Dicaffeoylquinic Acids Alleviates Oxidative Stress Injury In H9c2 Cardiomyocytes By Activating PI3K/Akt/HIF-1ɑ Signalling Pathway

BackgroundHeart failure(HF)is a common and highly prevalent disease in China and is the leading cause of death worldwide,which seriously affects the health,social,and economic development of the general public.HF is a complex of clinical syndromes and is mainly characterized by dyspnea,fatigue,and fluid.HF is caused by structural and/or functional abnormalities of the heart due to multiple causes,which impair ventricular systole and/or diastole.Most of patients develop chronic HF as the time goes,and treatment of HF symptoms is the main strategy.However,due to the poor drug compliance,the expected results cannot be achieved.Although the efficacies of various novel drugs on HF have been published,such as angiotensin receptor/neprilysin inhibitors(ARNI),angiotensin converting enzyme inhibitors(ACEI),angiotensin II receptor blocker(ARB),β-blocker,aldosterone receptor antagonist,sodium glucose cotransporter-2(SGLT2)inhibitor,and diuretics,no breakthroughs have been present yet.Cardiomyocyte apoptosis plays a key role in the etiology and pathogenesis of HF,in which oxidative stress is one of the major factors inducing apoptosis existing mainly in myocardial infarction,atherosclerosis and ischemia or reperfusion.Activation of the phosphorylated phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)signaling pathway is one of the important mechanisms of cardiomyocyte protection,which can promote cardiomyocyte cell survival by activating downstream targets.Among its downstream signals,hypoxia inducible factor-1(HIF-1),regulating hundreds of target genes in the hypoxic microenvironment thus maintaining cell survival,has been a hot topic of research in recent years.Dicaffeoylquinic acids(DCQAs)are important bioactive dietary polyphenols.The polyphenolic hydroxyl chemical structure it contains and its remarkable antioxidant,free radical scavenging,antiviral and other extensive biological activities have been a research hot for drug development,but the action mechanisms are not fully understood.ObjectiveIn this study,an in vitro oxidative stress model simulating the process of HF was constructed in H9c2 cardiomyocytes stimulated with TBHP;the anti-apoptosis activities of six natural DCQAs on H9c2 cardiomyocytes were screened,and the protective effect of DCQA through PI3K/Akt/HIF-1α signaling pathway was investigated,which would elaborate the mechanism of DCQAs protecting H9c2 as well as potential effect to HF.Methods1 Effects of DCQAs on the viability of H9c2 cell stimulated by TBHPAn in vitro oxidative stress model was established in H9c2 cells by TBHP induction to simulate HF.Anti-oxidative stress activity of six natural DCQAs were assessed by CCK-8assay,and the range of dose-dependent was screened for the most active compound.2 Bioinformatics analysis of 3,4-DCQA against HFTCMSP and Swiss Target Prediction database were used to obtain potential therapeutic targets of DCQAs,Dis Ge NET and Gene Cards database were used to screen the targets of HF.Protein-protein interaction(PPI)analysis was performed by STRING,and DAVID online platform was used to analyze GO and KEGG signaling pathway enrichment analysis.PPI network visualization was performed by Cytoscape software,and key targets were obtained based on topological data in PPI.Finally,binding analysis of active ingredient with potential therapeutic target was performed by molecular docking.3 Effects of 3,4-DCQA on PI3K/Akt/HIF-1ɑ pathway in H9c2 cells exposed to TBHPThe Western blot was used to determine the expressions of related proteins such as HIF-1ɑ,p-PI3 K,p-Akt,ROS,and Nrf2,as well as that of apoptosis related proteins including Caspase-3,Bax,and Bax.The q RT-PCR was used to determine the m RNA expressions of HIF-1ɑ,PI3 K,and Akt.4 PI3K-dependent effects of 3,4-DCQA on PI3K/Akt/HIF-1ɑ activationH9c2 cell viability in a state of PI3 K inhibition was studied using CCK-8 assay.The Western blot was used to determine the expressions of HIF-1ɑ,p-PI3 K,p-Akt,caspase-3,Bcl-2,and Bax.The q RT-PCR was used to determine the expressions of HIF-1ɑ,PI3 K,and Akt.5 PI3K-dependent effects of elevated PI3K/Akt/HIF-1ɑ activation by 3,4-DCQAH9c2 cells were pre-incubated with 3,4-DCQA alone.Western blot was used to determine the expressions of HIF-1ɑ,p-PI3 K,p-Akt,Caspase-3,Bcl-2,and Bax.The q RT-PCR was used to determine the expressions of HIF-1ɑ,PI3 K,and Akt.Result1 DCQAs increased the viability of H9c2 cell stimulated by TBHPDCQAs(5,10,and 20 μM)attenuated the TBHP-induced decrease of cell viabilities in a dose-dependent manner,in which 3,4-DCQA showed the best activity.The 3,4-DCQA showed no toxicicity on H9c2 cells in the range of 1-320 μM,and showed significant anti-apoptotic effects in the range of 20-160 μM.2 Bioinformatics analysis of 3,4-DCQA against HFA total of 13527 HF-related targets were collected from the database and 43 biological targets related to 3,4-DCQA.Among them,41 potential targets attributed for 3,4-DCQA to the treatment of HF.Five proteins including CASP3,CA9,AKR1B1,APP,and FYN located as the core targets,which are mainly involved in insulin signaling pathway,TNF signaling pathway,and PI3K/Akt signaling pathway.The top-ranked apoptosis protein Caspase-3 plays a central role in the execution phase of apoptosis.GO enrichment analysis showed that the BPs were mainly involved in cellular protein metabolic processes,myocardial hypertrophy,etc.CCs were mainly involved in cytoplasm,mitochondria,Golgi-associated vesicles,etc.MFs were mainly involved in carbonate dehydrogenase activity,3’,5’-cyclic-nucleotide phosphodiesterase activity,etc.KEGG pathway enrichment analysis showed that nitrogen metabolic pathway,AGE-RAGE signaling pathway in diabetic complications,lipid metabolic signaling pathway,and atherosclerotic signaling pathway were mainly involved.3 Effects of 3,4-DCQA on PI3K/Akt/HIF-1ɑ pathway in H9c2 cells exposed to TBHPThe 3,4-DCQA promoted the activation of PI3K/Akt/HIF-1ɑ pathway,reduced protein expressions of ROS,Nrf2,Caspase-3,and Bax,meanwhile increased expression of Bax.In q RT-PCR analysis,compared with TBHP group,the m RNA expressions of PI3 K,Akt,and HIF-1ɑ in H9c2 treated with 3,4-DCQA significantly increased.4 PI3K-dependent effects of 3,4-DCQA on PI3K/Akt/HIF-1ɑ activationLY294002 alone significantly reduced expressions of p-PI3 K,p-Akt,and HIF-1ɑ.The3,4-DCQA promoted p-PI3 K,p-Akt,and HIF-1ɑ protein expression.LY294002 significantly downregulated the expressions of p-PI3 K,p-Akt,and HIF-1ɑ increased by 3,4-DCQA.In q RT-PCR analysis,the m RNA expression of PI3 K,Akt,and HIF-1ɑ in H9c2 cells treated with 3,4-DCQA and LY294002 significantly increased.In addition,LY294002 significantly increased the expressions of Caspase-3 and Bax,and reduced Bcl-2 expression.LY294002 also significantly down-regulated the expressions of the Caspase-3 and Bax increased by3,4-DCQA,and reduced Bcl-2 expression.5 PI3K-dependent effects of elevated PI3K/Akt/HIF-1ɑ activation by 3,4-DCQAThe 3,4-DCQA increased expressions of p-PI3 K,p-Akt,p-Akt/Akt,and HIF-1ɑ.In q RT-PCR analysis,the m RNA expressions of p-PI3 K,p-Akt,and HIF-1ɑ in H9c2 cells pre-incubated with 3,4-DCQA and LY294002 were significantly increased when compared with TBHP group.The 3,4-DCQA inhibited the expressions of Caspase-3 and Bax,and increased Bcl-2 expression.ConclusionIn this study,we applied a cell-based oxidative stress model to mediate programmed cardiomyocyte death and thus mimic the process of HF,and found that 3,4-DCQA may protect H9c2 cells injured by TBHP by regulating the PI3K/Akt/HIF-1ɑ signaling pathway.Inhibition of oxidative stress/or programmed cardiomyocyte death will provide important basis for the development of new drugs for the prevention and treatment of HF.