Protective Effect Of Amentoflavone Against Myocardial Ischemia-reperfusion Injury

Objective: Through the establishment of SD rats myocardial ischemia/reperfusion injury(I/R)model,the protective effect of amentoflavone on I/R was studied in detail.Methods: 1.Microwave-assisted extraction(MAE)was used to extract Selaginella doederleinii.The extract was purified by D101 macroporous resin to obtain total biflavonoids from S.doederleinii.Total biflavonoids were separated by silica gel column chromatography to obtain amentoflavone(AME).The purity of AME was detected by high performance liquid chromatography(HPLC).Nuclear Magnetic Resonance(NMR)was used to identify the structure of AME.2.We established the model of I/R in rats myocardium and explored the protective effect of AME on I/R.SD rats were randomly divided into 6 groups(n=10):(1)Sham group,(2)I/R group,(3)Diltiazem(DIZ)group,(4)I/R+AME 25 mg/kg group,(5)I/R+AME 50 mg/kg group,(6)I/R+AME 100 mg/kg group.After 7 days of continuous administration,I/R model was established by ligating of the coronary artery in rats.The ST segments of the electrocardiogram of the rats were detected throughout,and then the blood of the abdominal aorta and the rats hearts were taken separately.The left ventricle of rat hearts were measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining method for the volume of myocardial infarction.Hematoxylin-eosin staining(HE)staining was used to observe the pathological changes of myocardium.Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transfer mediated d UTP biotin nick end labeling assay(TUNEL).The levels of LDH in serum were examined by lactate dehydrogrnase(LDH)release.The levels of CK-MB,IL-1?,IL-6 and TNF-? in serum were detected by ELISA.Expressions of the proteins in PI3K/AKT-NF-?B signaling pathways were measured using Western blot analysis.Results:1.After microwave-assisted extraction,147.21 g of the extract was obtained.The biflavonoids in S.doederleinii were separated by D101 macroporous,gel column chromatography and sephadex LH-20 to obtain 3.23 g of AME with purity 96.2%.2.The experimental results of AME on rat I/R model showed that AME could significantly reduce the volume of myocardial infarction and cardiomyocytes apoptosis in rats,and also reduce the levels of myocardial enzymes(LDH,CK-MB)and related inflammatory indexes in rat serum(p?0.05)including IL-1?,IL-6 and TNF-?.In addition,compared with the I/R group,AME treatment not only could increase the protein expression level of p-AKT,but also reduce the protein expression level of p-NF-?B in a dose-dependent manner.Conclusion: Based on the above results,AME could improve the myocardial injury induced by coronary artery ligation caused by I/R.Furthermore,AME could reduce cardiomyocyte apoptosis and the release of related inflammatory factors,thereby improving ventricular remodeling and myocardial tissue lesions,and it's potential mechanism may be related the regulation of PI3K/AKT-NF-?B signaling pathway.The results of this study were provided a new theoretical reference for the clinical application of AME in the prevention and treatment of I/R.