| Background:Ischemic heart disease(IHD)contains a series of diseases caused by the insufficiency of coronary blood supply.Today,it is one of the most major threats to human health and is the second cause of death in our country.At present,the main therapeutic method for IHD is the intra-coronary operation which can restore the blood perfusion.However,restoring blood flow also induces additional and irreversible deteriorations,which is called as "reperfusion injury".It is the key for treating IHD to find effectively intervening methods against reperfusion injury following myocardial ischemia.Endoplasmic reticulum-mitochondria interaction is a new research focus in recent years.The endoplasmic reticulum is specialized into the sarcoplasmic reticulum in cardiomyocytes.Sarcoplasmic reticulum-mitochondrial interaction plays a critical role in myocardial ischemia reperfusion injury.At present,the research on this target is still in the stage of exploring the pathological mechanism,and the research on new drug exploration and drug treatment mechanism based on it is still in blank.Salvianolic acids is the major active ingredients of Salvia Miltiorrhiza,a classical traditional Chinese herb,which has the effect of promoting blood circulation and removing blood stasis,and is widely used in the clinical treatment of IHD.Thus,introducing the sarcoplasmic reticulum-mitochondria interaction into the pharmacological study of salvianolic acids may have significance and bring a strong promotion for searching new drugs for IHD and elucidating the therapeutic mechanisms of "promoting blood circulation and removing blood stasis" Chinese herbs in IHD.Objective:To demonstrate that salvianolic acids exerts the protecting effect on ischemia-reperfusion injury through intervening the over-activation of sarcoplasmic reticulum mitochondria interaction.Method:Male SD rats(180-220 g)were randomly divided into sham operation group,ischemia-reperfusion model group,positive drug group(diltiazem hydrochloride 10 mg·kg-1),and total salvianolic acid treatment groups,including high dose group(40 mg·kg-1),medium dose group(20 mg·kg-1),and low dose group(10 mg·kg-1).Myocardium ischemia-reperfusion injury was initiated by a 30min ligation on the coronary left descending branch,which was then released to restore blood supply.The intervening drugs were given by intraperitoneal injection immediately after the ligation.Myocardial infarction area was measured with TTC-Evan’s Blue double staining,serum markers for myocardial injury(LDH,CK-MB,cTnI)were measured with biochemistry or ELISA assays,and myocardial morphology was observed with light microscopy(HE staining)and electron microscopy;these indexes were used to evaluate the therapeutic effects of total salvianolic acids on myocardial ischemiareperfusion.The contact between sarcoplasmic reticulum and mitochondria was determined by fluorescence co-localization assay and electron microscopy observation,and the formation of IP3R1-VDAC1-Grp75 sarcoplasmic reticulummitochondria calcium channel complex was determined by proximity ligation assay,and co-immunoprecipitation assay;these were used to observe the modulatory effect of total salvianolic acids on sarcoplasmic reticulum-mitochondria interaction.Mitochondrial calcium content,mPTP channel formation,mitochondrial membrane potential,and mitochondrial oxygen free radical content were measured by fluorescence spectrophotometries stained with Fura-2,Calcine AM,JC-1,and DCFHDA,respectively;myocardial GSH/GSSG ratio,MDA content,and protein carbonyl content were measured by colorimetry or ELISA;these measurements were used to evaluate the effect of salvianolic acids on the downstream of the sarcoplasmic reticulum-mitochondria interaction,further confirming the relationship between the protective effect of total salvianolic acids on myocardial ischemia-reperfusion and its modulation on sarcoplasmic reticulum-mitochondria interaction.Results:Ischemia-reperfusion induced significant increases in the myocardial infarction area and contents of serum LDH,CK-MB and cTnI(compared with sham operation group,all P<0.01);and these changes were significant inhibited by treatments of total salvianolic acids of high-dose and medium-dose,as well as treatment of diltiazem(compared with model group,P<0.01 or 0.05);morphologically,a series of pathological changes were shown in the model group,including myocardial necrotic contraction zone,cardiomyocytes and myocardial interstitium edema,and inflammatory cell infiltrations;treatments of total salvianolic acids and diltiazem ameliorated these injuries The results of electron microscopy showed that myofilaments were broken and dissolved,myofibrillar arrangement was disordered,subbasement membrane edema,mitochondrial size was different,mitochondrial membrane was broken,ridge was blurred,vacuolated and high-density particles were deposited in the model group.Fluorescence co-localization assay showed that in the model group,the localization correlation coefficient of ER-tracker and mito-tracker was significantly increased(compared with sham operation group,P<0.01);the results of electron microscopy showed that in model group the distance between the sarcoplasmic reticulum and mitochondria was significantly decreased,and the length of mitochondria associated sarcoplasmic reticulum membrane(MAM)was significantly increased(compared with sham operation group,P<0.01);treatments of high-dose and medium-dose total salvianolic acids and diltiazem significantly inhibited these changes(compared with model group,P<0.01 or 0.05).The proximity ligation and co-immunoprecipitation assays showed that the amounts of IP3R1-GRP75-VDAC1 channel complex were significantly enhanced in the model group(compared with sham operation group,P<0.01),and this increase was significantly inhibited by high-dose and medium-dose total salvianolic acid treatments and diltiazem treatment(compared with model group,P<0.01 or 0.05).In the model group,myocardial mitochondrial calcium content,mPTP amount,and oxygen free radical content were significantly increased,and mitochondrial membrane potential was significantly decreased(compared with sham operation group,P<0.01),additionally,myocardial GSH/GSSG ratio was decreased,MDA content,and protein carbonyl content were significantly increased(compared with sham operation group,P<0.01),total salvianolic acids of high-dose and medium-dose and diltiazem significantly ameliorated these changes(compared with model group,P<0.01 or 0.05).Conclusion:Salvianolic acids exert its protective effect on myocardial ischemiareperfusion injury through inhibiting the over-activation of sarcoplasmic reticulummitochondria interaction. |
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