Investigation Of PI3K/Akt Signaling Pathway In The Protection Effects On Oxidative Stress-Induced Cardiomyocytes Apotosis Of 3,5-dicaffeoylquinic Acid

BackgroundHeart failure is the end stage of multiple cardiovascular diseases.From the view of traditional Chinese medicine,heart failure emphasized the weakness of qi and yang,as well as phlegm,liquid and blood stasis,which is syndrome of deficiency ben and excessive biao.Improving blood circulation and dispersing stasis is one of the principles for treating heart failure.Ilex pubescens,a commonly used Chinese medicine,is great for detoxification,detumescence and promoting blood circulation.At present,Ilex pubescens is mainly used in treatment of coronary heart disease,heart failure,ect.Isochlorogenic acid A,also known as 3,5-dicaffeoylquinic acid(3,5-diCQA),is one of the main chemical constituents of Ilex pubescens,which has shown protective effects against oxidative stress in many diseases.Oxidative stress-induced apoptosis plays an important role in the development of heart failure.Therefore,3,5-diCQA may be the material basis of Ilex pubescens in the treatment of heart failure.ObjectThe objective of this study was to investigate the anti-apoptosis potential of 3,5-diCQA in cardiomyocyte cells under oxidative stress,explore its underlying mechanisms and provide a scientific basis for the anti-heart failure of Ilex pubescens.MethodA model of tert-butyl hydroperoxide(TBHP)-induced apoptosis in a cardiomyocyte cell line(H9C2)was established.Cell viabilities on cell lines were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium(MTT)assay.The apoptosis was measured by hoechst33342 and propidium iodide(PI)fluorescent staining.PI(in red)stained the regions of cell apoptosis;Hoechet33342(in blue)stained the nuclei.The Western blot was used to determine the expressions of related proteins such as p-PI3K:phosphorylated phosphatidylinositol-3-kinase(p-PI3K),phosphorylated Serine and Threonine kinase AKT(p-AKT),p-PTEN,Bcl-2,Bax,and caspase-3.Afterward,a PI3K inhibitor,LY294002,was applied to confirm the influence of the PI3K/Akt pathway on TBHP-treated cells of 3,5-diCQA.Then,H9C2 cells were pre-incubated with 3,5-diCQA alone to determine if the expression of activated PI3K/Akt signaling was mediated by 3,5-diCQA in H9C2 cells.ResultThe results showed that TBHP resulted in an increase in cardiomyocyte apoptosis,whereas 3,5-diCQA treatment protected cells from TBHP-induced apoptosis in a dose-dependent manner.Moreover,3,5-diCQA decreased expressions of Bax and caspase-3 but increased expression of Bcl-2,accompanying an increasing phosphorylation levels of PI3K and Akt in TBHP-treated cells,which are the key molecules mediating cell survival,whereas phosphatase and tensin homologue deleted on chromosome 10(PTEN)phosphorylation was unchanged.Importantly,pre-incubation with a PI3K inhibitor(LY294002)partly abolished the anti-apoptosis effects of 3,5-diCQA.Low cell viability and high apoptosis rate were manisfected in H9C2 cells pretreatment with LY294002.Further,3,5-diCQA enhanced the phosphorylation levels of PI3K and Akt in H9C2 cells directly,while LY294002 attenuated the effects of 3,5-diCQA on PI3K and Akt.ConclusionThis study suggested that 3,5-diCQA rescued myocardium from apoptosis by increasing the activation of the PI3K/Akt signaling pathway.3,5-DiCQA may be the pharmacological active ingredients of Ilex pubescens in treating heart failure.