Research On The Molecular Mechanism Of Resveratrol Against Hypoxia/reoxygenation Damage Of Cardiomyocytes Based On Mitochondrial Quality Control

ObjectiveDamaged mitochondria is the key factor in the development of myocardial ischemia/reperfusion injury(MIRI).Recent studies have found that the improvement of resveratrol on pathological lesions such as aging and metabolic syndrome,is related to its regulation of mitochondrial quality control and improvement of intracellular mitochondrial quality control.The mammalian Sirtuin protein family is a key regulator of cellular energy metabolism.In vitro and in vivo experiments have evidenced that resveratrol can activate Sirtuin proteins and participate in cellular energy metabolism through a Sirtuin-dependent pathway.Our previous studies have confirmed that resveratrol has a strong ability to protect cardiomyocytes from hypoxia/reoxygenation(H/R)injury.On this basis,the present study intends to explore the main mechanism of resveratrol on cultured neonatal rat cardiomyocytes for prevention and treatment of MIRI with hypoxia/reoxygenation injury,and to verify the hypothesis that "Resveratrol synergistically regulates mitochondrial dynamics,mitophagy and mitochondrial biogenesis through Sirtuin proteins to improve mitochondrial quality control and enhance MIRI tolerance",providing experimental basis for resveratrol against MIRI.Methods and ResultsWe isolated primary cardiomyocytes from neonatal rats with trypsin digestion and established an in vitro model of H/R by transferring cardiomyocytes to an incubator for 12 h of hypoxia and then placing back in the normoxic CO2 incubator for 12 h of reoxygenation.The cardiomyocytes were randomly divided into 5 groups:Control group,H/R group,5 ?M resveratrol pretreated group(Res-5 group),20 ?M resveratrol pretreated group(Res-20 group),and Sirtuin inhibitor group(Ex527 group).Cardiomyocytes were pretreated with resveratrol for 12 h,and the followed H/R treatment.Ex527 group was added Ex527(1 ?M)1 h before hypoxia.The study was divided into three parts to explore the reglatory mechanism of resveratrol on mitochondrial quality control of myocardial cells undergoing H/R injury.1.The research of resveratrol regulating mitochondrial quality in H/R injured cardiomyocytesMitochondrial membrane potential(MMP,??m)and cell ATP content measured by inverted fluorescence microscope and microplate reader,the activity of SOD detected by SOD detection kit-WST,MDA content in cells detected by MDA test kit were taken to assess the degree of myocardial cell mitochondrial damage in each grouo,and to analysis the effects of resveratrol on mitochondrial quality in H/R injured cardiomyocytes.The results indicated that:MMP of cardiomyocytes in H/R group decreased significantly compared with Control group(P<0.005),ATP content and the activity of SOD in cardiomyocytes fell about 55%(P<0.005)and 40%(P<0.05)respectively,and MDA content of cardiomyocytes in H/R group was significant higher than Control group(P<0.05),indicating that myocardial mitochondria suffered a great degree of damage after H/R treatment.In resveratrol pretreatment groups,MMP,ATP content and the activity of SOD were obviously improved(P<0.005 or P<0.05),MDA content wad reduced(P<0.05),there are significant difference compared with H/R group.These results indicated resceratrol pretreatment could strongly heighten mitochondrial antioxygenation and alleviate mitochondrial injuries induced by H/R.In Ex527 group,MMP and ATP content were evidently reduced after H/R treatment compared with Res-20 group(P<0.005).The SOD activity decreased by 51%and MDA content increased by 41%,which was significantly different from that of the Res-20 group(P<0.05).These results showed that Sirtuin proteins are involved in the protective effects of resveratrol on myocardial mitochondria treated by H/R.2.The effects of resveratrol on the regulation of Sirtl/Sirt3 in cardiomyocytes undergoing H/R injuryThe activity of Sirtl and Sirt3 in cardiomyocytes detected by Sirtl/Sirt3 deacetylase fluorometric assay kits,the protein expression level of Sirtl,Sirt3 and their downstream proteins FoxOl and Fox03a detected by Western Blot,the mRNA expression level of FoxOl and Fox03a detected by Real-Time PCR were measured in each group,to investigate the effect of resveratrol on the regulation of Sirtl and Sirt3 in myocardial cells injuried byH/R.The results showed that:The deacetylase activity and the protein expression Sirtl/Sirt3 of Res-20 group were markly higher than that of H/R group,and also significantly higher than that of Ex527 group(P<0.005 or P<0.05).These results confirmed the inhibitory effects of Ex527 on Sirtl and Sirt3,and showed that resveratrol could up-regulate the deacetylase activity and the protein expression levels of Sirtl/Sirt3 to activate Sirtl and Sirt3.Compared with H/R,resveratrol pretreatment raised Fox03a protein expression silightly,the protein expression level of FoxO1 was observably increased in Res-20 group(P<0.01).The mRNA expression levels of FoxO1 and Fox03a in hypoxic/reoxygenation-injured myocardium augmented significantly with increasing dose of resveratrol(P<0.005,P<0.01 or P<0.05).But the Sirtuin inhibitor,Ex527,could significantly weaken the upregulation of resveratrol on the protein expression of FoxO1 and FoxO3a(P<0.01 or P<0.05).The mRNA expression of FoxO1 and Fox03a in Ex527 group was evidently lower than that in Res-20 group(P<0.01).These results further demonstrated that resveratrol could activate Sirtl and Sirt3,and Sirtl/Sirt3-FoxO pathway participated in the protective effects of resveratrol on myocardial mitochondria in hypoxia/reoxygenation injury.3.Mitochondrial quality control regulated by Sirtl/Sirt3 is involved in the protective effects of resveratrol against myocardial hypoxia/reoxygenation injuryThe mRNA expression levels of Mfn1,Mfn2,Opal,Drp1,and Fis1 in myocardial cells were detected by Real-Time PCR to assess the state of mitochondrial fission and fusion in myocardial cells.The protein expression levels of LC3,PENK1 and Parkin in myocardial cells were detected by Western Blot,the number of mitochondrial autolysosomes in myocardial cells was assessed by laser confocal microscopy,and the degree of the interaction between p62 and Parkin was measured by Co-immunoprecipitation(Co-IP)to evaluate the activity of mitophagy in myocardial cells.The mitochondrial mass was observed by laser confocal microscopy,the mRNA level of PGC-1? was detected by Real-Time PCR to evaluate the activity of mitochondrial biosynthesis in myocardial cells,and finally to study the regulation of resveratrol on the mitochondrial quality in myocardial cells after hypoxia/reoxygenation.The results showed that:After pretreatment with resveratrol,the mRNA expression levels of Drp1,Mfn1,Mfn2,and Opal were significantly increased in hypoxic/reoxygenation-injured cardiomyocytes(P<0.005 or P<0.05).mRNA expression levels increased,Fis1 mRNA expression was higher than that of H/R group.Compared with H/R group,,the mRNA level of PGC-1?,a key regulator of mitochondrial biogenesis in cardiomyocytes,was significantly increased in the resveratrol pretreatment group(P<0.005 or P<0.01).After mitochondria were stained with red and green fluorescent probes,the degree of mitochondria network in the Res-20 group was greater than that in the H/R group,the number of functional mitochondria and the total number of mitochondria were both increased with the dose of resveratrol(P<0.05).These results illustrated that resveratrol could regulate mitochondrial dynamics by promoting mitochondrial fission-fusion and mitochondrial biosynthesis in cardiomyocytes treated by H/R.Compared with H/R group,the protein expression of LC3-? and Parkin in Res-20 group was significantly increased(P<0.05),the number of mitochondrial autolysosomes was significantly raised(P<0.01 or P<0.05),and the interaction between p62 and Parkin in cardiomyocytes of resveratrol pretreatment group strengthened significantly(P<0.05),indicating that resveratrol could induce mitophagy via Parkin-dependent pathway in cardiomyocytes injuried by H/R.In Ex527 group,the mRNAs expression Drp1,Mfn1,Mfn2,and Opal in cardiomyocytes was inhibited compared with Res-20 group;among them,the mRNA expression level of Mfii2 decreased more significantly(P<0.05).And the number of functional mitochondria and the total number of mitochondria were obviously less in cardiomyocytes added by Ex527 than that in Res-20 group(P<0.05).These results showed Sirtuin is involved in the regulation of resveratrol on mitochondrial dynamics in n myocardial cells after H/R injury.Compared with Res-20 group,the number of mitochondrial autolysosomes of cardiomyocytes in Ex527 group was drastically reduced(P<0.005),the protein expression of LC3-? and Parkin are significantly decreased(P<0.05),and the interaction between p62 and Parkin was markly weakened(P<0.01),illustrating that mitophagy mediated by Sirtl/Sirt3-Parkin pathway was an important mechanism of resveratrol regulating mitochondrial quality control in H/R cardiomyocytes.Conclusions1.Resveratrol could significantly increase MMP,ATP content and SOD activity,and obviously reduce MDA content in myocardial cells injuried by hypoxia/reoxygenation.It has a significant protective effect on myocardial mitochondria in myocardial cells suffering hypoxia/reoxygenation injury.Sirtuin-SOD could play an important regulatory role in the prevention of oxidative damage to mitochondria,it may be an important pathway of mitochondrial quality control.2.The protective effects of resveratrol on mitochondria in cardiomyocytes injured by hypoxia/reoxygenation might be related to activation of Sirt1/Sirt3?FoxO signaling pathway.3.Resveratrol could promote mitochondrial fission and fusion through Sirt1/Sirt3,accelerating the separations of damaged mitochondria from mitochondrial networks and promoting exchanges of mitochondrial content,to regulate mitochondrial quality.Its protective mechanism may be related to the upregulation of mRNA levels of mitochondrial fission and fusion key regulators mediatet ba Sirt1/Sirt3.4.The protective effect of resveratrol on mitochondria in hypoxia/reoxygenation cardiomyocytes is related to the Sirt1/Sirt3-Parkin signaling pathway,which could increase the interaction between p62 and Parkin,and then activate mitophagy to alleviates hypoxia/reoxygenation-induced mitochondrial damage.5.Resveratrol could regulate the mitochondrial quality through stimulation of mitochondrial biogenesis mediated by Sirtl/Sirt3 in hypoxia/reoxygenation injuried cardiomyocytes,and its mechanism may be related to the up-regulation of PGC-la expression.