The Mechanism Of Organic Acids Of Crataegus Pinnatifida Component Against Cardiomyocyte Apoptosis In Ischemia-reperfusion

Objective:To investigate whether the protective effect of organ acid of Crataegus pinnatifida on myocardial ischemia-reperfusion injury is achieved by inhibiting HIF-1α,inhibiting apoptosis and regulating the expression of apoptosis-related proteins Bcl-2 and Bax,and whether the mechanism of protective effect involve in PI3K/Akt,MAPK signaling pathway.Methods:In vitro experiments:1.H9c2 cardiomyocytes in logarithmic phase were randomly divided into 6 groups:Control,H₂O₂,H₂O₂+NAC and H₂O₂+organ acid of Crataegus pinnatifida（high,medium,low concentration）.After treated with H₂O₂ 100μmol/L for 2 h,the absorbance values of cell number were detected by MTS;The enzyme activity of Caspase 3 and Caspase 9 in the cells was determined by kit,and the apoptosis rate of the cells was determined.Western blot was used to detect H9c2.Expression of p-NF-κB/NF-κB protein levels in cells.2.Experimental were randomly divided into 6 groups:control group,H₂O₂ group,organic acid of Crataegus pinnatifida pretreatment group,ERK agonist group,JNK inhibitor group,PI3K inhibitor group.After treatment with inhibitor or agonist,H9c2cells were pretreated with Hawthorn organic acid for 2 h,and then added with H₂O₂ at100μmol/L for 2 h.The cell proliferation absorbance was measured by MTS method,and intracellular Caspase 3 was determined by kit.In vivo experiment:Normally reared SD rats were randomly divided into 4 groups:Sham group,I/R group,organic acid of Crataegus pinnatifida pretreatment group+I/R,organic acid of Crataegus pinnatifida administration blank group.Western blot was used to detect the expression of HIF-1α,Bcl-2 and Bax protein in myocardial tissue.Results:In vitro experiments:1.Mechanism of H₂O₂-induced H9c2 cardiomyocyte injury induced by organic acid of Crataegus pinnatifida in vitro model of MIRICompared with H₂O₂ group,H₂O₂+organic acid of Crataegus pinnatifida group increased the number of H9c2 cardiomyocytes,the effect were best at 6.25μg/ml and25μg/ml,and the H2O2+NAC group also had a significant effect.2.Effects of organ acid of Crataegus pinnatifida on apoptosis and related proteins of MIRI in vitro induced by H₂O₂-induced H9c2 cell injuryCompared with the Control group,the number of apoptotic cells were increased in the H₂O₂ group,the activities of Caspase 3 and Caspase 9 were enhanced,and the protein expression of p-NF-κB was enhanced.Compared with H2O2 group,the number of apoptotic cells in organ acid of Crataegus pinnatifida group were decreased,the activity of Caspase 3 and Caspase 9 enzyme activities were decreased,and decreased the p-NF-κB protein expression decreased.3.The effect of organic acid of Crataegus pinnatifida on PI3K/Akt,MAPK and JNK signaling pathways in MIRI model of H9c2 cardiomyocytes induced by H₂O₂ in vivoCompared with the Control group,the number of cells in the H₂O₂ group were decreased,and the activity of Caspase 3 was significantly increased.Compared with the H₂O₂ group,the number of cells in the organ acid of Crataegus pinnatifida group was increased,the activity of Caspase 3 The enzyme activity was significantly decreased.Compared with the organ acid of Crataegus pinnatifida group,the number of ERK agonists,JNK inhibitors,and PI3K inhibitor cells was significantly decreased,and the activity of Caspase 3 was significantly reduced.In vivo experiment:1.The effect of organic acid of Crataegus pinnatifida on in vivo model of myocardial ischemia-reperfusion injuryCompared with the Sham group,the protein expression of HIF-1αin the myocardial tissue of the model group was significantly increased;the protein expression of Bcl-2 was significantly decreased;the expression of Bax protein was significantly increased;the protein expression of HIF-1αwas significantly reduced in the drug-administered group compared with the model group;the protein expression of Bcl-2 was significantly increased;the protein expression of Bax was significantly decreased.Conclusions:The anti-myocardial ischemia-reperfusion injury of Organ acid of Crataegus pinnatifida may be related to the regulation of hypoxia-inducible factor HIF-1α,inhibition of apoptosis and apoptosis-related protein Bcl-2/Bax,and activation of PI3K/Akt and MAPK signaling pathways.