Hydroxysafflor Yellow A Against Myocardial Ischemia Reperfusion Injury By Improving Mitochondrial Dysfunction Via Activating Mdh1

Background:Acute myocardial infarction(AMI)refers to acute,severe and sustained myocardial ischemia,which is the most serious clinical coronary heart disease caused by atherosclerosis.Timely reperfusion therapy,including percutaneous coronary intervention(PCI)or fibrinolytic therapy,is the most effective treatment for AMI.But it also leads to myocardial ischemia reperfusion injury(MIRI).Inflammation,oxidative stress damage,Ca2+ overload,and mitochondrial dysfunction are the main pathogenesis of MIRI.Inhibiting inflammation,improving mitochondrial function and restoring energy metabolism are important in the treatment of MIRI.1.Mechanism Repositioning Based on Integrative Pharmacology:Anti-Inflammatory Effect of Safflower in Myocardial Ischemia Reperfusion InjuryObjective:Safflower is the dried tubular flower of Carthamus tinctorius L.,which is commonly used in clinical medicine.Safflower injection is mainly used for cardiovascular and cerebrovascular diseases in clinic.However,the mechanism and the effective components of safflower in the treatment of MIRI are not clear.Therefore,it is necessary to study on the mechanism of safflower in the treatment of MIRI.Based on the integrative pharmacology strategy,combining with the model of a mouse of left anterior descending(LAD)branch ligation and UPLC-QTOF-MS/MS,this study evaluated the mechanism of safflower treatment on MIRI in mice,in order to provide theoretical basis for the clinical application of safflower.Methods:(1)MIRI model was established based on C57BL/6 mice with LAD-ligated ligation.A total of 126 mice were randomly divided into six groups,including the sham group,model group,safflower low-dose group,medium-dose group,high-dose group(62.5 mg/kg,125 mg/kg,250 mg/kg,4 times/24 h,i.v.),and positive control metoprolol group(12.5 mg/kg/d,i.p.).Myocardial infarction size,echocardiography,apoptosis,LDH levels and SOD levels were all evaluated after 30 min of ischemia and 24 h of reperfusion.(2)Based on integrative pharmacology strategy,the mechanism of safflower treatment on MIRI were predicted,and the key components and hub targets were screened.(3)The mechanism verification was detected by qRT-PCR and western blotting.Results:(1)24 h of reperfusion,safflower(62.5,125 and 250 mg/kg)dose-dependently reduced myocardial infarction size,apoptosis,improved myocardial function,increased ejection fraction(EF%)and fractional shortening(FS%),reduced lactate dehydrogenase(LDH),and increased superoxide dismutase(SOD)level after MIRI.(2)Based on integrated pharmacological strategy,literature review and UPLC-QTOF-MS/MS,11 hub components and 31 key targets were screened.(3)Safflower alleviated inflammatory effects by downregulating the expression of NFκB1,IL6,IL-1β,IL-18,TNFα,and MCP-1 and upregulating NFκBia,and markedly increased the expression of phosphorylated PI3K,AKT,PKCε,HIF1α,VEGFA,ERK1/2,and Bcl2,and decreased the level of BAX and phosphorylated p65.Safflower shows a significant cardioprotective effect by activating multiple inflammation-related signaling pathways,including the NFκB,HIF-1α,MAPK,TNF,and PI3K/AKT signaling pathway.These findings provided valuable insights into the clinical applications of safflower.2.Hydroxysafflor yellow A against Myocardial Ischemia Reperfusion injury by improving Mitochondrial Dysfunction via activating MdhlObjective:Hydroxysafflor yellow A(HSYA),as a characteristic natural ingredient extracted from safflower,has significant pharmacological activities,such as antioxidant,antiinflammatory,anticoagulant,and anticancer effects.However,the unclear mechanism and target have been severely hampering the clinical applications of HSYA during the treatment of cardiovascular and cerebrovascular disease.This study aims to explore the direct target and related mechanism of HSYA in the treatment of MIRI.Methods:(1)Myocardial infarction size,echocardiography,apoptosis,the level of SOD and LDH were evaluated after 24 h of reperfusion based on the LAD-ligated model of C57BL/6 mice in the acute phase of IR injury.Echocardiography and the area of myocardial fibrosis were detected after 30 min of ischemia and 28 d of reperfusion throughout the chronic phase.Based on hypoxia/reoxygenation(H/R)model,cell viability,mitochondrial membrane potential,mitochondrial permeability transition pore(mPTP)and reactive oxygen species(ROS)level were detected.(2)Isotopic tandem orthogonal proteolysis activity-based protein profiling(isoTOP-ABPP)was used to screen the protective mechanism of HSYA,and a novel pharmacological target were discovered for HSYA in treatment of MIRI.In vivo and in vitro energy metabolism indicators were detected based on the LAD-liganded model in C57BL/6 mice and H9c2 in H/R model;(3)Protein silencing was performed on the protein in the TCA cycle,and the target protein of HSYA was Malate dehydrogenase 1(Mdhl).The covalent binding of HSYA with the Mdhl target protein can increase the thermal stability of the Mdhl protein,and its target site is the 137 of cysteine binding site on the Mdhl protein.Cell viability,mitochondrial-membrane potential,mPTP,glycolysis and oxidative phosphorylation were detected after siRNA transfection.Results:(1)After 24 h of reperfusion,HSYA significantly improved the cardiac function of MIR-injured mice,reduced myocardial infarction and cell apoptosis,increased SOD level,reduced the level of LDH,and inhibited the expression of inflammatory factor,such as IL-6,IL-1β and MCP-1.After 28 d of reperfusion,HSYA significantly improved cardiac function after MIRI and inhibited myocardial fibrosis injury;HSYA significantly improved cell viability,mitochondrial membrane potential and mPTP,and decreased ROS after H/R injury on H9c2 cell;(2)Based on isoTOP-ABPP,we found that the mechanism of action of HSYA in the treatment of MIR injury were mainly developed by tricarboxylic acid cycle.The results of energy metabolism in vivo suggested that HSYA could significantly improve the production of ATP and NAD+/NADH after MIRI.And HSYA could also significantly improve the mitochondrial energy metabolism and glycolysis after H/R injury;(3)malate dehydrogenase 1(Mdhl)was discovered as the target of HSYA.Mdhl-siRNA transfection could reverse the myocardial protective effect of HSYA.Innovation:1.We employed an integrative pharmacological strategy to explore the mechanism of action of safflower in improving MIR injury in mice.We characterized 79 chemical components of safflower.Among them,56 chemical compounds,including 11 key ingredients,may ameliorate MIR injury partially by interacting with 31 hub candidate targets,mainly through an "inflammation-immune" system.Further studies are needed more systematic efficacy evaluations and mechanistic explorations.2.Based on chemical proteomics,HSYA exerts protective effects against MIR injury in vivo and in through energy metabolism related signaling pathways mediated by Mdhl selectively binding to the Cys137 residue,revealing the mechanism of action of HSYA in treating MIRI in mice from the perspective of mitochondrial energy metabolism.