Puerarin Alleviates The Hypoxic Injury Of Cardiomyocytes H9c2 By Regulating PI3K/AKT And AMPK Signaling Pathways Via Sirtl

BackgroundHypoxia-induced cardiomyocytes could promote the peroxidation progress of cell membrane,the generation of reactive oxygen radicals,resulting in the injury of cardiomyocytes.In addition,it was found that the changes of electrolyte,cell volume and the component of cell membrane phospholipids.Furthermore,the function of cardiomyocytes could also alter.Previosus research found that puerarin has the cytoprotective effect on multiple cell types.The cytoprotective effect of puerarin may be achieved by regulating the PI3K/AKT and AMPK signaling pathway.It has been reported that the activations of PI3K/AKT and AMPK signaling pathway could be associated with a modulatory manner dependent on the expression of Sirtl.At present,there is no clear study on the effect of puerarin on Sirtl in PI3K./AKT and AMPK signaling pathways,especially the protective effect of puerarin on hypoxia-induced myocardial cell injury.ObjectiveThe protective effect of puerarin on cardiac cells was investigated through in vitro injury experiment of cardiac cells induced by hypoxia.Moreover,phosphotylinosital 3 kinase(PI3K)/Protein kinase B(PKA/AKT)and adenosine monophosphate activated protein(AMPK)signaling pathways were also explored.Further mechanism study of puerarin inhibiting apoptosis and improving the cell viability was also designed.On the basis of the above research,the regulatory effect of puerarin against pro-apoptotic proteins(Cleaved/Pro-caspase-9 and Cleaved/Pro-caspase-3)and upregulation effect of puerarin on anti-apoptotic protein(Bcl-2)were also analyzed by Western blot method.In addition,the effect of hypoxia on Sirtl,cell viability,and apotosis were studied.We regarded PI3K/AKT and AMPK signaling pathway as the main research objects to explore the regulatory of puerarin on the activation of PI3K/AKT and AMPK signaling pathway.The role of Sirtl in the activation of PI3K/AKT and AMPK signaling pathway was also explored.The purpose of the research was to study the mechanism of purerarin inhibiting hypoxia-induced injury in cardiomyocytes.Moreover,our research are supposed to provide experimental evidence for puerarin application in improving hypoxia-induced myocardial cell injury.Methods(1)Chapter 1:Puerarin inhibited hypoxia-induced myocardial cell injury.Myocardial cell was isolated from rat embryo,used to establish the hypoxia-induced injury experimental model.In this study,we confirmed the application time of hypoxia treatment.We also determined the effects of hypoxia-induction on apoptosis and apoptosis related-factors.We pretreated H9c2 cells with puerarin in different concentrations(0,20,40,60,80,and 100 μM.We finally determined the pretreatment concentration of puerarin in 60 μM which was nontoxic from the cell vability results.Myocardial cell damage was induced by hypoxia after puerarin pretreatment with a concentration in 60 pM in order to explore the inhibitory effect of puerarin on hypoxia-induced injury in myocardial cells.Further,we the regulatory effect of puerarin on the expression of apotosis related-proteins(Bcl-2,Pro-Caspase-9,Pro-Caspase-3,Cleaved-Caspase-9,and Cleaved-Caspase-3).The phosphorylation of PI3K,AKT,and AMPK was also analyzed in our experiment.In this chapter,the cell viability was determined by cell counting kit-8 method(CCK-8).The cell apoptosis was determined by Annexin V-Fluorescein isothiocyannate/Propidium iodide(Annex V-FITC/PI)method.The expression of apoptotic factors(Bcl-2,Pro-Caspase-9,Pro-Caspase-3,Cleaved-Caspase-9,and Cleaved-Caspase-3)at protein level was analyzed by Western blot.The phosphorylation progress of PI3K,AKT,and AMPK was also evaluated by Western blot method.(2)Chapter 2:Puerarin reduced the myocardial cell injury by regulating Sirtl.Based on the research in chapter 2,we further explored the regulatory effect of puerarin on Sirtl in hypoxia-induced myocardial cell injury.Specifically,the expression of Sirtl was silenced by si-Sirtl in H9c2 cells in order to verify its role in the progress of cell viability and apoptosis,the expression of apoptosis-related factors(Bcl-2,Pro-Caspase-9,Pro-Caspase-3,Cleaved-Caspase-9,and Cleaved-Caspase-3)taken as indicators.In the experiments of this chapter,the expression of Sirtl at the mRNA level was determined by real-time quantitative PCR.The expression of Sirtl at the protein level was analyzed by Western blot,and the expression levels of Bcl-2,Pro-Caspase-9,Pro-Caspase-3,Cleaved-Caspase-9,and Cleaved-Caspase-3 were also determined by Western blot method.(3)Chapter 3:Puerarin regulated PI3K/AKT and AMPK pathways via Sirt1.In this chapter,we further explored the role of puerarin in the regulation of PI3K/AKT signaling pathway and AMPK pathway via Sirt1.In the experiments of this chapter,the expression of Sirtl was silenced after myocardial cells being pretreated with 60μM puerarin for 48 hours,the myocardial cells injury being subsequently induced by hypoxia.The expression of related-proteins(PI3K,p-PI3K,AKT,p-AKT,AMPK,and p-AMPK)in PI3K/AKT and AMPK pathway was determined by Western blot method to clarify the effect of puerarin on phosphorylation of PI3K,AKT,and AMPK.Furthermore,the activation effect of puerarin on PI3K/AKT and AMPK pathway was investigated to verify that the puerarin was involved in regulating PI3K/AKT pathway and AMPK signaling pathway via Sirt1.In addition,the inhibitors and activators of PI3K/AKT and AMPK pathways were used to verify whether Puerarin regulated Sirt2/PI3K/AKT/AMPK signaling to protect H9c2 cells against hypoxia induced damage.Results(1)Chapter 1:Puerarin inhibited hypoxia-induced myocardial cell injury.The myocardial cells H9c2 were treated with hypoxia for 0,12,24,and 48 hours,respectively.The results from CCK-8 showed that the cell viability of H9c2 was significantly reduced after hypoxia treatment compared with the cell viability of H9c2 in the Control group(P<0.05).The cell viability of H9c2 decreased to 50%after the cell induced by hypoxia for 24 hours.While the cell viability of H9c2 was lower than 50%after the cells induced by hypoxia for 48 hours(P<0.001).The cell viability of H9c2 cells was negatively correlated with the hypoxia treatment time in the range of the time we explored.Therefore,the time of hypoxia treatment for 24 hours was choosen for subsequent experiment.The results showed that the the cell apoptosis rate significantly increased after hypoxia treatment compared with the H9c2 cells’ in the Control group(P<0.001).The results of Western blot showed that the expression of anti-poptotic factor Bcl-2 was downregulated by hypoxia treatment.We further analyzed the effect of hypoxia treatment on the expression of pro-poptotic factors.The results showed that the ratio of Cleaved-Caspase-9 and Pro-Caspase-9 increased after hypoxia treatment.The ratio of Cleaved-Caspase-3 and Pro-Caspase-3 was also increased by hypoxia treatment.After treating H9c2 cells with puerarin at different concentrations(0,20,40,60,80,and 100 μM),we could discover that the cell viability was observably reduced by puerarin at 80 and 100 μM(P<0.05).We can conclude that the puerarin was toxic for H9c2 cells at higher concentration,while non-toxic at lower concentration like 20-60 μM which was no significant stimulation and inhibitation for cell viability.Therefore,the concentration of puerarin selected in subsequent experiments was 60 μM.The pre-protective effect of puerarin on hypoxia-induced cells showed that hypoxia treatment significantly reduced the cell viability(P<0.01),while the cell viability was significantly increased after 60 μM puerarin pretreatment.We could conclude that puerarin pretreatment improved the cell viability which reduced by hypoxia.Hypoxia treatment significantly promoted the cell apoptosis(P<0.001).The hypoxia-induced cell apoptosis was alleviated by 60μM puerarin pretreatment(P<0.05).The results of Western blot showed that the expression of anti-apoptotic factor Bcl-2 was upregulated in the H9c2 cells by puerarin compared with the hypoxia-induced cells’(P<0.05),with the expression of Cleaved-Caspase-9 and Cleaved-Caspase-3 inhibited(P<0.01).The results of PI3K/AKT and AMPK study showed that the puerarin activated the phosphorylation of PI3K and AKT inhibited by hypoxia(P<0.05).The above results showed that the puerarin could reserve the inactivation of hypoxia on PI3K/AKT signaling pathway.Similary,puerarin also promoted the phosphorylation of AMPK(P<0.001).(2)Chapter 2:Puerarin reduced the myocardial cell injury by regulating Sirtl.The results of qRT-PCR showed that hypoxia treatment reduced the expression of Sirtl at mRNA level compared with the Control group(P<0.05).Compared with expression of Sirtl in the hypoxia group,puerarin pretreatment significantly increased the expression of Sirtl(P<0.001).Western blot results showed that the expression of Sirtl at protein level was dramatically reduced by hypoxia compared with the Control group(P<0.05).After treated with puerarin,the expression of Sirtl was up-regulated compared with the hypoxia group(P<0.01).In addition,the relative expression of Sirtl at mRNA level was inhibited after si-Sirtl transfected into H9c2 cells.The results of Western blot also showed that the expression of Sirtl at protein level was significantly reduced after si-Sirtl transfected into H9c2 cells(P<0.01).Moreover,the cell viability was reduced by silencing the expression of Sirtl in the H9c2 cells compared with that of the negative control group(P<0.05).Further study showed that the apoptosis rate of H9c2 cells was significantly increased by inhibiting the expression of Sirtl after H9c2 cells treated with puerarin compared(P<0.01).Analysis of the relative expression of apoptotic related-factors at protein level showed that inhibition of Sirtl significantly reduced the expression of anti-apoptotic factor at protein level(P<0.05).The proportion of Cleaved-Caspase-9 in the Pro-Caspase-9 was promoted(P<0.01).Similarly the proportion of Cleaved-Caspase-3 in the Pro-Caspase-3 was also promoted(P<0.05).(3)Chapter 3:Puerarin regulated PI3K/AKT and AMPK pathways via Sirtl.We further studied whether puerarin could regulate the activation of PI3K/AKT pathway through Sirt1.The results showed that the phosphorylation of PI3K was significantly inhibited by inhibiting the expression of Sirtl compared with that of negative control group(P<0.001).The phosphorylation of AKT was significantly reduced compared with the negative control group(P<0.001).The phosphorylation of AMPK was also significantly reduced compared with the negative control group(P<0.001).The inhibitors of PI3K/AKT and AMPK pathway(LY294002 and Dorsomorphin)significantly suppressed cell viability(P<0.05),induced apoptosis(P<0.01)and involved in regulation apoptosis-related protein expression(P<0.01 or P<0.001).Of contrast,the activators of PBK/AKT and AMPK pathway(IGF-1 and A-769662)promoted cell viability(P<0.001),inhibited apoptosis(P<0.001)and acted the adverse impacts on apoptosis-related protein expressions(P<0.01 or P<0.001).ConclusionsIn this study,we could conclusion that puerarin played a protective role in hypoxia-induced myocardial cells by regulating PI3K/AKT and AMPK signaling pathways via Sirt1.In addition,we verified the role of Sirtl in the regulation of puerarin on hypoxia-induced myocardial cells.We obtained the following main conclusions:(1)Puerarin pretreatment improved the hypoxia-induced myocardial cell injury,promoted the expression of anti-apoptotic factor Bcl-2,inhibited the expression of pro-apoptotic factors(Caspase-9 and Caspase-3),and finally improved cell viability and inhibited apoptosis.(2)Puerarin may play a protective effect on myocardial cells by activating PI3K/AKT and AMPK signaling pathways;(3)Puerarin pretreatment promoted the expression of Sirtl and further inhibited the apoptosis induced by hypoxia.(4)Puerarin could alleviated the inactivation of hypoxia on PI3K/AKT and AMPK signaling pathways by up-regulating the expression of Sirt1.