| Coronary atherosclerotic heart disease is a serious disease to harm human’s health,andmyocardial infarction is the "first dangerous killer".The key to solve the problem is reflowthe criminal vascular as soon as possible. But myocardial ischemia and reperfusion can usuallymake the second damage, which made ischemic myocardium progressive seriously. So how toreduce ischemia-reperfusion injury had been focus in by medical scientist. Looking for a safe,effective drug is very important. Ginsen is a kind of traditional Chinese medicine. It canprotect ischemic myocardium, and have the fouction of antioxidant, antineoplastic andimproving immunity.25-hydroxyl-Protopanaxatriol (25-OH-PPT), we code it T19，it’schemical name is20(R)-dammarane-3beta，6alpha，12beta，20，25-pentol，molecular formulais C30H54O5，molecular weight is494.It Is a kind of gensin monomer which was extractedfrom the leaves and stem from gensin,it’s belong to Protopanaxatriol and is a ginsengenin.And then whether25-OH-PPT can be a safe, effective drug for protecting myocardialischemia-reperfusion injury is what we need to study. The present study attempted toinvestigate the protection effects and its underlying mechanism of25-OH-PPT on myocardialischemia/reperfusion injury.First, to investigate the protection effect and its possible mechanisms of25-hydroxyl-Protopanaxatriol on myocardial ischemia/reperfusion injury (MI/RI) inexperimental rats in vivo. Second, the model of H2O2-induced H9c2cardiomyocytesoxidative stress injuries was adopted, in order to explore protection effect of it. Experimentsare divided into the following three parts:Part one Protective effect of25-OH-PPT pretreatment on experimentalmyocardial ischemia/reperfusion injury in rats Objective To investigate the protective effect of25-OH-PPT on MI/RI in Wistar rats invivo and correlation with25-OH-PPT dosages. To investigate the effect of inhibitor of PI3K(LY294002) on MI/RI.Methods(1) Adult Wistar rats, weight180to250g were randomly divided into the followingseven groups (n=20): Sham operation group (sham group); I/R group; T19a group: I/R+T19low dosage; T19b group: I/R+T19middle dosage; T19c group: I/R+T19large dosage;Inhibiter of PI3K group: I/R+T19large dosage+LY294002; Sandard drug group: I/R+Trapidil. The dosages of T19groups were5,10,20mg/kg/d were given by intragastricadministration seven days. Inhibiter of PI3K group:0.3mg/kg LY294002was given byintraperitoneal injection30min before20mg/kg/d dosage of T19was given by intragastricadministration of the last day. The dosages of trapidil was15mg/kg/d.(2) The MI/R animal model was constructed by left anterior descending coronary artery(LAD) ligation，Rats were subjected to30min of myocardial ischemia followed by24h ofreperfusion.(3) Electrocardiogram of reperfusion24h, echocardiogram before ischemia andreperfusion24h, Blood serum for CK-MB, LDH, AST, SOD, MDA, CAT and GSH-px wereestimated. The area of myocardial infarction was estimated by Nitrotetrazolium blue chloridestaining. Structural changes within the left ventricular anterior wall (LVAW) from rat heartswere observed under an optical microscope and transmission electron microscope. Theapoptosis of cardiac myocyte was detected by terminal deoxynucleotidyl transferase-mediateddUTP-biotin nick end labeling (TUNEL) method.Results(1) After24h of reperfusion, compared with the Sham group, the ST segment ofelectrocardiogram in all dosages of T19were descended. T19low dosage group had nosignificant difference (P>0.05), high and middle groups had obviously descended (P<0.01, P<0.01), The degree of descend has a direct-proportion relation with the amount of dosage.(2) Compared with the I/R group, the areas of myocardial infarction in all dosages ofT19were decreased，T19low dosage group had no significant difference (P>0.05), middle and high groups had obviously descended (P<0.05, P<0.05), The degree of decreasing had adirect-proportion relation with the amount of dosage.(3) The results of echocardiogram before ischemia in all groups had no difference. After24h of reperfusion, compared with the I/R group, T19low dosage group had tendency ofincreasing left ventricular ejection fraction (LVEF), decreasing left ventricular end diastolicvolume（LVEDV）and ventricular end systolic volume（LVESV）(P>0.05), middle and highgroups obviously increased LVEF (P<0.01), decreasing LVEDV and LVESV (P<0.01,P<0.01), The degree of change had a direct-proportion relation with the amount of dosage.(4) After24h of reperfusion, compared with the I/R group, T19low dosage group hadtendency of decreasing the content of CK-MB,LDH and AST, but had no significantdifference (P>0.05), middle and high groups had obviously descended CK-MB,LDH and ASTin blood serum,(P<0.05, P<0.05, P<0.05), The degree of change had a direct-proportionrelation with the amount of dosage.(5) After24h of reperfusion, compared with the I/R group the T19high dosage group canincreased the content of SOD, CAT,GSH-Px (P<0.05, P<0.05, P<0.01) and decreased thecontent of MDA (P<0.05), The low and middle group had no different change in the contentof SOD,CAT,MDA (P>0.05), but obviously increased the content of GSH-Px (P<0.05,P<0.01).(6) After24h of reperfusion, compared with the I/R group, the change of myocardiumpathology, under optical microscope and transmission electron microscope and apoptosis ofmyocardial cell detection with TUNEL method: The T19high and middle dosage groups canobviously decreased necrosis myocardial cell, inflammatory cell and apoptotic index.(P<0.01)The degree of decreased had a direct-proportion relation with the amount of dosage. The lowdosage group had no significantly decreased above (P>0.05).The standard drug trapidil can protect the myocardium in MI/RI, the role wasequivalency to middle dosage group. But role of protect the myocardium in MI/RI wereblocked in inhibitor group.Conclusion25-OH-PPT had a protective effect on myocardial ischemia/reperfusioninjury model in rats，the low dosage had no significant effect and the middle and high dosages had significant effect and related to correlation with dosages.PI3K/Akt pathway participatedthe protective effect of25-OH PPT on myocardial ischemia/reperfusion injury model in rats.Part two Study of mechanism of25-OH-PPT’s protective effect onexperimental myocardial ischemia/reperfusion injury in rats actived byPI3K/Akt pathwayObjective: To investigate the mechanism of PI3K/Akt pathway actived on the protectiveeffect of25-OH-PPT on experimental myocardial ischemia/reperfusion injury in ratsMethods(1) Adult Wistar rats, weight180to250g were randomly divided into the following fivegroups (n=10): sham operation group (sham group); I/R group; T19group: I/R+T19;Inhibiterof PI3K group: I/R+T19+LY294002; Standard drug group:I/R+Trapidil. The dosage of T19group was20mg/kg/d, was given by intragastric administration seven days. Inhibiter of PI3Kgroup:0.3mg/kg LY294002was given by intraperitoneal injection30min before20mg/kg/ddosage of T19was given by intragastric administration the last day. Trapidil dosage was15mg/kg/d.(2) The MI/RI animal model was constructed by left anterior descending coronary artery(LAD) ligation, Rats were subjected to30min of myocardial ischemia followed by24h ofreperfusion. Heart and blood serum were detected.(3) The expression of Akt, p-Akt(Ser-473), eNOS, p-eNOS(Ser-1177), Bcl-2, Bax andCaspase-3were extracted from the myocardium after or24h of reperfusion by Western Blotand immunohistochemisty. The content of NO of all groups were detected by nitrationrecovery method.Results(1) With using Western Blot and immunohistochemisty，we found the expression of Aktand eNOS had no significant difference in all groups. Compared with the I/R group,T19group can obviously increased the expression of p-Akt and p-eNOS (P<0.01), LY294002group can obviously decreased the expression of p-Akt and p-eNOS vs T19group (P<0.01) (2) Compared with the I/R group, T19group can obviously increased the content ofNO(P<0.01), Compared with the T19group: LY294002group can obviously decreased it (P<0.01).(3) With using Western Blot and immunohistochemisty, compared with the sham group,I/R group obviously up-regulate the expression of Caspase-3and Bax (P<0.01, P<0.01)anddown-regulate the expression of Bcl-2(P<0.01). Compared with the I/R group, T19groupcan obviously down-regulate the expression of Caspase-3and Bax (P<0.01, P<0.01) andup-regulate the expression of Bcl-2(P<0.01), LY294002group can obviously up-regulate theexpression of Caspase-3and Bax (P<0.01, P<0.01) and down-regulate the expression ofBcl-2(P<0.01) vs T19group.Compared with the I/R group,Trapidil group can obviously increased the expression ofpAkt and peNOS (P<0.01), obviously increased the content of NO (P<0.05),obviouslydown-regulate the expression of Caspase-3and Bax (P<0.01, P<0.01) and up-regulate theexpression of Bcl-2(P<0.01).Conclusion(1)25-OH-PPT reduced myocardial ischemia/reperfusion injury in experimental ratsmay be relate to p-Akt and p-eNOS.(2)25-OH-PPT had anti-apoptotic effect maybe relate to up-regulate the expression ofanti-apoptotic protein Bcl-2, down-regulate the expression of apoptotic protein Bax andCaspase-3.(3) PI3K/Akt pathway had important effect in protecting of myocardial ischemia/reperfusion injury in rats. It’s mechanism maybe related to the expression the apoptotic proteinwhich activied by PI3K/Akt pathway.(4)25-OH-PPT can activated PI3K/Akt pathway to protect myocardial cell endotheliumfounction reduce apoptotic cell and reduce myocardial ischemia/reperfusion injury. Part three Study of protective effect of25-OH-PPT on H2O2-induced H9c2cadiocytes injury and underlying mechanismObjective To investigate protective effect and underlying mechanism of25-OH-PPT onH2O2-induced H9c2cadiocytes injury.Methods(1) H9c2cells were be randomly divided into the following seven groups (n=6)Control group, H2O2group, H2O2+T19(low, middle and high) dosage group, H2O2+T19high+LY294002group，Standard drug:Trapidil group. The dosage of T19were31.25,62.5and125μg/ml. H9c2cells were pretreated with different concentrations of T19and30μg/mltrapidil for4hours and then exposed to250μM H2O2incubation for6hours. H2O2+T19high+LY294002group: LY294002was given to the H9c2cells1h,then gave125μg/ml T19for4h,and then gave250μM H2O2for6h.(1) The cell viability was measured by MTT assay to determine the effects of T19. Cellmorphology was observed under microscope. Flow cytometry analysis of Annexin Ⅴ/PI stainingand Hoechst33258were used to assess the effects of T19on H2O2-induced H9c2cellapoptosis.(2) H9c2cells were be randomly divided into the following five groups (n=6) with theformer method of administration.Control group,H2O2group,H2O2+T19high group,H2O2+T19high+LY294002group, Standard drug Trapidil group. The dosage of T19high group was125μg/ml.(4) The expression and activation of Akt, p-Akt(Ser-473),eNOS,p-eNOS（Ser-1177）,Bcl-2,Bax and Caspase-3were extracted from H9c2cells after H2O2by Westernblot.Results(1) Different concentrations of T19pretreated H9c2cells could significantly reduce theapoptotic rate by enhancing antioxidative activity. And its protective effect correlated withdosages to a certain extent. After giving the LY294002, the protective effect of25-OH-PPTwere blocked.(2) Compared with H2O2group, T19group can obviously up-regulate the expression ofp-Akt and p-eNOS (P<0.01), LY294002group can obviously down-regulate the expression of p-Akt and p-eNOS vs T19group (P<0.01). Compared with H2O2group,T19group canobviously up-regulate the expression of Bcl-2(P<0.01) and down-regulate the expression ofBax and Caspase-3(P<0.01), LY294002group can obviously down-regulate the expressionof Bcl-2vs T19group (P<0.01) and up-regulate the expression of Bax and Caspase-3(P<0.01).Trapidil group was equivalency to the T19group.Conclusions:25-OH-PPT had a protective effect against H2O2-induced H9c2cadiocytesinjury. The underlying mechanism maybe was relate to activate PI3K/Akt pathway to regulatethe apoptotic proteins and reduce apoptotic cells. |
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