Flavonoid C-glycosides From Semen Ziziphi Spinosae Alleviate The Myocardial Ischemia-reperfusion Injury Through Inhibiting GSK3? In Rats

BackgroundAcute myocardial infarction(AMI)is one of the leading causes of death and has become a major public health problem in many countries.The mechanism of myocardial ischemia-reperfusion injury is complicated.It is considered that free radical generation,oxidative stress,mitochondrial damage and energy depletion are the main causes of myocardial cell damage.Inhibiting the phosphorylation of glycogen synthase kinase-3?(GSK3?)plays a key role in protecting myocardial ischemia-reperfusion injury,and can achieve myocardial protection by inducing autophagy and activating multiple cell survival pathways.Flavonoid C-glycosides isolated from Semen Ziziphi Spinosae(Suan Zao Ren),including spinosin(SP)and 6'''-feruloylspinosin(FS),are natural antioxidants with free radical scavenging and antioxidant effects.Whether they are involved in the protection of myocardial ischemia-reperfusion injury is still unclear.MethodsMyocardial ischemia was induced by ligation of the left anterior descending coronary artery(LAD).After 30 minutes ischemia,the blood flow was resumed to induce reperfusion for 1 hour.Animals were randomly divided into four groups:sham operation group,model with vehicle control group,SP and FS administration group.SP or FS(5mg/kg)was intraperitoneally injected 30 minutes before LAD ligation.Serum levels of cardiac troponin 1(cTnI)was detected by ELISA,and lactate dehydrogenase(LDH)was detected by colorimetry after reperfusion.Paraffin sections were made from heart tissue and protein and RNA were extracted.The myocardial tissue damage was detected by hematoxylin-cosin(HE)staining,and the apoptosis rate was determined by TUN EL assay.Western blot and fluorescence quantitative PCR were used to detect the protein and mRNA expressions of each myocardial sample to explore the potential molecular mechanism.ResultsElectrocardiogram displays elevated ST segment dorsal arch and high T wave,indicating the success of model.SP and FS can significantly reduce the release of cTnI and LDH,and decrease the apoptosis rate after ischemia and reperfusion.The HE staining of model rats showed that some myocardial cells had edema and necrosis,accompanied by local hemorrhage and neutrophil infiltration,which could be significantly alleviated after the administration of flavonoid C-glycosides.In addition,these two flavonoids also increased the expression of LC3B-? and decreased the level of p62,indicating that they promoted autophagic flow.Molecular mechanism studies suggest that both SP and FS could decrease the phosphorylation of GSK3 at Tyr216 and increased the peroxisome proliferator-activated receptor y coactivator-la(PGC-la),accompanied by up-regulation of downstream signals erythropoies-derived nuclear factor-associated factor-2(Nrf2)and heme oxygenase 1(HO-1).Compared with SP,FS administration more obviously reduced pGSK3 and increased the expression of PGC-l?,Nrf2 and HO-1,suggesting FS is superior to SP in the regulation of cell protection signals.ConclusionsFlavonoid C-glycosides of Suanzaoren(SP and FS)could reduce the releases of cTnl and LDH,alleviate the myocardial tissue damage and apoptosis,and promote cell autophagy in myocardial ischemia-reperfusion rats.These effects may be related to inhibition of phosphorylation of GSK30 at tyrosine 216 and activation of the PGC-la/Nrf2/HO-1 pathway.Our study suggests that SP and FS may protectcardiomyocytes from acute cardiac ischemia by GSK3? inhibition-mediatedautophagy stimulation and the PGC-la/Nrf2/HO-1 pathway.This study not only provides a better understanding of the cardioprotective mechanism,but also updates the current knowledge about the cardioprotective effects of the active components of Semen Ziziphis Spinosae.It provides a preliminary basis for the clinical development of new drugs for patients with myocardial infarction.