| BackgroudWith the changed of social environment and diet structure, the mordity and motality of cardiovascular disease appears the trend that increased by years, the episode age is gradually younger, especially coronary heart disease and acute myocardial infarction and cardiac failure were the major reason that the mordity and motality increased of Cardiovascular disease, also as a major reason of cardiac sudden death. Cardiovascular disease is the most common cause of mortality in high-income countries. The global statistic datas expressed that the motality lead by cardiovascular disease and circulation system diseases have already increased 1/3 from 1999 to 2010 by the World Health Organization, in the year 2015 that one person dead by cardiovascular disease in every three people, Therefore, it is act as an urgent problem for mankinds to preventing and treatment cardiovascular disease.Sirt1 is the closest homologue of the yeast(Saccharomyces cerevisiae) silent information regulator-2( Sir2), a nicotinamide adenine dinucleotide( NAD+)-dependent protein deacetylase. In mammals, seven members of the Sir2 homologues have been found and identified as Sirt1-7. Sirt1 has been the most extensively investigated sirtuin. Sirt1 expressed in all organs of the body including the brain, heart, liver, pancreas, skeletal muscle, spleen, and adipose tissues and it exists in both the nucleus and cytoplasm with dominant expression in the nucleus. Sirt1 mediated deaceylation has significant impact on the activity of many proteins, resulting in the regulation of a number of proteins and their translation which play important roles in the biological process including oxidative stress, metabolism, cell proliferation, and genomic stability. Therefore, Sirt1 has been linked to aging, metabolic diseases, neurodegenerative diseases, cancer, and cardiovascular dysfunction.The PI3k/Akt pathway is important not only in the development of cancers but also for signaling in normal cells. This pathway is known to play a key role in numerous cellular functions including proliferation, adhesion, migration, invasion, metabolism, and survival. Based on the above situation, depend on the research progress at home and abroad we may propose such questions: resveratrol whether promote the expression of Sirt1, and protect myocardial cells during ischemia reperfusion injury? Resveratrol protect myocardial cells during ischemia reperfusion injury dependent on upregulation PI3k-Akt pathway. Objective1. To detect whether Sirt1 protect myocardial cells in the process of ischemia reperfusion injury.2. To explore the influence of Sirt1 and PI3k-Akt pathway, to clear the mechanism of sirt1 protect myocardial cells from ischemia reperfusion injury. Methods1. H9c2 Cardiac cell line randomly divided into control group and hypoxia reoxygenationgroup, the hypoxia reoxygenation group as well divided into three group, one group cultured with 25 ?M resveratrol medium, the other incubated in medium with 25 ?M resveratrol and 1 ?M EX527, the last have no treatment, after cultured 2 d-3 d treat with hypoxia following reoxygenation, the control group curtuled with normal medium and have nothing to done.2. Using immunofluorescent labeling with TUNEL, H9c2 cells apoptosia was observed with a laser confocal microscop and calculated the rate of H9c2 cells apoptosis.3. Using immunofluorescent labeling with DHE, H9c2 cells ROS levels was observed with a laser confocal microscop and calculated the rate of H9c2 cells ROS levels.4. Using colorimetric MTT test detected the cell viability of each group.5. Using immunofluorescent labeling with JC-1, H9c2 cells mitochondrial membrane potential and calculated the rate of red light with green light.6. Using antibody immunofluorescent observe the expression of Sirt1 in the nucleus.7. Using Western blotting detection the express levels of Sirt1 and p-Akt protains.8. Using Western blotting detection the express levels of Sirt1 and p-Akt protains after added in LY294002, the specific inhibitor of Akt. Results1. Compared with control group( 0.240 ± 0.007), the rate of apoptosis was obviously increased in the hypoxia reoxygenation group( 0.487 ± 0.013), compared with hypoxia reoxygenation group( 0.487 ± 0.013), the rate of apoptosis was obviously decreased in the activated of Sirt1 group( 0.255 ± 0.010), on the contray, the rate of apoptosis of Sirt1 inhibition group( 0.569 ± 0.010) was decreased significantly,( P < 0.001).2. Compared with control group( 0.222 ± 0.004), the levels of ROS in intracellular was significantly increased in the hypoxia reoxygenation group( 0.372 ± 0.020), compared with hypoxia reoxygenation group( 0.370 ± 0.020), the levels of ROS in intracellular was obviously decreased in the activated of Sirt1 group( 0.256 ± 0.026), on the contray, the levels of ROS in intracellular was significantly increased in the Sirt1 inhibition group( 0.593 ± 0.018),( P < 0.001).3. Compared with control group( 0.778 ± 0.007), cell viability was obviously decreased in the hypoxia reoxygenation group( 0.455 ± 0.002), compared with hypoxia reoxygenation group( 0.455 ± 0.002), cell viability was obviously increased in the activated of Sirt1 group( 0.587 ± 0.005), on the contray, in the Sirt1 inhibition group( 0.240 ± 0.009), the cell viability was the lowest,( P < 0.001).4. Compared with control group( 3.822 ± 0.007), mitochondrial membrane potential was significantly decreased in the hypoxia reoxygenation group( 2.579 ± 0.006), compared with hypoxia reoxygenation group( 2.579 ± 0.006), mitochondrial membrane potential was significantly increased in the activated of Sirt1 group( 3.904 ± 0.042), on the contray, mitochondrial membrane potential was significantly decreased in the Sirt1 inhibition group( 1.517 ± 0.037),( P < 0.001).5. Compared with control group( 0.333 ± 0.026), the expression of Sirt1 in the nucleus in the activated of Sirt1 group( 0.574 ± 0.018) was the most,( P < 0.001).6. The protein expression of Sirt1 and p-Akt were shown in grey value ratio. Sirt1 and p-Akt expression of control group were repectively( 1.697 ± 0.020) and( 0.0137 ± 0.0005), and the hypoxia reoxygenation group were repectively( 2.141 ± 0.015) and( 0.0156 ± 0.0004), compared with control group, the protein expression of Sirt1 was obviously decreased, but the were no statistical significance of the protein expression of p-Akt, Sirt1 and p-Akt expression of the activated of Sirt1 group were repectively( 3.434 ± 0.010) and( 0.0227 ± 0.0009), compared with hypoxia reoxygenation group( 2.141 ± 0.015) and( 0.0156 ± 0.0004), the protein expression of Sirt1 and p-Akt were all increased,( P < 0.001). Sirt1 and p-Akt expression of the Sirt1 inhibition group were repectively( 1.530 ± 0.030) and( 0.0120 ± 0.0004), compared with hypoxia reoxygenation group, the protein expression of Sirt1 and p-Akt were all decreased( P < 0.001).7. The levels expression of Akt could been inhibition of LY294002, the protein expression of p-Akt the grey value ratio was( 2.353 ± 0.059), added in LY294002, the protein expression of p-Akt the grey value ratio was( 1.548 ± 0.021), they were statistics significance between these two groups( P < 0.05). At the same time, the protein expression of Sirt1 also significantly decreased, the protein expression of Sirt1 the grey value ratio was( 2.355 ± 0.262), when added in LY294002 the protein expression of Sirt1 the grey value ratio was( 1.214 ± 0.151), they were statistics significance between these two groups( P < 0.05). Conclusion1. Hypoxia then reoxygenation could inmitation the ischemia reperfusion injury of myocardial infarction, and the rate of apoptosis in myocardial cells was obviously increased.2. Sirt1 alleviate H9c2 cells in ischemia reperfusion injury, PI3k-Akt pathway involved in ischemia reperfusion injury that have great benefit effect in protect H9c2 cells.3. It is also an important theory foundation and new target for prevention and treatment CHD. |
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