Study On The Treatment Of Acute Myocardial Ischemia Reperfusion Injury In Rats By Zhu Riheng Drop Pills Based On Network Pharmacology

Objective:Based on the network pharmacology method,the intervention effect of Zhu Riheng drop Pills on myocardial ischemia-reperfusion injury in rats was studied.Methods:Experiment 1:A network pharmacology study of the basic prescription（Sanwei Sandalwood Powder）for the treatment of coronary heart disease.The chemical composition of sanwei sandalwood powder is collected and sorted out by document mining and retrieval of multiple databases of Chinese medicinal materials,and then analyzed and screened by OB（Oral bioavailability）and DL（drug-like Drug affinity）analysis.By using the online platforms such as PharmMapper,SWISS and Superpred,the potential targets of the compounds were predicted;OMIM database was used to collect CHD related targets.Protein interaction analysis with STRING;Pathway enrichment of key targets was conducted by DAVID online tool;Cytoscape software was applied to establish the network model of"medicinal herbs-compounds-target-pathway",and then the network model was analyzed topologically;Some results of network model analysis were verified by Schrodinger molecular docking technology.Experiment 2:The intervention effect of Zhu Riheng drop pills on myocardial ischemia-reperfusion injury in rats and its experimental verification in vivo.Rats were randomly divided into:normal group（Control）,model group（Model）,Guangzao group（GZ）,Guangzao-Roudoukou group（GZ-RDK）,Sanwei Sandalwood group（SWTX）and Zhu Riheng drop Pills group（ZRH）.Each dosage group continuous lavage after 10 days,in addition to the normal group,both groups through the ligation/reperfusion rat heart LAD（Left coronary anterior descending branch）induced by myocardial ischemia reperfusion injury;The electrocardiogram of the II lead was used to observe the changes of electrocardiogram in each group.The blood biochemical indicators CK（Creatine kinase）,LDH（Lactate dehydrogenase）were detected by abdominal aorta blood sampling.Evans Blue/TTC staining was used to observe the ischemic risk zone and infarct zone,respectively;hematoxylin and eosin（HE）staining was used to observe the pathological changes of myocardial tissue;qRT-PCR method was used to detect the gene expression of target Aktl,Bim and PIK3R1 by network pharmacology;Western The protein expression of Aktl,p-Akt1,FoxO3a,p-FoxO3a and Bim was detected by blot.Experiment 3:The drug monomer inhibited the oxidative damage of rat H9c2 myocardial cells induced by H₂O₂.Based on the results of network pharmacology,the main active monomers of Zhu Riheng drop pills for coronary heart disease were selected for cell experiments.900mmol·L^-1H₂O₂ induced the establishment of myocardial oxidative damage model,MTT method observation model group,model+PI3K/Akt inhibitor group（LY294002）,model+each active monomer group(gallogen（G3）,quercetin（G8）,methyleugenol（R2）,macelignan（R7）,santol（T1）,model+monomer+PI3K/Akt inhibitor group on cell viability;Flow cytometry was used to detect apoptosis;Immunofluorescence was used to detect the distribution of FoxO3a protein.Results:experiment 1:The results of network pharmacology showed that the main biological pathways related to the pathogenesis of coronary heart disease were stress reaction,angiogenesis,immune inflammatory reaction,cell injury,apoptosis and necrosis,endocrine regulation,cell division and proliferation,atherosclerosis and instability of arterial plaque.According to the network topological analysis,the main active ingredients of sanwei sandalwood powder in the treatment of CHD include gallogen,（-）-chebulic acid,safrole and macelignan in nutmeg,and alpha-santalene and santol in sandalwood.Cellular tumor antigen p53,Adenosine receptor A1,RAC-alpha serine/threonine-protein kinase,E3ubiquitin-binding enzyme Mdm2（E3 ubiquitin-Targets such as protein ligase Mdm2）,prothrombin,and Retinoic acid receptor RXR-alpha may be key targets for Sanwei Sandalwood in the treatment of cardiovascular disease;The results of pathway enrichment showed that the PI3K-Akt signaling pathway（P=1.45E-04）and FoxO signaling pathway（P=3.75E-04）were the top two signaling pathways with significant difference（they were upstream and downstream pathways）,suggesting that the PI3K-Akt-FoxO signaling pathway was involved in the myocardial protective effect of Sanwei Sandalwood Powder and Zhu Riheng drop pills.experiment 2:Compared with the normal group,the electrocardiogram of the model group showed a significant elevation in the ST segment（0.40±0.05 mV,P<0.01）.Compared with the model group,the ST segments of each administration group were reduced to varying degrees,of which Sanwei Sandalwood Powder The group（0.14±0.05mV,P<0.01）was the most significant decrease,Zhu Riheng drop pills Group（0.18±0.05mV,P<0.01）,Guangzao-Roudoukou Group（0.21±0.07mV,P<0.01）and Guangzao Group（0.27±0.06mV,P<0.01）second;Compared with the normal group,the CK and LDH values in the model group were significantly increased（P<0.01）.Compared with the model group,the CK values of the Guangzao and Sanwei Sandalwood Powder groups were significantly lower（P<0.05）.The CK value of the cardamom and Zhu Riheng drop pills group was lower than that of the model group,but there was no significant difference（P>0.05）.Compared with the model group,except for the jujube-administered group,the other drug-administered groups were all.It can significantly reduce the LDH value（P<0.01 or P<0.05）,among which the Sanwei Sandalwood powder group is the most obvious,and the Guangzao-Roudoukou and Zhu Riheng drop pills are the second.The results of Evans blue/TTC staining showed that the myocardial ischemia and infarct size were the largest in the model group,and the ischemic and infarct size were significantly reduced in each administration group.Zhu Riheng drop pills and Sanwei Sandalwood Powder Group were superior to Guangzao-Roudoukou Medicine pair,jujube group;The results of HE staining showed that the myocardial cells in the model group were disordered,broken and dissolved,the tissue was loose,the myocardial fiber space was enlarged,the interstitial edema was increased,and the inflammatory cells were infiltrated.Among them,the myocardial structure of the Zhu Rihang drop pills and Sanwei Sandalwood powder groups were relatively complete,with less damage.The results of qRT-PCR showed that compared with the normal group,the expression of Bim in the model group was significantly increased（P<0.01）,and the expression level of Bim was significantly decreased in each administration group（P<0.01 or P<0.05）.Zhu Riheng drop pills was most significant in the drug-administered group（P<0.01）.Compared with the normal group,the expression of Aktl and PIK3R1 in the model group was weak（P<0.01）,and the expression levels of Aktl and PIK3R1 were significantly increased in each drug-administered group,among which Zhu Riheng drop pills the expression of Aktl was the strongest in the pill-administered group（P<0.01）,the expression of PIK3R1 was the strongest in the Sanwei Sandalwood Powder group（P<0.01）,and the expression in the Guangzao group was the weakest（P<0.01）.Western blot results showed that compared with the model group,each of the drug-administered groups significantly increased the ratio of p-Akt1/Akt1,p-FoxO3a/FoxO3a,Zhu Riheng drop pills were the most significant,and Sanwei Sandalwood Powder was the second;The expression of Bim protein in the other drug-administered groups was significantly different from that in the model group.Zhu Riheng drop pills were the strongest,followed by Sanwei Sandalwood Powder and Guangzao.experiment 3:PI3K/Akt inhibitors can reverse the protective effect of drug treatment on H₂O₂-induced H9c2 cardiomyocytes.Compared with H₂O₂+monomer group,the survival rate of H₂O₂+monomer+LY294002 group was significantly lower（P<0.01 or P<0.05）.The apoptosis rate of cardiomyocytes was significantly increased（P<0.01 or P<0.05）,and the expression level of FoxO3a in cytoplasm was decreased（P<0.01）.Conclusion:Through the analysis of the"medicine-compound-target-pathway"network topology and related target network topology parameters,it was found that Zhu Riheng drop pills foundation Sanwei Sandalwood Powder can treat coronary heart disease and stress response PI3K/Akt signaling pathway and FoxO signaling pathway.Correlation,and then in vivo myocardial ischemia-reperfusion injury in rats,qRT-PCR,Western blot results verify that Zhu Riheng drop pills can regulate the network pharmacology prediction of Akt1,PIK3R1,Bim gene expression and Aktl,p-Akt1 Protein expression of FoxO3a,p-FoxO3a,Bim;H₂O₂-induced H9c2 cardiomyocyte oxidative damage in vitro,also confirmed that PI3K/Akt/FoxO signaling pathway is involved in the protective effect of Zhu Riheng drop pills on oxidative damage of cardiomyocytes.