Research On The Partner Proteomics Of Hes1 And Its Mediated Cardioprotective Effects Of Apigenin And Astragaloside ? Against Anoxia/reoxygenation Injury

Chapter 1 IntroductionThe present study focused on Notch 1 signaling pathway and its downstream target Hes1,we established the anoxia/reoxygenation(A/R),ischemic preconditioning(IPC)and ischemic postconditioning(IPost)models in vitro.Then we detected the protein expression of Notchl intracellular domain(N1ICD)and Hes1,the activity of CPK and LDH,the cell viability,in order to verify the potential roles of Notchl signaling pathway and its downstream target Hesl in protecting cardiomyocytes against A/R injury.We constructed adenoviral vector and infected H9c2 cells,using tandem affinity purification(TAP)and liquid chromatography electrospray ionisation tandem mass spectrometry(LC-ESI-MS/MS)of proteomics techniques to screen the differential expression partner-proteins of Hesl,which is a downstream target of Notchl signaling pathway.Using co-immunoprecipitation(Co-IP)assay to demonstrate whether Hesl protein interacted with VDAC1 protein,which is one of the differential expression partner-proteins of Hesl.Finally,we infected H9c2 cells by using adenoviral vector,then evaluated the cell viability,the protein expression of Hesl and VDAC1,ROS,??m and cell apoptosis,with the aim at demonstrating the cardioprotective effects of Notch 1/Hes1/VDAC 1 signaling pathway against A/R injury.We determinated the cell viability,the expression of Hesl protein,the activity of CPK and LDH,reactive oxygen species(ROS),mitochondrial membrane potential(??m),the opening of mitochondrial permeability transition pore(mPTP),the activity of caspase-3 and cell apoptosis,trying to investigate whether the cardioprotective effects of apigenin(AP)and astragaloside ?(ASI)against A/R injury were associated with Notch1/Hes1 signaling pathway.In order to investigae the molecular mechanisms of Notchl/Hesl signaling pathway to protect cardiomyocytes against A/R injury,and provide the theoretical basis for the clinical therapies for ischemic heart disease(IHD).Chapter 2 Notchl signaling pathway protects cardiomyocytes against A/R injuryObjectives:To verify whether the Notch1 signaling pathway can protect cardiomyocytes against A/R injury in IPC and IPost.Methods:To establish the A/R,IPC and IPost models in H9c2 cells,using y-secretase inhibitor(DAPT)to inhibit the Notchl signaling pathway,the N1ICD protein expression was detected by Western blotting,the activity of CPK and LDH was measured by colorimetric method,and the cell viability was determined by MTS assay.Results:IPC and IPost could up-regulate the protein expression of N1ICD,decrease the activities of CPK and LDH,increase the cell viability.However,after pretreatment with DAPT,the protein expression of N1ICD was down-regulated,the activities of CPK and LDH were increased,and the cell viability was decreased.Conclusions:Notchl signaling pathway plays a role in IPC and IPost cardioprotection against A/R injury.Chapter 3 Cardioprotection against A/R injury by Hesl:a downstream target of Notchl signaling pathwayObjectives:To demonstrate whether Hesl,a downstream target of Notchl signaling pathway,has the cardioprotection against A/R injury in IPC and IPost,and confirm the endogenous cardioprotective effects of Notch1/Hes1 signaling pathwayMethods:To infect H9c2 cells by using Hes1-shRNA adenoviral vector(AD-Hes1-shRNA),then the A/R,IPC and IPost models were established in H9c2 cells,the expression of Hesl protein was detected by Western blotting,the activities of CPK and LDH were measured by colorimetric method,the cell viability was determined by MTS assay.Results:IPC and IPost could up-regulate the protein expression of Hesl,decrease the activities of CPK and LDH,increase the cell viability.However,when infected by AD-Hes1-shRNA,the protein expression of Hesl was down-regulated,the activities of CPK and LDH were increased,and the cell viability was decreased.Conclusions:Hesl,a downstream target of Notchl signaling pathway,might protect cardiomyocytes from A/R injury in IPC and IPost.Chapter 4 Screen the differential expression partner-proteins of HeslObjectives:To screen the differential expression partner-proteins of Hesl,a downstream target of Notch1 signaling pathway.Methods:To construct adenoviral vector AD-NTAP/Hesl,which contains TAP tag,and infect H9c2 cells,then establish the A/R model,detect the exogenous protein expression of Hesl,the activities of CPK and LDH,the cell viability,testify the biological functions of AD-NTAP/Hes1.Using the proteomics techniques(TAP and LC-ESI-MS/MS)to screen the differential expression partner-proteins of Hesl and demonstrate whether Hesl protein could interact with VDAC1 protein by Co-IP assay.Results:AD-NTAP/Hes1,which was constructed successfully,could activate the exogenous protein expression of Hesl,decrease the activities of CPK and LDH,increase the cell viability.There were 88 partner-proteins of Hesl.VDAC1,one of the differential expression partner-proteins of Hesl,was selected for the following research.Co-IP assay confirmed that Hesl protein interacted with VDAC1 proteinConclusions:AD-NTAP/Hesl was constructed successfully.AD-NTAP/Hesl could activate the exogenous protein expression of Hesl to protect cardiomyocytes from A/R,and VDAC1 is one of the differential expression partner-proteins of Hes1.Chapter 5 The cardioprotection of Notchl/Hes1/NDAC1 signaling pathway against A/R injuryObjectives:To demonstrate the cardioprotective effects of Notch1/Hesl/VDAC1 signaling pathway against A/R injury.Methods:To infect H9c2 cells by AD-NTAP/Hesl and AD-Hes1-shRNA respectively,establish the A/R,IPC and IPost models.The expression of Hesl and VDAC1 protein was detected by Western blotting,the levels of intracellular ROS,??m and cell apoptosis were evaluated by flow cytometric analysis.Results:AD-NTAP/Hesl could up-regulate the protein expression of Hesl,down-regulate the protein expression of VDAC1,reduce the ROS generation,stabilize the ??m,inhibit the cell apoptosis.While AD-Hes1-shRNA could down-regulate the protein expression of Hesl,up-regulate the protein expression of VDAC1,increase the ROS generation,unbalance the ??m,increase the cell apoptosis,attenuate the cardioprotection of IPC and IPost.Conclusions:Notch1/Hes1 signaling pathway can directly down-regulate the protein expression of VDAC1,improve the A/R injury.The cardioprotection of IPC and IPost was associated with Notch1/Hesl/VDAC1 signaling pathway.Chapter 6 Hesl mediated the cardioprotection of AP and ASI against A/R injuryObjectives:To investigate whether the cardioprotective effects of AP and ASI against A/R injury in the primary neonatal Sprague Dawley(SD)rat cardiomyocytes were associated with Notch1/Hes1 signaling pathway.Methods:Pretreated with AP,ASI and DAPT in the primary neonatal SD rat cardiomyocytes,established the A/R model,then detected the cell viability,the expression of Hesl protein,the activities of CPK and LDH,ROS,??m,the opening of mPTP,the activity of caspase-3 and cell apoptosis,respectively.Results:AP and ASI protected cardiomyocytes from A/R injury in a dose-dependent manner,could effectively increase the cell viability,decreased the activities of CPK and LDH,reduce the ROS generation,stabilize the ??m,inhibit the mPTP opening and activity of caspase-3,finally decrease the cell apoptosis in cardiomyocytes.AP and ASI could up-regulate the protein expression of Hesl,while most of these cardioprotective effects were abolished by DAPT,except of ROS generation.Conclusions:AP and ASI have the cardioprotective effects against A/R injury,which were mediated by Hesl.