Therapeutic Effect And Mechanism Of Novel Tetracyclic Diterpenoids On Myocardial Ischemia

ObjectiveMyocardial ischemia has high morbidity and high mortality,which is a major problem in current clinical treatment,and the research and development of its therapeutic drugs is very important.A lot of previous studies have proved that isosteviol has a good protective effect on the myocardium and can treat myocardial ischemia.In the early stage of our research group,a series of tetracyclic diterpenoid isosteviol derivatives were synthesized using isosteviol as the parent nucleus.KFH-08 with stronger cardioprotective effect was screened out.However,it is unknown whether KFH-08 has a protective effect on myocardial ischemia.Therefore,this study aimed to explore whether KFH-08 has a protective effect on myocardial ischemia and its possible pharmacological mechanism,including:（1）To explore the protective effect of KFH-08 on chronic myocardial ischemia in rats.（2）To explore whether the mechanism of KFH-08 protecting myocardial ischemia depends on promoting angiogenesis.（3）To explore whether the mechanism of KFH-08 protecting myocardial ischemia is through anti-oxidative stress injury.Research contents1、To explore whether KFH-08 has a protective effect on myocardial ischemia.In this study,a rat model of chronic myocardial ischemia was established by ligating the left anterior descending coronary artery,and the rats were administered for 4 weeks.The protective effects of KFH-08 on cardiac function and myocardial function in rats with myocardial ischemia were evaluated by pathological analysis（HE staining）and ultrasound detection.2、To explore whether KFH-08 protects myocardial ischemia by promotingangiogenesis signaling pathway.In vitro,the effect of KFH-08 on the proliferation of HUVEC cells was evaluated by the method of detecting cell viability by CCK-8.In vivo,using Tg [flk: EGFP] transgenic zebrafish embryos to evaluate the proangiogenic effect of KFH-08 on VRI inhibition of intersegmental angiogenesis in zebrafish.The RNA seq detection results of different experimental groups were analyzed to find the molecular mechanism of KFH-08 protecting myocardial ischemia.At the same time,RT-q PCR was used to verify the expression level of target genes in the RNA seq results.Finally,the administration of combined signaling pathway inhibitors was used to verify whether KFH-08 activates the signaling pathway related to VEGF.3、To explore whether KFH-08 protects myocardial ischemia through antioxidantstress signaling pathway.In vitro,the protective effect of KFH-08 against oxidative stress injury caused by t-BHP was evaluated by the method of detecting cell viability by CCK-8,and the effect of KFH-08 on ROS was verified by DCFH-DA.In vivo,AB-WT zebrafish were used to construct models of oxidative stress injury induced by VRI and tert-butyl hydroperoxide,respectively,to evaluate the protective effect of KFH-08 on oxidative stress injury.The expression levels of genes related to oxidative stress injury in the RNA Seq results were verified by RT-q PCR.Results1.To explore the protective effect of KFH-08 on chronic myocardial ischemia in rats,a rat myocardial ischemia model was successfully constructed by ligating the left anterior descending coronary artery.KFH-08 has a protective effect on chronic myocardial ischemia in rats and significantly improves cardiac function.After drug treatment,the difference between ventricular end-systole and end-diastole,ejection fraction,fractional shortening,heart rate and cardiac output of rat hearts recovered significantly,and the upper and lower ventricular walls were serrated.After hematoxylin-eosin staining,the results also showed that:KFH-08 significantly improved the morphology of cardiomyocytes and protected the myocardium.2.In vitro,KFH-08 can promote the proliferation of HUVEC.In vivo,in the model of VRI-induced vascular injury,KFH-08 was determined to have an angiogenesis-promoting effect,and the optimal concentration of the experiment was 200 μmol/L.So as to verify the effect of KFH-08 on the expression of genes related to angiogenesis signaling pathway,the RNA seq results showed that KFH-08 activated the expression of genes related to angiogenesis pathway（vegfaa,vegfba,flt1,kdr,notch1 a,dll4,etv2,hif1 aa,mapk8ip2,ndrg4）.The results of RNA seq were further verified by RT-q PCR,showing that KFH-08 significantly increased the expression of related genes.And through the study of signaling pathway inhibitors,it was found that the mechanism of KFH-08’s effect indeed depends on the VEGF angiogenesisrelated signaling pathway.3.In vitro,KFH-08 has a significant protective effect on tert-butyl hydroperoxideinduced H9C2 oxidative stress injury,and significantly reduces the number of reactive oxygen species.In vivo,in the t-BHP oxidative stress injury model and the VRI oxidative stress injury model,the amount of reactive oxygen species in zebrafish was significantly reduced after KFH-08 administration,which has the effect of protecting against oxidative stress injury.And when the results of RNA seq were verified by RT-q PCR,it was also found that KFH-08 significantly increased the expression of genes related to oxidative stress（pax10,barx1 and steap3）.Conclusions1.KFH-08 has a significant protective effect on myocardial ischemia.2.The molecular mechanism of KFH-08 protecting myocardial ischemia depends on the pro-angiogenic signaling pathway activated by VEGF/Notch/Etv2.3.The molecular mechanism of KFH-08 protection against myocardial ischemia depends on the anti-oxidative stress signaling pathway activated by pax10/barx1/steap3.