| Background Ischemic heart disease(IHD)remains a public health concern with high morbidity and mortality throughout the world.The primary therapeutic strategy after the ischemic insult is timely recovery of blood supply.However,reperfusion can aggravate myocardial injury and cardiac dysfunction caused by ischemia.Therefore,prevention and treatment of myocardial ischemia/reperfusion(MI/R)injury is an important mean to treat IHD.In contrast to western medicine,traditional Chinese medicine(TCM)uses a holistic and synergistic approach to restore the balance of Yin-Yang of body energy.In clinical practice,Salvia Miltiorrhiza(SM)-Dalbergia Odorifera(DO)or the prescription of these two herbs are widely used in China,Japan and Korea for the treatment of IHD.However,bottlenecks still exist in international promotion and secondary development of SM-DO because the synergistic actions of the multiple components were unknown,the scientific connotation and mechanism were unclear.The emergence of metabonomics,a powerful new technology that assesses holistic metabolic profiles through a nontargeted approach,is well coincided with the integrity and systemic feature of TCM,which usually involves multicomponent,multipathway,and multitarget treatments.Metabonomics provides powerful technical support for the evaluation of the efficacy and mechanism of TCM,which helps to systematically and profoundly reveal the scientific connotation of TCM,and helps to comprehensively and deeply develop the theory of TCM.Objectives 1.To investigate the protective effect of aqueous extract of Salvia Miltiorrhiza(SM),aqueous extract of Dalbergia Odorifera(DOA),volatile oil of Dalbergia Odorifera(DOO),SM+DOA and SM+DOO on MI/R injury in rats.2.To search potential biomarkers of MI/R injury in SM+DOO,to analyze protective mechanism of SM+DOO based on metabonomics.3.To verify the cardioprotective effect mechanism of SM+DOO.Methods 1.The 80 SD rats were divided randomly into sham operation(Sham)group,model(Model)group and diltiazem(Diltiazem)group,SM group,DOA group,DOO group,SM+DOA group,SM+DOO group,n=10.Rats were intragastric administrated with homologous drugs for 7 days,and then subjected to 30 minutes of ischemia followed by 180 minutes of reperfusion.The ST-segment elevation and heart rate variation of electrocardiogram were observed.The area of myocardial infarction and histological tissue were assessed.Meanwhile,the level of creatine kinase-MB(CK-MB),lactate dehydrogenase(LDH),tumor necrosis factor(TNF-α)and interleukin-6(IL-6)in serum,the level of superoxide dismutase(SOD)and malondialdehyde(MDA)in serum and myocardial tissue,the morphological analysis of apoptotic damage were assessed after 180 minutes reperfusion.2.Serum samples of Sham group,Model group,SM+DOO group were detected by metabonomics technique based on gas chromatography time-of-flight mass spectrometry(GC-TOF-MS).The raw data obtained were preprocessed and then using SMICA14.1 software to conduct multivariate statistical analysis including principal component analysis(PCA),partial least square-discriminant analysis(PLS-DA),orthogonalpartial least square-discriminant analysis(OPLS-DA),thus the cardioprotective effect of SM+DOO on MI/R injury rats was evaluated comprehensively.According to the results of OPLS-DA,the potential biomarkers were screened based on variable importance in projection(VIP)and t test,and the metabolic pathway were analyzed by Metabo Analyst 3.0.3.Biochemical indicators such as lactic acid(LD),non-esterified fatty acid(NEFA),citroyl synthetase(CS),acetyl coenzyme A/coenzyme A(Ac Co A/Co A),adenosine triphosphate(ATP),Na+K+-ATPase,Ca++-ATPase and Ca++Mg++-ATPase were assessed.Then,Pearson correlation analysis method was used to explore the inherent relationship between these important biochemical indicators and endogenous biomarkers,and to evaluate the protective effects of SM+DOO on MI/R injury in rats.In addition,Western Blot was used to detect the expression levels of AMP-activated protein kinase(AMPK),acetyl Co A carboxylase(ACC),carnitine palmitoyl transterase-1(CPT-1),pyruvate dehydrogenase kinase(PDK)and pyruvate dehydrogenase complex(PDC),and to verify the cardioprotective mechanism of SM+DOO on MI/R injury rats.Results 1.The administration groups had different cardioprotective effects on MI/R injury in rats,of which SM+DOO can improve the electrocardiogram of rats,reduce infarct size,improve myocardial tissue injury,decrease the levels of CK-MB and LDH,decrease oxidative stress and inflammatory response inhibition caused by MI/R injury,and reduce the number of myocardial apoptosis.Compared with SM group,SM+DOO group showed a significant difference.2.The results of the PCA and PLS-DA showed that the Sham,Model,and SM+DOO pretreatment groups were separated clearly,and the SM+DOO group was much closer to the Sham group than the Model group,which suggested that SM+DOO could restore the pathological process of MI/R injury in rats.In the results of OPLS-DA,compared with Sham group,the levels of lactic acid,3-hydroxybutyric acid, phosphate,unknown,palmitoleic acid,heptadecanoic acid,stearic acid and arachidonic acid showed an increasing trend in Model group,while malic acid,citric acid,creatine and phenylalanine showed a decreasing trend,suggesting that these 12 endogenous substances are potential biomarkers for MI/R injury.These endogenous substances received a different degree of recovery in the SM+DOO group of rats,and the level of callback of 3-hydroxybutyric acid,citric acid,palmitoleic acid,heptadecanoic acid and arachidonic acid were statistically different from that of the Model group.Metabolic pathway analysis showed that SM+DOO could inhibit the uptake and oxidation of fatty acid by decreasing the content of arachidonic acid,heptadecanoic acid,palmitoleic acid,stearic acid and 3-hydroxybutyric acid;and could promote the oxidative use of glucose by increasing the content of malic acid,citric acid and phenylalanine(the formation of fumaric acid,involved in TCA cycle)in the tricarboxylic acid(TCA)cycle to improve the energy metabolism and protect ischemic myocardium.3.The consistency of the biochemical indicators results and metabonomics results affirmed the cardioprotective effect of SM+DOO from classical pharmacological methods and systems biology methods;Western Blot experiments showed that SM+DOO could decrease the level of fatty acid β-oxidation via the AMPK/ACC/CPT-1 pathway,and promote glucose oxidation via the PDK/PDC pathway.SM+DOO play a role on cardioprotective by reducing myocardial oxygen consumption,improving energy metabolism and protecting ischemic myocardium.Conclusions SM,DO and SM-DO have a significant role in myocardial protection,especially with the best cardioprotective effect of SM+DOO.It was suggested that there is a synergistic effect between SM and DOO,and it could be concluded that DOO can enhance the therapeutic effect of SM by exerting the function of "guiding the main bioactive compounds to the site of the disease,and adjusting all potency of herbs to work together and synergistically".The results of metabonomics showed that MI/R injury model was established successfully and SM+DOO pretreatment was effective.According to metabonomics,12 biomarkers related to MI/R injury were screened,while SM+DOO mainly regulated TCA cycle and β-oxidative metabolism of fatty acids,so as to improve energy metabolism.In addition,SM+DOO pretreatment attenuated MI/R injury by AMPK/ACC/CPT-1 and PDK/PDC pathway,which were consistent with the results of metabonomics analysis.This study advances the knowledge of the overall efficacy and mechanism of SM-DO in the treatment of IHD,and makes basic contributions to the application of metabolomics technology for TCM research. |
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