High-Throughput Screening Of Natural Compound Libraries To Identify The Improvement Of Myocardial Ischemia/Reperfusion Injury By Oroxin A And Its Mechanism Of Action

Objective: Myocardial ischemia/reperfusion injury(MI/RI)is a serious pathophysiological process associated with cardiovascular disease.Oroxin A(OA)is A kind of natural flavonoid glycoside with various biological activities.However,its effect on the pathophysiological process of MI/RI has not been reported.The purpose of this study was to find drugs that can effectively reduce MI/RI through high-throughput drug screening.Methods: 1.The Oxygen and glucose deprivation/reperfusion(OGD/R)model of rat cardiomyocytes(H9c2)were established in vitro to simulate MI/RI in rats.High-throughput drug screening and Cell counting kit-8(CCK-8)detection of 2661 natural compounds were carried out to determine the drug OA,which can effectively reduce rat MI/RI.2.In order to determine the best intervention concentration of OA on H9c2 cell OGD/R model,the concentration gradient of cell administration was set,and the effect of different concentration on cell viability was detected by CCK-8.3.The effects of OA on OGD/R-induced H9c2 cell injury,apoptosis,pyroptosis and inflammation were investigated by cell morphological observation,Hoechst 33258,WB and ELISA experiments.Results: 1.By applying different concentrations of OA in the OGD/R model of H9c2 cells,the optimal concentration of OA was determined to be 10μM.2.Cell experiments showed that OA could significantly reduce OGD/R induced H9c2 cell injury,apoptosis,pyroptosis and inflammation.Conclusion: OA is an effective drug to treat MI/RI,which provides a new target for clinical treatment of MI/RI.Objective: The purpose of this study was to determine whether OA can alleviate inflammation and pyroptosis induced by MI/RI at the experimental level in vivo,and to provide a new therapeutic regimen for the treatment of MI/RI.Methods: Under general anesthesia and ventilator-assisted ventilation,the MI/RI rat model was established by ligating the Left anterior descending(LAD)coronary artery.The rats were randomly divided into three groups: sham operation group,positive control group and OA intervention group.After 72 hours,left ventricular end-diastolic and end-systolic diameters and left ventricular function were measured by echocardiography.TTC staining was used to evaluate the area of myocardial infarction.HE staining,Tunel staining and immunohistochemistry were used to evaluate the degree of myocardial tissue injury,apoptosis and inflammatory response.The levels of TNF-α,IL-6,IL-18,IL-1β and IL-18 in myocardial tissue and serum were determined by enzyme-linked immunosorbent assay(ELISA).ELISA was used to detect the levels of serum c Tn I,c Tn T,CK-MB,LDH and AST.Western blotting and RT-q PCR were used to detect the protein expression and m RNA transcription levels of NLRP3,Caspase 1,and GSDMD in myocardial tissue.To investigate the effects of OA on MI/RI,apoptosis,pyroptosis,inflammatory response and cardiac function in rats.Results: The rat model of MI/RI were successfully constructed.Compared with the positive control group,OA could reduce the infarct size and can improve left ventricular systolic function after MI/RI in rats.OA can alleviate myocardial injury and inflammatory cell infiltration after MI/RI in rats.OA can reduce the release of pro-inflammatory factors in myocardium and serum and reduce myocardial injury and the release of myocardial enzymes after MI/RI in rats also.Furthermore OA could inhibit the expression and m RNA transcription of NLRP3,Caspase 1 and GSDMD in myocardial tissue after MI/RI in rats.Conclusion: OA can reduce inflammation response and inhibit pyroptosis during MI/RI,and is an effective drug in the treatment of MI/RI injury.Background: It was found in the early stage of the project that OA can improve MI/RI by inhibiting pyroptosis.This study intends to explore the target and molecular mechanism of OA inhibiting pyroptosis and regulating MI/RI through network pharmacology and molecular docking.Methods: The potential targets of OA were predicted by Swiss Target Prediction and Pharm Mapper,and the targets related to pyroptosis and MI/RI were obtained from OMIM,TTD,Gene Cards and Dis Ge NET databases.The KEGG enrichment analysis of rendezvous target is analyzed in Metascape database.Finally,molecular docking was carried out to verify the binding force between the core target and OA.Results: KEGG analysis showed that there were two pyroptosis-related pathways,"lipid and atherosclerosis" and "nod-like receptor signal pathway",as well as six nuclear molecules such as ABCA1,ABCG1,Nrf2,IP3 R,Yop J and PLCB.Molecular docking shows that the binding energies of OA and core molecules are less than 0kcal/mol,and the binding energy of Nrf2 and Nrf2 is the lowest.Conclusion: OA acts on Nrf2 and enhances the anti-oxidative stress and anti-pyroptosis function of Nrf2,thus alleviating MI/RI.