is irreversible pathway of ischemia/reperfusion induced injury.As a result,how to improve reperfusion conditions, prevent intracellular calcium overload play a key role in treat ischemic reperfusion injury treatment. So far, it is not completely clear on mechanism related to intracellular calcium overload during ischemia/reperfusion, and lack of effective way in inhibiting intracellular calcium overload in clinical practice.Wnt/frizzled pathway is regarded as important regulatory target for various cardiovascular diseases. It has been confirmed that the activation of Wnt/ frizzled pathway after acute myocardial infarction, accompanied by a series of pathological process. Among the member of Wnt family, Wnt5a/frizzled-2 pathway is considered to be related with intracellular calcium metabolism closely, the activation of Wnt/frizzled pathway can trigger the release of calcium by Wnt/Ca2+ pathway. And Ca2+ sensitive enzyme such as protein kinase C and Ca2+/calmodulin dependent kinase Ⅱ are activated either.In our previous study, Wnt5a/frizzled-2 can be stably expressed in myocardial cells of rat and its H9C2 cell lines, on physiological conditions, the expression of Wnt5a and frizzled-2 was a relatively low level;and transfection H9C2 cells with plasmid containing total frizzled-2 gene can significantly promote the expression of Wnt5a and frizzled-2 on level of both gene and protein in H9C2 cells, and intracellular calcium concentration was also increased significantly in transfected H9C2 cells.We also found that, in vivo and in vitro, ischemia/reperfusion can increase the expression of frizzled-2 and Wnt5a on level of gene and protein, and to downregulate the expression of frizzled-2 and Wnt5a on level of gene and protein through pretreatment of cell Stealth RNAi, can significantly reduce the calcium level in H/R cells. Our previous research demonstrated that:the activation of Wnt5a/Frizzled-2 pathway is one of the potential mechanisms on H/R induced calcium overload of myocardial cell.Resveratrol is a natural antioxidant polyphenolic compounds.However, in recent years, the role of resveratrol on cardiovascular protection has attracted more and more attention. It is generally believed that the cardiovascular protective effects of resveratrol is achieved through its preconditioning effect, and research on its postconditioning effct is very few. In addition, there is controversy on the relationship between dosage and its effects of cardiovascular protection. Previous studies showed that resveratrol pretreatment can inhibit H2O2 induced calcium overload in myocardial cell of neonatal rat and reduce the injury and apoptosis of cells, but the potential mechanism is not reported. Therefore, in this experiment, we established Hypoxia/reoxygenation model in vitro, for simulation of myocardial ischemia reperfusion injury at the level of cell, to study effect of resveratrol post-treatment on H/R-induced calcium overload in H9C2 myocardial cells and its potential mechanisms.ObjectivesBased on the above analysis, we intend to discuss from the cell level:1.H9C2 myocardial hypoxia reoxygenation model is established for simulating ischemia reperfusion injury, to explore the effect of different concentrations of resveratrol post treatment on hypoxia reoxygenation induced calcium overload of myocardial cell,as well as cell injury and apoptosis.2. H9C2 myocardial hypoxia reoxygenation model is established for simulating ischemia reperfusion injury, to study the potential mechanisms on protective effect of resveratrol on hypoxia reoxygenation induced myocardial cells calcium overload. Provide a new strategy for preventing myocardial cells from intracellular calcium overload.3. Through pathway intervention, to investigate the effects of resveratrol treatment on intracellular calcium concentration, for further discussing the mechanisms of resveratrol on intra-cellular calcium overload.MethodsThe first part of the experiment:we first developed H9C2 rat myocardial cells in vivo, and established hypoxia reoxygenation model, the experiments were divided into 5 groups:control group, hypoxia reoxygenation group:hypoxia for3h,before reoxygenation 4h, reveratrol post-treatment group:H/R cells was post-treated with resveratrol(5,25,100umol/l). cell growth were observed under inverted microscope, intracellular calcium fluorescence intensity was detected by laser confocal microscopy, after cells were incubated with Fluo-3, apoptosis rate of H9C2 cells was detected by flow cytometry, activity of apoptosis protease-3(Caspase-3) was observed by apoptosis protease-3 kit.The second part of the experiment:we first developed H9C2 rat myocardial cells in vivo, and established hypoxia reoxygenation model, the experiments were divided into 5 groups:control group, hypoxia reoxygenation group:hypoxia for3h,before reoxygenation 4h, reveratrol post-treatment group:H/R cells was post-treated with Resveratrol(5,25,100umol/l).Expression of Frizzled-2 and Wnt5a gene were detected by quantitative Real-time Polymerase Chain Reaction(RT-qPCR). Expression of Frizzled-2 and Wnt5a potein were detected by Western Blotting.The third part of the experiment:Firstly,Frizzled-2 gene was synthesized, and Combined with Iipo2000, pEGFP-Cl was used as a carrier, then H9C2 cells were transfected with pEGFP-Cl-Frizzled-2 plasmid. the experiments were divided into 3 groups:control group, Plasmid group:H9C2 cells were merely subjected to Frizzled-2 gene stable transfection, Plasmid+Resveratrol group:Frizzled-2 gene-transfected H9C2 cells were post-treated with Res (10umol/l). Expression of Frizzled-2 and Wnt5a gene were detected by quantitative Real time Polymerase Chain Reaction (RT-qPCR). Expression of Frizzled-2 and Wnt5a potein were detected by Western Blotting, intracellular calcium fluorescence intensity was detected by laser confocal microscopy, after cells were incubated with Fluo-3.The data were analyzed using the SPSS 13.0 statistical software (SPSS, Chicago, IL, USA) and are expressed as the means±SD. Differences were considered to be statistically significant if the p value was less than 0.05.Results1. We cultured H9C2 cells, established a cell model of hypoxia/ reoxygenation, Intracellular calcium fluorescence intensity was detected by laser confocal microscopy, after cells were incubated with Fluo-3. Compared with the control group, intracellular calcium fluorescence intensity of H/R group(hypoxia for 3h, before reoxygenation for 4h) is significantly decreased (p<0.001). After post-treated with different concentrations of Resveratrol (5,25,100 umol /I) on hypoxia/reoxygenation cells, intracellular calcium fluorescence intensity of Resveratrol post treatment groups are significantly decreased respectively, Compared with the H/R group (p<0.001).And among three groups post-treated with different concentrations of Resveratrol, with the increasing of RES concentration, intracellular Ca2+fluorescence intensity is decreased, (Figure 1-2, Tablel-1).Activity of H9C2 myocardial cells was detected by CCK-8 kit, Compared with the control group,cell availity of hypoxia reoxygenation group is significantly decreased (p<0.001). After post-treated with different concentrations of Resveratrol (5,25,100umol/l) on hypoxia reoxygenation cell,cell viability of Resveratrol post-treatment groups are significantly increased respectively, Compared with the H/R group (p<0.001). And among three groups post-treated with different concentrations of Resveratrol, with the increasing of RES concentration, cell viability is decreased, (Figure 1-3, Table 1-2).In order to observe the effect of Resveratrol on injury of H/R cells, level of LDH was detected. Compared with the control group, level of LDH of hypoxia reoxygenation group is significantly increased (p<0.001). After post-treated with different concentrations of Resveratrol (5,25,100umol/l) on hypoxia/reoxygenation cell, level of LDH of Resveratrol post-treatment groups are significantly decreased respectively, Compared with the H/R group (p<0.01). And among three groups post-treated with different concentrations of Resveratrol, with the increasing of RES concentration, level of LDH is decreased (Figurel-4, Tablel-3).Using way of Alexa Fluor 488 Annexin YFITe/Pl double staining, apoptosis rate of H9C2 myocardial cells was observed by flow cytometry(Figure 1-5,Tablel-4).Compared with the control group, apoptosis rate of hypoxia reoxygenation group is significantly increased (p<0.01). After post-treated with different concentrations of Resveratrol (5,25,100umol/l) on hypoxia/reoxygenation cell, apoptosis rate of Resveratrol post-treatment groups are significantly decreased respectively, Compared with the H/R group (p<0. 01). apoptosis protease-3(Caspase-3) kit was used to detected activity of Caspase-3 in different groups(Figurel-6,Tablel-5).Compared with the control group, activity of Caspase-3 in hypoxia reoxygenation group is significantly increased (p<0.01). After post-treated with different concentrations of Resveratrol (5,25,100umol/l) on hypoxia reoxygenation cell, activity of Caspase-3 in Resveratrol post-treatment groups are significantly decreased respectively, Compared with the H/R group (p<0. 01).2. We cultured H9C2 cells, established a cell model of hypoxia/reoxygenation, Expression of Frizzled-2 and Wnt5a gene were detected by quantitative Real-time Polymerase Chain Reaction(RT-qPCR). Compared with the control group,expression of Frizzled-2 and Wnt5a gene in hypoxia reoxygenation group is elevated 5.876±0.180 and 3.468±0.180 times respectively, expression of Frizzled-2 and Wnt5a gene in hypoxia reoxygenation group is significantly increased, Compared with the control group, (p<0.01). After post-treated with different concentrations of Resveratrol (5,25,100umol/l) on hypoxia/re- oxygenation cell, expression of Frizzled-2 gene of different groups are elevated 4.238±0.118,2.550±0.187 and 2.263±0.117 times respectively, and expression of Wnt5a gene of different groups are elevated 2.398±0.092,1.993±0.153,1.627±0.116 times respectively,expression of Frizzled-2 and Wnt5a gene in post-treatment groups are significantly decreased respectively, compared with the H/R group, (p<0.01). And among three groups post-treated with different concentrations of Resveratrol, with the increasing of RES concentration, expression of both Frizzled-2 and Wnt5a gene are decreased (Figure2-1,Table2-1). Expression of Frizzled-2 and Wnt5a protein were detected by quantitative Western Bloting. Expression of Frizzled-2 and Wnt5a protein in control group is 0.203±0.017,0.407±0.025 respectively,expression of Frizzled-2 and Wnt5a protein in hypoxia/reoxygenation group is 1.142± 0.072,0.915±0.071 respectively, expression of Frizzled-2 and Wnt5a protein in hypoxia reoxygenation group is significantly increased, Compared with the control group, (p<0.01). After post-treated with different concentrations of Resveratrol (5,25,100umol/l) on hypoxia reoxygenation cell, expression of Frizzled-2 protein of different groups are 0.829±0.023,0.505±0.037,0.402±0.026 respectively, and expression of Wnt5a protein of different groups are 0.707±0.050,0.601± 0.037,0.556±0.039, expression of Frizzled-2 and Wnt5a protein in post-treatment groups are significantly decreased respectively, compared with the H/R group, (p<0. 01). And among three groups post-treated with different concentrations of Resveratrol, with the increasing of RES concentration, expression of both Frizzled-2 and Wnt5a protein are decreased (Figure2-2,Table2-2)3. We synthesized Frizzled-2 gene,and used pEGFP-C1 as a carrier,then Combined with lipo2000, H9C2 cells were transfected with pEGFP-C1-Frizzled-2 plasmid, Expression of Frizzled-2 and Wnt5a gene of different groups were detected by quantitative Real-time Polymerase Chain Reaction (Figure3-2,Table3-1) Compared with the control group,expression of Frizzled-2 and Wnt5a gene in plasmid group is elevated 6.798±0.311,3.003±0.217 times respectively, expression of Frizzled-2 and Wnt5a gene in plasmid group is significantly increased,Compared with the control group, (p<0.01). After post-treated with different concentrations of Resveratrol (10umol/l) on Frizzled-2 gene transfected cell, expression of Frizzled-2 and Wnt5a gene in post-treatment is 4.040±0.249,1.844±0.197 times respectively, Compared with the plasmid group, expression of Frizzled-2 and Wnt5a gene in post-treatment group is significantly decreased respectively, compared with plasmid group, (p<0.01). Expression of Frizzled-2 and Wnt5a protein were detected by quantitative Western Bloting. Compared with the control group,expression of Frizzled-2 and Wnt5a protein in plasmid group is 1.295±0.028 and 0.801±0.019 respectively, expression of Frizzled-2 and Wnt5a protein in plasmid group is significantly increased respectively, Compared with the control group, (p<0.01). After post-treated with different concentrations of Resveratrol (10umol/l) on Frizzled-2 gene transfected cell, expression of Frizzled-2 and Wnt5a protein in post-treatment group is 0.801±0.017,0.602±0.013 respectively, Compared with the plasmid group,expression of Frizzled-2 and Wnt5a protein in RES-treatment group is significantly decreased, (p<0.01), (Figure3-3,Table3-2).Intracellular calcium fluores cence intensity was detected by laser confocal microscopy, after cells were incubated with Fluo-3 (Figure3-4,Table3-3),Compared with the control group(0.0100±0.0008), intracellular calcium fluorescence intensity of Plasmid group(0.0288±0.0021) is significantly increased, (p<0.01), intracellular calcium fluorescence intensity of post-treatment is 0.0182±0.0012, Compared with Plasmid group, intracellular calcium fluorescence intensity of RES-treatment is significantly decreased, (p<0.01).Conclusions1. In this study, established a cell model of hypoxia/reoxygenation,for simulating I/R injury,and cells were post treated with different concentration of Resveratrol, results demonstrated that Resveratrol post treatment can suppress of H/R induced intracellular calcium overload, Resveratrol post treatment can protect myocardial cells from necrosis and apoptosis,during I/R,and the same cardio-protective effect may be achieved in higher dosage.2. We established a cell model of hypoxia/reoxygenation,for simulating I/R injury,and cells were post treated with different concentration of Resveratrol,results showed that the level of expression of Frizzled-2/Wnt5a pathway in normal H9C2 cells is low.after hypoxia reoxygenation injury, expression of Frizzled-2/Wnt5a is upregulated, and the same effect may be achieved in higher dosage.3. In this study, We synthesized pEGFP-C1-Frizzled-2 plasmid, and Combined with Hpo2000, H9C2 cells were transfected with pEGFP-C1-Frizzled-2 plasmid,and treatment with Resveratrol on frizzled-2 gene transfected H9C2 cells.results demonstrated that post-treatment with Resveratrol can downregulate expression of Frizzled-2/Wnt5a pathway in frizzled-2 gene transfected H9C2 cells,and suppress of intracellular calcium concentration in frizzled-2 gene transfected H9C2 cells,we conclude that Resveratrol can attenuates hypoxia/reoxygenation induced calcium overload by inhibiting the Wnt5a/Frizzled-2 pathway. |
PDF Full Text Request