Research On Protein Expression Of Naoxintong Formula In Anti-myocardial Ischemia Based On The Theory Of "Naoxin Tongzhi"

Objective:The theory of "simultaneous treatment of brain and heart" has guiding significance for the treatment of cardiovascular and cerebrovascular diseases in clinic.Naoxintong prescription summarized on the basis of "simultaneous treatment of brain and heart" has remarkable curative effect on cardiovascular and cerebrovascular diseases in clinic.Therefore,this study explores the therapeutic effect of Naoxintong prescription on heart and brain injury under myocardial ischemia,and explores the mechanism of Naoxintong prescription by using proteomics method.Finally,western blotting is used to verify the protein expression.In order to provide scientific basis for Naoxintong prescription in treating myocardial ischemia,and provide reference for rational application of Naoxintong prescriptione in clinic.Research contents:Study 1:Pharmacodynamic study,which aims to explore the damage of heart and brain caused by myocardial ischemia,and determine the best dose and the best time by giving different concentrations of drugs and measuring the related indexes of heart and brain at different times.Study 2:Proteomics study,which aims to explore the effect of Naoxintong prescription on protein expression in heart and brain under myocardial ischemia.Study 3:Proteomics data screening study,aiming at excavating the association of protein expression between different tissues and making clear the associated proteins.Study 4:To verify the experimental study,which aims to further clarify the expression of related proteins in the heart and brain under the condition of myocardial ischemia treated by Naoxintong prescription.Method:1.Pharmacodynamic study:120 male SD rats,SPF grade,were randomly divided into sham operation group,left anterior descending coronary artery ligation(LAD)model group,positive captopril group(10.125 mg/kg),Naoxintong low dose group(110 mg/kg),Naoxintong middle dose group(220 mg/kg)and Naoxintong high dose group(440 mg/kg).Except for the sham operation group,all rats in other groups used the method of ligating the left anterior descending coronary artery to establish myocardial ischemia model.In the sham operation group,only threading was performed without ligation.After the model was established,the rats were administered continuously.The myocardial infarction area,hemodynamics,myocardial enzyme spectrum(AST,ALT,LDH,CK)and brain water content were measured on 3d and 14d,respectively,to determine the influence of myocardial ischemia on heart and brain,and the best dose and time of administration.2.Proteomics study:Rats were divided into sham operation group,model group and drug administration group,the myocardial ischemia model was established according to pharmacodynamic method,and the best drug dosage was continuously stomach feeding,after the best administration time,the myocardial tissue,cortical and hippocampal tissue of rats were extracted,the tissues were broken and centrifuged to obtain tissue extract.The protein concentration of each tissue extract was determined by BCA method,then the quality control of protein was carried out by gel electrophoresis technology.Finally,the protein was extracted,quantified,digested and desalted,the protein analysis was carried out by nano-upgrade reverse phase chromatography Orbitrap Fusion Lumos.The Uniprot_RAT(April 20,2019 Download)database was searched,then the original mass spectrometry file was processed by MaxQuant software.Lastly,the differences were analyzed by GO,KEGG and STRING.3.Screening study of proteomics data:Analyze the expression of differential proteins in different tissues and explore the association of proteins among every tissues.The callback of proteins in myocardial tissue,cortical and hippocampal tissue was analyzed,the up-regulation or down-regulation of proteins expression in model group and sham operation group,administration group and model were compared,then the action pathway of differential proteins was analyzed by KEGG.Excavating the connection of proteins among tissues,analyzing the expression of proteins with callback in myocardial tissue,cortical and hippocampal tissue,and analyzing the action pathway of differential proteins by KEGG,Finally identify the related proteins in myocardial tissue,cortical and hippocampal tissue.4.Verification experimental study:The differential protein data obtained from analysis were verified by WB,and the associated proteins in myocardial,cortical and hippocampal tissue were verified by Western Blot with P-actin as internal reference protein.Results:1.Pharmacodynamic research:3 d,results of myocardium infarction area:The myocardium infarction area of rats in Naoxintong high dose group decreased significantly(P<0.05).Hemodynamic results:Left ventricular diastolic pressure(LVSP)and left ventricular end diastolic pressure(LVEDP)in Naoxintong low dose group were significantly decreased(P<0.05).The results of myocardium enzyme spectrum showed that LDH in Naoxintong low dose group was significantly decreased(P<0.05),while the contents of ALT,AST,CK and LDH in Naoxintong middle dose group and Naoxintong high dose were significantly decreased(P<0.05).Results of brain water content:the brain water content of rats in each administration group of Naoxintong decreased,but there was no significant difference(P>0.05).14 d,myocardium infarction area:The myocardium infarction area of Naoxintong middle dose group and Naoxintong high dose group decreased significantly(P<0.05).Hemodynamic results:The left ventricular diastolic pressure(LVSP)and left ventricular end diastolic pressure(LVEDP)in Naoxintong low dose group decreased with statistical significance(P<0.05),while those in Naoxintong middle dose group decreased with statistical significance(P<0.01).The results of myocardium enzyme spectrum showed that the contents of ALT,AST and LDH in Naoxintong low dose group were significantly decreased(P<0.05),the contents of ALT,AST and LDH in Naoxintong middle dose group were significantly decreased(P<0.01),the contents of ALT,AST,CK and LDH in Naoxintong high dose group were significantly decreased(P<0.001).Results of brain water content:the brain water content of rats in Naoxintong middle dose group and Naoxintong high dose group decreased significantly(P<0.05).2.Proteomics research:In myocardial tissue,compared with the sham operation group,there were 176 down-regulated proteins and 50 up-regulated proteins in the model group.Compared with the model group,there were 81 down-regulated proteins and 136 up-regulated proteins in the administration group.The action pathways of these proteins mainly focus on metabolic pathway,parkinson's disease,huntington's disease,oxidative phosphorylation and alzheimer's disease.In cortical tissue,compared with sham operation group,there were 140 down-regulated proteins and 125 up-regulated proteins in model group.Compared with the model group,there were 66 down-regulated proteins and 73 up-regulated proteins in the administration group.The action pathways of these proteins mainly focus on lupus erythematosus,peroxisome,fatty acid biosynthesis,hypertrophic cardiomyopathy and myocardium contraction.In hippocampal tissue,compared with sham operation group,there were 90 down-regulated proteins and 152 up-regulated proteins in model group.Compared with the model group,there were 76 down-regulated proteins and 58 up-regulated proteins in the administration group.The action pathways of these proteins mainly focus on life-span regulation pathway,RNA transport,ribosome biogenesis in eukaryotes,cocaine addiction and tryptophan metabolism.3.Screening of proteomics data:Differential protein SCAI is expressed in myocardial and hippocampal tissue.The expression of NADH dehydrogenase(ubiquinone)1? complex 9 in myocardial tissue is up-callback,in cortical tissue,it is down-regulated in model group and administration group.The expression of mitochondrial NADH dehydrogenase(ubiquinone)ferritin 4 in myocardial tissue is up-callback,and the expression of mitochondrial NADH dehydrogenase(ubiquinone)in hippocampal tissue has no difference in model group,but it is down-regulated after administration.Among them,NADH dehydrogenase(ubiquinone)1?complex 9 and mitochondrial NADH dehydrogenase(ubiquinone)ferritin 4 have common action pathways:parkinson's disease,alzheimer's disease,huntington's disease,thermogenesis,oxidative phosphorylation and nonalcoholic fatty liver disease.4.Verify the experimental results:In myocardial tissue:compared with sham operation group,the relative expression of SCAI increased significantly(P<0.01),while the relative expressions of B2RYW3_RAT and NDUS4_RAT decreased significantly(P<0.01).Compared with the model group,the relative expression of SCAI decreased significantly(P<0.001),while the relative expressions of B2RYW3_RAT and NDUS4_RAT increased significantly(P<0.01).In cortical tissue:compared with sham operation group,the relative expression of SCAI protein increased significantly(P<0.05),while the relative expression of B2RYW3_RAT and NDUS4_RAT protein decreased significantly(P<0.01).Compared with the model group,the relative expression of SCAI protein and NDUS4 RAT protein decreased slightly(P>0.05),the relative expression of B2RYW3_RAT protein increased slightly(P>0.05).In hippocampal tissue,compared with sham operation group,the relative expression of SCAI protein in model group decreased significantly(P<0.05),while the relative expression of B2RYW3_RAT protein and NDUS4_RAT protein increased slightly(P>0.05),Comppared with the model group,the relative expression of SCAI protein increased significantly(P<0.001),B2RYW3_RAT protein increased slightly(P>0.05)and NDUS4_RAT protein decreased significantly(P<0.001).Conclusion:1.Myocardial ischemia has a certain degree of damage to both heart and brain,and the damage is relieved after administration.The best dose and time of administration were determined.2.There are 443 differential proteins in myocardial tissue,404 in cortical tissue and 376 in hippocampal tissue.The action pathways of these proteins mainly focus on metabolic pathway,parkinson's disease,huntington's disease,oxidative phosphorylation and alzheimer's disease.3.The differentially expressed proteins related to each tissue are SCAI proteins,B2RYW3_RAT proteins and NDUS4_RAT proteins,and their action pathways are similar to parkinson's disease,alzheimer's disease,huntington's disease,thermogenesis,oxidative phosphorylation and nonalcoholic fatty liver disease.4.WB verified that the expression of differential protein SCAI proteins and mitochondrial NADH dehydrogenase(ubiquinone)ferritin 4 in myocardial and hippocampal tissue was basically consistent with the data of proteomics,which could be used as target protein of Naoxintong prescription for treating myocardial ischemia with the simultaneous treatment of brain and heart.