| Percutaneous coronary intervention(PCI) is a critical part of the intervention of acute myocardial infarction(AMI) patients. However, myocardial tissue suffers from myocardial ischemia/reperfusion(I/R) injury during revascularization, which results in poor clinical outcomes. Apoptosis of cardiomyocytes is known to be an important mechanism of I/R injury. Accumulating evidence has shown that apoptosis of cardiomyocytes is suppressed by glycogen synthase kinase-3β(GSK-3β). Therefore manipulation of GSK-3β is a promising strategy for myocardial protection in I/R injury. The GSK-3 family of serine/threonine kinases plays an important role in various pathologic processes of heart, including pressure overload and ischemic injury. It is comprised of two isoforms(α and β) which are encoded by distinct genes and are ubiquitously expressed. Numerous studies have shown that GSK-3β, but not GSK-3α is cardioprotective.Micro RNA(mi RNA) is a class of endogenous noncoding RNA, which has about 20~25 nucleotides. mi RNAs binding target m RNA by base complementary pairing, degradation or blocking translation of target m RNA.Various studies have shown that increased mi R-199a-5p expression is responsible for pathophysiological alterations contributing to and promoting the development of heart diseases including atrial fibrillation and heart failure. Therefore, therapeutic interventions targeting mi R-199a-5p represent promising strategies for the treatment of I/R injury.Statins were found to prevent heart failure, acute coronary syndrome(ACS), and cardiovascular risk. Numerous studies have demonstrated the cardioprotection against myocardial I/R injury of statins in animal experiments and clinical studies. However, the major molecular mechanisms of the cardioprotective effect of statins have not been completely elucidated. Therefore, we hypothesized that cardioprotection by statins may be associated with inhibition of apoptosis in myocardial I/R injury through mi R-199a-5p /GSK-3β pathway.In this study, through the establishment of cardiomyocytes OGD/R injury Simulated I/R damage. Then examined the effects of atorvastatin on H9c2 myocardial cells and neonatal rat cardiac ventricular cardiomyocytes by detecting changes in the m RNA and protein expression levels of GSK-3β. Moreover, we firstly observed the influence of atorvastatin( Ator) on mi R-199a-5p expression and its influence on GSK-3β expression. We further analyzed the mechanism involved in the effects of atorvastatin on myocardial I/R injury to provide a theoretical basis and new intervention targets for the prevention and treatment of myocardial I/R injury in the clinic. Part 1 The effects and mechanism of atorvastatin preconditioning onmyocardial oxygen-glucose deprivation/reperfusion(OGD/R) injury in ratsObjective: To investigate the protective effect of atorvastatin preconditioning on myocardial oxygen-glucose deprivation/reperfusion(OGD/R) injury in rats, and whether the protection of atorvastatin on myocardial is targeting GSK-3β.Methods:Neonatal rat cardiac cardiomyocytes were prepared from 1 to 3 day-old neonatal SD rat. To establish an in vitro model of I/R, the neonatal rat cardiac cardiomyocytes were treated with oxygen–glucose deprivation 6 hours and followed by 1hour recovery.Neonatal rat cardiac cardiomyocytes were randomly assigned to 5 groups as following:1) control group: cells were cultured in DMEM high glucose medium containing 10% FBS and under 5%CO2,37℃.2) OGD/R group: cells were treated with 6 h OGD followed by 1 h reperfusion.3) Ator group: using the Ator(1μM) incubated the cells in DMEM high glucose medium for 3h and using PBS to clean the cell, then OGD/R.4) Li Cl+Ator+OGD/R group: Using Li Cl(1μM) incubated the cells in DMEM high glucose medium for 1h and using PBS to clean the cells. Replacing Ator incubated the cells for 3h, using PBS to clean the cells, and then OGD/R.5) Li Cl+ OGD/R group: Using Li Cl(1 μ M) incubated the cells in DMEM high glucose medium for 1h and using PBS to clean the cells, and then OGD/R.After reperfusion for 1 h, the cell viability was evaluated using the MTS and lactic dehydrogenase(LDH) release assays. Western blot was used to detect the apoptosis related protein expression and GSK-3 beta protein expression. Real-time quantitative PCR was used to detect the expression of GSK-3 beta m RNA and mi R-199a-5p.Results:1 The effect of atorvastatin preconditioning on the expression of GSK-3beta protein and m RNA in primary myocardial cellsWestern Blot uesd to detect the expression of GSK-3beta protein, the results show that compared with the control group, the expression of GSK-3 beta protein in OGD/R group was significantly decreased, and the two groups had significant difference. The expression level of GSK-3 protein in atorvastatin preconditioning group was significantly higher than that in OGD/R group.Real-time quantitative PCR was used to detect the expression of GSK-3 beta m RNA and beta-actin used as a reference. The results show that compared with the control group, the expression of GSK-3 beta m RNA level in myocardial cells of OGD/R group decreased significantly, the difference between two groups was significant(P < 0.05). GSK-3 beta m RNA expression level in atorvastatin preconditioning group was higher than in OGD/R group(P < 0.05).2 Effect of preconditioning with atorvastatin on the viability of primary myocardial cellsThe viability of cardiomyocytes was determined by MTS, and the myocardial viability was decreased by OGD/R. Compared with the OGD/R group, the myocardial cell viability was significantly increased in the atorvastatin preconditioning group(P < 0.05). To join the GSK-3 beta inhibitor Li Cl and Ator pretreated cells, Li Cl abolished the protective effect of Ator on cardiomyocytes(P < 0.05). Compared with OGD/R group, the cell viability was not significant after the addition of Li Cl(P > 0.05).3 Effect of preconditioning with atorvastatin on the LDH release of primary myocardial cellsTo detect the content of LDH in the culture medium of primary myocardial cells, compared with control group, the level of LDH in OGD/R group was increased significantly, and there was significant difference between the two groups(P<0.05). The LDH level in the atorvastatin preconditioning group was significantly lower than that in the OGD/R group(P<0.05). Li Cl and atorvastatin preconditioning cell group, Li Cl abolished the effect of atorvastatin preconditioning on reducing the release of LDH(P<0.05). Compared with OGD/R group, Li Cl group was not significantly affected the LDH(P > 0.05).4 Effects of Ator preconditioning on the apoptosis related protein of Caspase9, B-cell lymphoma-2(Bcl-2), cytochrome C(C Cyto) in primary myocardialWestern blot was used to detect the apoptosis related protein expression. Compared with the control group, expression of caspase9, Bcl-2 and cyto C protein in OGD/R group increased significantly. The expression of caspase9, Bcl-2 and cyto C protein in Ator group was lower than that in OGD/R group. Compared with the Ator group the expression of Caspase9 Bcl-2 and Cyto C was increased in Li Cl +Ator group. The effect of atorvastatin preconditioning on OGD/R of primary myocardial cells was explained by Li Cl.5 Effect of atorvastatin preconditioning on the expression of mi R-199a-5p in primary myocardial cellsWe further examined the effect of OGD/R on mi R-199a-5p expressions of primary myocardial cells with and without atorvastatin preconditioning. Real-time quantitative PCR was used to detect the expression of mi R-199a-5p m RNA and U6 was used as a reference, mi R-199a-5p m RNA level was significantly increased after OGD/R compared to the control group(P < 0.05), whereas preconditioning with atorvastatin significantly reserved it(P < 0.05).Conclusions:1 Atorvastatin protects primary myocardial cells against OGD/R injury.2 Atorvastatin protects primary myocardial cells against OGD/R injury by up-regulation of GSK-3beta.3 mi R-199a-5p m RNA level was increased after OGD/R and reduced in preconditioning of atorvastatin, so we need a further study about effect of mi R-199a-5p on atorvastatin preconditioning in primary myocardial cells. Part 2 Atorvastatin preconditioning against H9C2 myocardial cellsischemia/reperfusion injury through up-regulation of GSK-3 betaObjective: To investigate whether the protection of atorvastatin preconditioning on H9C2 myocardial cells oxygen-glucose deprivation/reperfusion(OGD/R) injury is targeting GSK-3β,and the Effect of mi R-199a-5p on atorvastatin preconditioning.Methods:Use H9C2 myocardial cells. To establish an in vitro model of I/R, the H9C2 myocardial cells were treated with oxygen–glucose deprivation 6 hours and followed by 1hour recovery.H9C2 myocardial cells were randomly assigned to 5 groups as following:1) control group: cells were cultured in DMEM high glucose medium containing 10% FBS and under 5%CO2, 37℃.2) OGD/R group: cells were treated with 6 h OGD followed by 1 h reperfusion.3) Ator group: using the Ator(1μM) incubated the cells in DMEM high glucose medium for 3h and using PBS to clean the cell, then OGD/R.4) Li Cl+Ator+OGD/R group: Using Li Cl(1μM) incubated the cells in DMEM high glucose medium for 1h and using PBS to clean the cells. Replacing Ator incubated the cells for 3h, using PBS to clean the cells, and then OGD/R.5) Li Cl+ OGD/R group: Using Li Cl(1 μ M) incubated the cells in DMEM high glucose medium for 1h and using PBS to clean the cells, and then OGD/R.After reperfusion for 1 h, the cell viability was evaluated using the MTS and lactic dehydrogenase(LDH) release assays. Western blot was used to detect the apoptosis related protein expression and GSK-3 beta protein expression. Real-time quantitative PCR was used to detect the expression of GSK-3 beta m RNA and mi R-199a-5p.Results:1 The effect of atorvastatin preconditioning on the expression of GSK-3beta protein and m RNA in H9C2 myocardial cellsWestern Blot uesd to detect the expression of GSK-3beta protein, the results show that compared with the control group, the expression of GSK-3 beta protein in OGD/R group was significantly decreased, and the two groups had significant difference. The expression level of GSK-3 protein in atorvastatin preconditioning group was significantly higher than that in OGD/R group.Real-time quantitative PCR was used to detect the expression of GSK-3 beta m RNA and beta-actin used as a reference. The results show that compared with the control group, the expression of GSK-3 beta m RNA level in myocardial cells of OGD/R group decreased significantly, the difference between two groups was significant(P < 0.05). GSK-3 beta m RNA expression level in atorvastatin preconditioning group was higher than in OGD/R group(P < 0.05).2 Effect of preconditioning with atorvastatin on the viability of H9C2 myocardial cellsThe viability of H9C2 myocardial cells was determined by MTS, and the cell viability was decreased in OGD/R group. Compared with the OGD/R group, the cell viability was significantly increased in the atorvastatin preconditioning group(P < 0.05). To join the GSK-3 beta inhibitor Li Cl and Ator pretreated cells, Li Cl abolished the protective effect of Ator on cardiomyocytes(P < 0.05). Compared with OGD/R group, the cell viability was not significant after the addition of Li Cl(P > 0.05).3 Effect of preconditioning with atorvastatin on the LDH release of H9C2 myocardial cellsTo detect the content of LDH in the culture medium of H9C2 myocardial cells, compared with control group, the level of LDH in OGD/R group was increased significantly, and there was significant difference between the two groups(P < 0.05). The LDH level in the atorvastatin preconditioning group was significantly lower than that in the OGD/R group(P < 0.05). Li Cl and atorvastatin preconditioning cell group, Li Cl abolished the effect of atorvastatin preconditioning on reducing the release of LDH(P < 0.05). Compared with OGD/R group, Li Cl group was not significantly affected the LDH(P > 0.05).4 Effects of Ator preconditioning on the apoptosis related protein of Caspase9, B-cell lymphoma-2(Bcl-2), cytochrome C(C Cyto) in H9C2 myocardial cellsWestern blot was used to detect the apoptosis related protein expression. Compared with the control group, expression of caspase9, Bcl-2 and cyto C protein in OGD/R group increased significantly. The expression of caspase9, Bcl-2 and cyto C protein in Ator group was lower than that in OGD/R group. Compared with the Ator group the expression of Caspase9 Bcl-2 and Cyto C was increased in Li Cl +Ator group. The effect of atorvastatin preconditioning on OGD/R of primary myocardial cells was explained by Li Cl.5 Effect of atorvastatin preconditioning on the expression of mi R-199a-5p in H9C2 myocardial cellsWe further examined the effect of OGD/R on mi R-199a-5p expressions of H9C2 myocardial cells with and without atorvastatin preconditioning. Real-time quantitative PCR was used to detect the expression of mi R-199a-5p m RNA and U6 was used as a reference, mi R-199a-5p m RNA level was significantly increased after OGD/R compared to the control group(p<0.05), whereas preconditioning with atorvastatin significantly reserved it(p<0.05).Conclusions:1 Atorvastatin protects H9C2 myocardial cells against OGD/R injury.2 Atorvastatin protects H9C2 myocardial cells against OGD/R injury by up-regulation of GSK-3beta.3 mi R-199a-5p m RNA level was increased after OGD/R and reduced in preconditioning of atorvastatin. GSK-3beta was reduced after OGD/R and increased in preconditioning of atorvastatin. So we need a further study about whether mi R-199a-5p is targeting GSK-3beta and effect of mi R-199a-5p on atorvastatin preconditioning in primary myocardial cells. Part 3 Atorvastatin precondition protects myocardium againstischemia-reperfusion injury by inhibiting mi R-199a-5pObjective: To investigate the relationship between mi R-199a-5p and GSK-3beta, and the role of mi R-199a-5p in Atorvastatin precondition protects myocardium against ischemia-reperfusion injury.Methods:Use H9C2 myocardial cells. Transfection cells with mi R-199a-5p inhibitor(20 n M) for 24 h.H9C2 myocardial cells were randomly assigned to 5 groups as following:1) control group: cells were cultured in DMEM high glucose medium without FBS and under 5%CO2,37℃.2) OGD/R group: cells were treated with 6 h OGD followed by 1 h reperfusion.3) mi R-199a-5p inhibitor + OGD/R group: Transfection cells with mi R-199a-5p inhibitor(20 n M) for 24 h and than OGD/R.4) mi R-199a-5p inhibitor group: Transfection cells with mi R-199a-5p inhibitor(20 n M) for 24 h and than cultured in DMEM high glucose medium without FBS and under 5%CO2, 37℃.Real-time quantitative PCR was used to detect of transfection efficiency. Cell viability was evaluated using the MTS and lactic dehydrogenase(LDH) release assays. Western blot was used to detect the apoptosis related protein expression and GSK-3 beta protein expression. Real-time quantitative PCR was used to detect the expression of GSK-3 beta m RNA and mi R-199a-5p. The expression of GSK-3 beta protein was detected by immunofluorescence.Results: 1 H9C2 myocardial cells transfection efficiencyMi R-199a-5p expression was detected by RT-PCR, which to make sure the success of transfection. Compared with control group and negative transfection group, transfected mir-199a-5p mimic was increased the expression of mir-199a-5p and transfected mir-199a-5p inhibitor lead to decreased expression of mir-199a-5p in H9c2 cells, both with significant difference(P < 0.05). 2 Effect of cell transfection on gene expression 2.1 Effect of transfection of mi R-199a-5p mimic and mir-199a-5p inhibitor on the level of GSK-3 beta m RNA.Compared with control group and negative transfection group, transfected mir-199a-5p mimic led to a decrease expression of GSK-3 beta m RNA, and transfected mir-199a-5p inhibitor led to a increase expression of GSK-3beta m RNA, both of which have significant difference(P < 0.05). 2.2 Effect of transfection of mi R-199a-5p mimic and mir-199a-5p inhibitor on the GSK-3 beta protein level.Compared with control group and negative transfection group, transfected mir-199a-5p mimic led to a decrease expression of GSK-3 beta protein level, and transfected mir-199a-5p inhibitor led to a increase expression of GSK-3beta protein level, both of which have significant difference. mi R-199a-5p regulates gene expression of GSK-3 beta and inhibits mi R-199a-5p restore the activity of GSK-3 beta. 3 Effect of mi R-199a-5p inhibitor on the viability of H9C2 myocardial cells.The viability of H9C2 myocardial cells was determined by MTS, and the cell viability was decreased in OGD/R group(P < 0.05). Compared with the OGD/R group, the cell viability was significantly increased in the mi R-199a-5p inhibitor+OGD/R group(P < 0.05). Compared with conrtol group, cell activity has no significant changes mi R-199a-5p inhibitor group(P > 0.05). 4 Effect of mi R-199a-5p inhibitor on the LDH release of H9C2 myocardial cells.To detect the content of LDH in the culture medium of H9C2 myocardial cells, compared with control group, the level of LDH in OGD/R group was increased significantly, and there was significant difference between the two groups(P<0.05). The LDH level in the mi R-199a-5p inhibitor+OGD/R group was significantly lower than that in the OGD/R group(P < 0.05). Compared with conrtol group, LDH has no significant changes mi R-199a-5p inhibitor group(P > 0.05). 5 Effects of mi R-199a-5p inhibitor on the apoptosis related protein of Caspase9, B-cell lymphoma-2(Bcl-2), cytochrome C(C Cyto) in H9C2 myocardial cells.Western blot was used to detect the apoptosis related protein expression. Compared with the control group, expression of caspase9, Bcl-2 and cyto C protein in OGD/R group increased significantly. The expression of caspase9, Bcl-2 and cyto C protein in mi R-199a-5p inhibitor+OGD/R group was lower than that in OGD/R group. 6 Effect of mi R-199a-5p inhibitor on the expression of GSK-3 beta protein in H9C2 myocardial cellsWestern blot was used to detect the GSK-3 beta protein expression. The results showed that, compared with the control group GSK-3 beta protein level decreased significantly in OGD/R group, both of which have significant difference. The expression of GSK-3 beta protein in mi R-199a-5p inhibitor+OGD/R group was lower than that in OGD/R group.Transfected mir-199a-5p inhibitor up-regulation GSK-3beta protein expression.Immunofluorescence showed that compared with control group, GSK-3 beta fluorescence decreased significantly in OGD/R group. GSK-3 beta fluorescence was enhanced in both mi R-199a-5p inhibitor+ OGD/R group and mi R-199a-5p inhibitor group.Conclusions:1 GSK-3 beta is the target gene of mi R-199a-5p.2 Inhibiting of mi R-199a-5p protects H9C2 myocardial cells against OGD/R injury.3 Inhibiting of mi R-199a-5p protects H9C2 myocardial cells against OGD/R injury by targeting GSK-3 beta.4 Atorvastatin protects H9C2 myocardial cells against OGD/R injury by inhibiting mi R-199a-5p. |
PDF Full Text Request