The Effects Of Yixin-Shu On Myocardial Ischemia/Reperfusion Injury And Underlying Mechanisms

Objective:Positive evidence from clinical trials has obtained growing acceptance of traditional Chinese medicine（TCM）for cardiac diseases treatment.Here,we investigated the effects of YiXin-Shu（YXS）,an antioxidant-enriched TCM formula,on myocardial ischemia/reperfusion（MI/R）injury and its underlying mechanisms.Methods:8-week old male C57BL/6 mice were fed with high-cholesterol diets for 8 weeks,and then randomly assigned to the following groups:sham,vehicle（saline）,YXS-1(60mg·kg^-1·d^-1),or YXS-2(120 mg·kg^-1·d^-1)by gavage for 1 week.In-vivo cardiac-specific silencing of LXRα,ERα,PPARαand PPARβwas used by direct myocardial injection of siRNA.These mice were subjected to ischemia for 30 min and then reperfusion for 3 hours（for apoptosis and oxidative stress assay）or 24 hours（for infarct size,viable myocardial metabolism,and cardiac function assays）.Myocardial infarct size was evaluated using Evans blue-TTC double staining.¹⁸F-FDG Micro-PET/CT scanning was used to detect viable myocardial metabolism.Cardiac function was assessed by echocardiographic measurements.In addition,the mechanism of cardioprotective effects of YXS on MI/R injury was determined by TUNEL,caspases activity assay,transmission electron microscopy,western blot,immunohistochemistry,enzyme-linked immuno sorbent assay and quantitative PCR.Results:YXS pretreating significantly reduced myocardial infarct size,improved both viable myocardium metabolism and cardiac function in hypercholesterolemic mice.Mechanically,YXS attenuated myocardial apoptosis via inhibiting the mitochondrial mediated apoptosis pathway（as demonstrated by inhibition of mitochondrial swelling,cytochrome c release and Caspase-9 activity,and normalization of Bcl-2 and Bax expression）without altering the endoplasmic reticulum-stress and death receptor death pathways.In addition,YXS decreased oxidative/nitrative stress（as demonstrated by decrease of superoxide and nitrotyrosine content and normalization of pro-and anti-oxidant enzyme levels）.Interestingly,pretreatment of YXS upregulated the expression of endogenous metabolic nuclear receptors including LXRα,ERα,PPARαand PPARβ,and in-vivo cardiac-specific silencing of LXRαsignificantly blunted the cardio-protective effects of YXS.Conclusion:these data suggest that YXS is a cardioprotective TCM in mitigating MI/R injury via upregulating LXRαexpression and suppressing the mitochondrial mediated apoptosis and oxidative stress,therefore providing a rationale for future clinical trials and clinical applications.