Caffeic Acid O-nitro Phenethyl Ester Alleviates Rat MIRI And Its Cardiac Metabolites Analysis

Myocardial ischemic injury,a variety of reasons caused coronary blood flow decreased and the supply of blood to the heart blocked,results in insufficient myocardial oxygen supply and metabolite clearance,and then leads to cardiac dysfunction or myocardial cell death.Reperfusion after ischemia to restore blood flow is the most direct treatment,however,sometimes reperfusion may increase tissue structure,function damage and even irreversible damage,resulting in myocardial ischemia-reperfusion injury?MIRI?.Oxidative stress,inflammatory reaction,myocardial fibrosis and apoptosis caused by oxygen free radicals?ROS?and calcium overload are the main pathogenesis.Therefore,the study of anti-MIRI drugs for the cause of the disease possesses positive clinical significance.Caffeic acid phenethyl ester?CAPE?,a major phenolic active ingredient of propolis,belongs to flavonoids and has a wide range of pharmacological activities such as anti-inflammatory,anti-oxidation,anti-tumor and so on.There was literature reported that flavonoids like curcumin could increase antioxidant capacity by up-regulating silent information regulator-1?SIRT1?signaling to attenuate MIRI.Other flavonoids including CAPE have yet to be studied with regard as SIRT1 activation.Caffeic acid p-nitro phenethyl ester?CAPE-pNO₂?,a CAPE analogue with para nitro replace,was determined to be an intensifier of macrophage phagocytosis,anti-platelet agent,and a protector of myocardial ischemia and diabetic nephropathy injury with more potent capacity in our lab studies.And our previous research has shown that the nitro introduction can increase the NO content in myocardial tissue of the MIRI rats,but the NO source is unclear.It's still need further verification that the NO acceleration is directly metabolized from nitro substituted compounds or indirectly derived from nitric oxide synthase?eNOS?.In this study,we firstly synthesized caffeic acid o-nitro phenethyl ester?CAPE-oNO₂,CAPE substituted with ortho-nitro group?and obtained the Chinese patent.According to the SYBYL molecular simulation software,CAPE-oNO₂ has higher solubility and lower viscera toxicity than CAPE.In this paper,we choose CAPE-oNO₂ to investigate the cardioprotective potential and the possible mechanism on MIRI based on the animal experiments and cell experiments.In animal experiments,the left anterior descending coronary artery?LAD?ligation of rats resulted in myocardial ischemia.After ischemia for 30 min,the LAD was allowed to recover blood flow for 2 h to establis an acute rat MIRI model.SIRT1inhibitor nicotinamide?NAM?or eNOS inhibitor N-nitro-L-arginine methyl ester?L-NAME?was administered via tail vein 30 min before ligation,while the drug or vehicle was given 15 min prior to ligation.The rats were randomly divided into 8groups:the Control group;the IR group;the CAPE?0.1 mg/kg?group;the CAPE-oNO₂?0.1 mg/kg?group;NAM?1 mg/kg?+CAPE-oNO₂?0.1 mg/kg?group;NAM?1mg/kg?+Vehicle group;L-NAM?1 mg/kg?+CAPE-oNO₂?0.1 mg/kg?group;L-NAME?1 mg/kg?+Vehicle group.TTC staining,HE staining,immunohistochemical staining and sirius red staining were used to determine the myocardial infarct size,histopathological changes,TNF-?expression and collagen content in cardiac tissue,respectively;the content of serum myocardial enzymes?HBDH,LDH,CK,CK-MB and IMA?and the levels of t-SOD,MDA,MPO and NO in heart tissue were measured by kit;the expression of related proteins in myocardial tissue was analyzed by western blot;the metabolism of CAPE and CAPE-oNO₂ in the myocardial tissue of MIRI rats was detected by LC-MS.The results showed that CAPE and CAPE-o NO₂ could significantly decrease myocardial infarct size,alleviate the disorder of myocardial cells and inflammatory cell aggregation caused by ischemia,reduce the TNF-?expression and collagen deposition?P<0.05?.Moreover,CAPE and CAPE-oNO₂ could reduce MDA content and MPO activity while increase t-SOD activity and NO content?P<0.05?.Western blotting showed that the expressions of SIRT1 and eNOS were up-regulated by CAPE and CAPE-oNO₂,consequently affecting the downstream signaling pathway NF-?B to exert cardioprotection effects,including that the oxidative stress was ameliorated by PGC-1?/Nrf2/SOD1 system activating;the myocardial inflammation was decreased by the inhibitions of I?B?P65 phosphorylation and the of TNF-??IL-6expression;the myofibrosis was relieved by down-regulating the TGF-?1,p-Smad2/3,Collagen expression;the cardiomyocytes apoptosis was reduced by up-regulating Bcl-2expression and down-regulating the Bax pression.It is noteworthy that CAPE-oNO₂presented the stronger activity on anti-oxidant,anti-inflammatory,anti-fibrotic and anti-apoptotic effect than CAPE?P<0.05?,whereas the CAPE-oNO₂-mediated cardioprotection effect was significantly remitted in the presence of NAM or L-NAME.There was a remarkable difference between the metabolic products and ways of two drugs.CAPE-oNO₂ possessed the metabolic product and way of denitrification.In cell experiments,H9c2 cardiomyocytes was induced hypoxia by oxygen-glucose deprived culture,and reoxygenation was performed for 4h after 2h of hypoxia to establish a cell MIRI model.The inhibitors were given at the beginning of hypoxia while the drug and vehicle were given at the beginning of reoxygenation.The logarithmic phase cells were divided into 10 groups:Control group,HR group,CAPE group?10,20,40?M?,CAPE-oNO₂ group?10,20,40?M?,NAM?40?M?+CAPE-oNO₂?40?M?group,L-NAME?40?M?+CAPE-oNO₂?40?M?group.The survival rate of cardiomyocytes was determined by MTT assay.Apoptosis and intracellular reactive oxygen species?ROS?were detected by AO staining and ROS staining respectively.The results showed that the survival rate of cardiomyocytes was significantly decreased after HR procedure,but CAPE and CAPE-oNO₂ treatments could effectively increase the cell viability,reduce HR-induced cell apoptosis and ROS production in a dose-dependent manner?P<0.05?.Western blot analysis showed that the expression of SIRT1,eNOS,PGC-1?,SOD1 and Bcl-2 were significantly increased,while p-I?B/I?B,p-P65/P65 and Bax were significantly decreased in CAPE and CAPE-oNO₂ groups?P<0.05?.Similarly,CAPE-oNO₂ also showed a better cardioprotection effect on MIRI than CAPE,whereas the presence of inhibitor attenuated the therapeutic effect of CAPE-oNO₂.In summary,both animal experiments and cell experiments have shown that CAPE and CAPE-oNO₂ can effectively relieve MIRI and may be candidates for clinical treatment to improve and prevent myocardial damage.Its mechanism may be to increase the protein expression of SIRT1 and eNOS,affect downstream signaling pathway of NF-?B,produce anti-oxidative stress,anti-inflammation,anti-myocardial fibrosis and anti-apoptotic effect,exert myocardial protection.Nitro-substituted CAPE can significantly improve the anti-MIRI activity,which may be due to a greater degree of regulation of eNOS and directly denitrification of CAPE-oNO₂ to elevate the NO level.And it also may be one of the reasons of CAPE-oNO₂ better than CAPE that two drugs have different metabolites and ways in the heart tissue.