| Myocardial ischemia reperfusion injury(MIRI) is a clinically commonpathophysiological phenomenon. It usually occurs when the blood supply to themyocardial tissue is recovered after it is interrupted a certain time. This is not onlydisable to restore normal heart function, but also it can increase heart dysfunctionand structural damage, and even lead to irreversible damage. Studies have shownthat ischemia and reperfusion can cause myocardial apoptosis. Apoptosis is one ofthe characteristics of MIRI, and is closely related to delayed myocardial death. So itis important to develope the effective drugs so as to preventing myocardial ischemiareperfusion injury.Ginsenoside Rb3and Rb2Combination contains plenty of Ginsenosides Rb3and little of Ginsenosides Rb2. Ginsenosides Rb3and Ginsenosides Rb2are isomers,they have the same sapogenin, four sugar, of which three six-carbon sugars areglucose, and a five-carbon sugar, five-carbon sugar of ginsenosides Rb3is xylose,while ginsenosides Rb2is pyranoid arabinose. the structural difference of both istiny and some physical and chemical properties are similar. Our research shows thatGinsenosides Rb3has a good protective effects on MIRI and can probablydeveloped into a class of drugs, but because separation process of ginsenosides Rb2is complex and increase the cost of development significantly, Ginsenosides Rb3isnot suitable for industrial production. Extraction process of Ginsenoside Rb3andRb2Combination is simplified and reduce the cost, so it is more appropriate forindustrial production and may be developed as drugs. Therefore, this studyoberserves the effects and mechanisms of G-Rb3/Rb2on myocardial ischemia reperfusion injury, and compare with G-Rb3, so as to providing an experimentalbasis for the development and utilization of the composition.MIRI model was established by ligation of the left anterior descending coronaryartery for30min and reperfused for120min in rats. to probe the effect of G-Rb3/Rb2and G-Rb3on MIRI and compare with their effects; the cell apoptosis model wasestablished by hydrogen peroxide (H2O2,200μmo1/L) to oberserve the effect ofG-Rb3/Rb2and G-Rb3on cardiomyocyte apoptosis and compare with their effects;specific inhibition of PI3K agent LY294002was used to observe the effects ofG-Rb3/Rb2on PI3K/AKT signaling pathway.1. The effect of G-Rb3/Rb2on myocardial ischemia reperfusion injury in rats1.1Experimental methodsThe myocardial ischemia reperfusion injury model was established by ligationof the left anterior descending coronary artery for30min and reperfused for120minin rats. The experiment include five groups: sham operation group, myocardialischemia reperfusion model group, G-Rb3/Rb2group, G-Rb3group, Diltiazemgroup. The following indicators were detected: Hemodynamics include HR, SBP,DBP, LVSP, LVEDP and±dp/dtmax; the myocardial infarct size; the myocardialenzymes include AST, CK-MB and LDH; Oxidative stress such as SOD, MDA,GSH-Px and CAT in myocardial；Serum inflammatory factors such as TNFαandIL-6; myocardial histopathology and ultrastructure changes were observed; theapoptosis rate was assayed by TUNEL kit; the protein expression of Caspase-3,Bcl-2and Bax were detected by immunohistochemistry; the level of Caspase-3,Bcl-2and Bax gene were determined by QPCR.1.2Experimental resultsG-Rb3/Rb2and G-Rb3can significantly accelerate HR and increase SBP, DBP,LVSP,±dp/dtmax and decrease LVEDP had no statistically significant. G-Rb3/Rb2and G-Rb3can reduce myocardial infarct size and inhibit the activities of AST, LDHand CK-MB. G-Rb3/Rb2and G-Rb3can reduce MDA content, increase theactivities of SOD, GSH-Px and CAT in Myocardial tissue. G-Rb3/Rb2and G-Rb3 can reduce the content of TNF-α and IL-6. Myocardial histopathology andultrastructural observation results shown that G-Rb3/Rb2and G-Rb3can reducemyocardial pathological changes, but the myocardial pathological changes is moreobviously in ultrastructural observation. TUNEL results showed that G-Rb3/Rb2andG-Rb3can significantly inhibit myocardial apoptosis; The immunohistochemicalresults showed that G-Rb3/Rb2and G-Rb3can decrease the expression of Caspase-3and Bax, increase the expression of Bcl-2; QPCR results showed that G-Rb3/Rb2and G-Rb3can decrease the level of Caspase-3mRNA and Bax mRNA, increase thelevel of Bcl-2mRNA and decrease the ratio of Bax/Bcl-2. The result show thatG-Rb3/Rb2and G-Rb3have better protection on myocardial ischemia reperfusioninjury, its mechanism may be related to oxidative stress, anti-inflammation, resistingcell apoptosis.2. Effect and mechanism of G-Rb3/Rb2on cardiomyocytes apoptosis2.1Experimental methodsThe cardiomyocytes were subjected with H2O2200μmol/L for120min to induceapoptosis. G-Rb3/Rb20,25,50,100,200,400,800and1000μg/mL were added intothe primary cultured cardiomyocytes, myocardial cell survival rate were detected byMTT. The cardiomyocytes were divided into normal control group, H2O2modelgroup, G-Rb3/Rb2100,200μg/mL group, G-Rb3100,200μg/mL group. H2O2200μmol/L was added in when drugs had been added in cardiomyocytes for24h,normal control group didn’t treat with H2O2.The following indicators were detected:The cell morphology, the rate of survival cells, the activity of LDH, GSH-px, CATand SOD, the level of MDA, the rate of apoptosis, the activity of Caspase-3,Caspase-8, Caspase-9, the protein expression of FADD, Fals, Apaf-1, Cytc, AKT,p-AKT, Bcl-2and Bax.2.2Experimental resultsG-Rb3/Rb2100,200μg/mL had no effect on myocardial cell survival as theexperimental concentration.G-Rb3/Rb2100,200μg/mL and G-Rb3100,200μg/mLcan reduce the changes of oxidative injury, increase the cardiomyocytes survival rate, and decrease the activities of LDH, G-Rb3/Rb2and G-Rb3can reduce MDA level,increase the activity of SOD, GSH-Px and CAT. G-Rb3/Rb2and G-Rb3can inhibitapoptosis significantly. G-Rb3/Rb2and G-Rb3can decrease the activity and proteinexpression of Caspase-3, Caspase-8and Caspase-9. G-Rb3/Rb2and G-Rb3candecrease the protein expression of FADD, Fals, Apaf-1, Cytc and Bax, increase theexpression of Bcl-2and p-AKT.3. G-Rb3/Rb2inhibit cardiomyocytes apoptosis by activition of PI3K/AKT signaltransduction pathway3.1Experimental methodsThe cardiomyocytes were subjected with H2O2200μmol/L for120min to induceapoptosis. The cardiomyocytes were divided into normal control group, H2O2model group, G-Rb3/Rb2group, G-Rb3/Rb2+LY294002group, model+LY294002group.H2O2200μmol/L was added in when drugs had been added in cardiomyocytesfor24h, while LY294002was add in cardiomyocytes earlier than drugs for20min,normal control group didn’t treat with H2O2.The following indicators were detected:the activity of LDH, The protein expression of PTEN, AKT, p-AKT, GSK-3β,p-GSK-3β, Bcl-2and Bax.3.2Experimental resultsG-Rb3/Rb2can decrease the activities of LDH. LY294002can increase theactivitIies of LDH; G-Rb3/Rb2can decrease the protein expression of PTEN, butThis action had nothing to do with LY294002. G-Rb3/Rb2can increase the proteinexpression of p-AKT and p-GSK-3β.When LY294002was add in, the proteinexpression of p-AKT and p-GSK-3β decreased significantly, and had no differenceswith model group. G-Rb3/Rb2can increase the protein expression of Bcl-2anddecrease the protein expression of Bax, the protein expression of Bcl-2decrease andthe protein expression of Bax increase when LY294002was added in.4. Experimental results4.1G-Rb3/Rb2and G-Rb3have protective effect on myocardial ischemiareperfusion injury in rats, this protective effect may be related to anti-oxidant, anti-inflammatory, anti-cardiomyocyte apoptosis.4.2Cardiomyocytes experiments show that G-Rb3/Rb2and G-Rb3can inhibitcardiomyocyte apoptosis by antioxidant stress and affecting endogenous, exogenouspathway of apoptosis and PI3K/AKT pathway, further protect myocardialischemia-reperfusion injury.4.3G-Rb3/Rb2and G-Rb3can activate PI3K/AKT signaling pathway by decreasingPTEN and improve the AKT phosphorylation activity, activate anti-apoptotic geneGSK-3β, Bcl-2, inhibit pro-apoptotic Bax genes.4.4G-Rb3/Rb2and G-Rb3have the same protective effect on ischemia-reperfusioninjury, the study suggests that G-Rb3/Rb2has the value of preventing and treatingMIRI. |
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