The Mechanism Of PI3K/AKT And ERK1/2 Signaling Pathway In The Treatment Of Ischemic Heart Failure In Cardiac Shock Wave Therapy

Objective:To establish an ischemic heart failure model in SD rats.Survival SD rats were treated with CSWT,PI3K/Akt and ERK1/2 signalling blockers LY294002 and PD98059 at 4 weeks post-surgery,and CSWT inhibition was verified from the whole animal level.To research the mechanism of cardiomyocyte apoptosis induced by cardiac insults of PI3K/Akt and ERK1/2 signaling pathways and to investigate the mechanisms of cardiomyocyte apoptosis induced by cardiac insults,Whether the two signaling pathways PI3K/Akt and ERK1/2 have synergistic effects in the treatment of ischemic heart failure by CSWT.Method:1.Ninety-eight adult male SD rats were randomly selected and weighed 220-250 g.Ninety rats were operated on thoracotomy.The left anterior descending coronary artery was ligated at high level to establish a rat model of ischemic heart failure.Four weeks after surgery,survivors were randomly assigned to HF group(8 cases),HF +CSWT group(8 cases),HF+LY294002 group(8 cases),HF+PD98059 group(8 cases),HF+LY294002+PD98059 group(8 cases),HF+CSWT+LY294002 group(8 cases),HF + CSWT + PD98059 group(8 cases),HF+CSWT+LY294002 + PD98059 group(8 cases),and 8 cases SD rats participated in the experiment as sham operation group,Among them,the cardiac shock wave was intervened 4 weeks after surgery,and the shock wave energy was 0.24 mJ/mm2,200 pulses at a time,and the frequency was 60 beats/min,three days per week,once a day for 4 weeks,in which the inhibitor LY294002 was intraperitoneally administered 100 mg/kg,Inhibitor PD98059 was administered intravenously 10 mg/kg once a week.2.High frequency ultrasonography was used to evaluate the cardiac function of rats before surgery,at the fourth and eighth weeks after surgery.NT-proBNP levels were measured before and after 4 and 8 weeks after operation.The percentage of myocardial infarct was assessed by TTC staining.TUNEL method was used to determine the apoptosis index of cardiomyocytes.mRNA expression levels of Bcl-2,Bax and Casepase-3 in cardiac tissues were detected by RT-PCR,The expression levels of Bcl-2,Bax,Casepase-3,p-AKT and p-ERKl/2 proteins in cardiac tissues were detected by immunohistochemistry.Result:1.Sixty seven SD rats completed the acute myocardial infarction model with a success rate of 74.4%.Five rats died of anesthesia during anesthesia,fifteen died of ventricular fibrillation,and three died of heart rupture.Sixty-four heart failure model rats survived after 4 weeks,the survival rate was 71.1%,and 3 died of infection and heart failure.2.At 4 weeks after operation,high-frequency echocardiography showed that left ventricular enlargement,left ventricular wall thinning,and interventricular septal amplitude were significantly attenuated in HF rats.LVEDV and LVESV were significantly larger than before surgery(P<0.05),and FS was significantly decreased(P<0.05),LVEF was significantly reduced(P<0.05).At 8 weeks after operation,echocardiography was performed,compared with HF group,LVEDV and LVESV in HF+CSWT group were reduced(6.11 ±0.35 vs 9.21 ±0.31,4.10±0.37 vs 7.14±0.32,P<0.05),and LVEF and FS were significantly increased.(62.85±2.65 vs 36.84±4.75,42.15±2.16 vs 22.11 ±2.17,P<0.05),and the LVEDV and LVESV in the HF+CSWT+LY294002 group were significantly larger than those in the HF+CSWT group(7.54±0.33 vs 6.11±0.35,5.47±0.33 vs 4.10±0.37,P<0.05),LVEF and FS were significantly decreased(45.77±2.47 vs 62.85±2.65,P<0.05),and LVESV increased in HF+CSWT+PD98059 group(4.59±0.25 vs 4.10±0.37,P<0.05),LVEF decreased(54.25±3.80 vs 62.85±2.65,P<0.05),Compared with HF+CSWT+LY294002 group and HF+CSWT+PD98059 group,HF+CSWT+LY294002+PD98059 group LVEF Significantly decreased(38.07±2.73 vs 45.77±2.47,38.07±2.73 vs 54.25±3.80,all P<0.05).3.Serum nt-probnp levels in rats were determined before and after surgery 4 weeks and 8 weeks respectively.Compared with the preoperative period,the level of nt-probnp in SD rats in HF group 4 weeks after surgery was significantly increased(30.18±1.18 vs 9.78±1.13,P<0.05).At 8 weeks postoperatively,serum NT-proBNP levels in the HF+CSWT group were significantly lower than those in the HF group(18.19±2.98 vs 33.79±3.85,P<0.05).Compared with the HF+CSWT group,the HF+CSWT+LY294002 group NT-proBNP levels and HF+CSWT+PD98059 groups were significantly higher(30.68±2.25 vs 18.19±2.98,22.39±2.57 vs 18.19±2.98,all P<0.05).Compared with HF+LY294002+PD98059 group,NT-proBNP levels in HF+CSWT+LY29002+PD98059 group decreased(35.37±2.71 vs 64.65±7.63,P<0.05).Compared with HF+CSWT+LY294002 group and HF+CSWT+PD98059 group,serum NT-proBNP levels in HF+CSWT+LY29002+PD98059 group were significantly higher(35.37±2.71 vs 30.68±2.25,35.37±2.71 vs 22.39±2.57,all P<0.05),and NT-proBNP levels in HF+CSWT+LY294002 group were higher than those in HF+CSWT+PD98059 group(30.68±2.25 vs 22.39±2.57,all P<0.05).4.TTC staining was used to measure the myocardial infarction area in IHF rats.Compared with the control group,the area of myocardial infarction in HF group was significantly enlarged(24.24±1.45 vs 0,P<0.05).Compared with HF group,the area of myocardial infarction of HF+CSWT group was significantly reduced(13.69±0.94 vs 24.24±1.45,P<0.05),and the infarcted area of HF+LY294002 group,HF+PD98059 group was enlarged(42.57±2.82 vs 24.24±1.45,32.96±4.24 vs 24.24 +1.45,P<0.05).5.The apoptosis index of rat myocardial cells was determined by TUNEL assay.Compared with the sham group,the apoptosis index of HF group increased(24.89±2.97 vs 1.16±0.20,P<0.05).Compared with HF group,the apoptosis index of HF+CSWT group was significantly reduced(14.95 + 0.91 vs.24.89 + 2.97,P<0.05),and the apoptosis index of HF+LY294002 group,HF+PD98059 group was significantly increased(44.48±2.94 vs 24.89±2.97,34.77±2.45 vs 24.89 + 2.97,P<0.05).Compared with HF+CSWT group,the apoptosis index of HF+CSWT+LY294002 group,HF+CSWT+PD98059 group was increased(22.84±1.12 vs 14.95±0.91,19.13±1.86 vs 14.95±0.91,all P<0.05).Compared with HF+CSWT+LY294002 group,HF+CSWT+PD98059 group,the apoptosis index of HF+CSWT+LY29002+PD98059 group was significantly increased(27.29±1.22 vs 22.84±1.12,27.29±1.22 vs 19.13±1.86,P<0.05),and the apoptosis index of HF+CSWT+LY294002 group was higher than HF+CSWT+PD98059 group(22.84±1.12 vs 19.13±1.86,P<0.05).6.The results of RT-PCR showed that the mRNA expression level of anti-apoptotic gene bcl-2 was higher than that in HF+CSWT group,HF+CCSWT+LY294002 group and HF+CSWT+PD98059 group(1.14±0.03 vs 0.49±0.02,1.14±0.03 vs 0.62±0.04,1.14±0.03 vs 0.71±0.03,P<0.05).The mRNA expression levels of bcl-2 in HF+LY294002+PD98059 group were significantly lower than those in HF+LY294002 group and HF+PD98059 group(0.11±0.02 vs 0.23±0.04,0.11±0.02 vs 0.34±0.03,all P<0.05).Compared with HF+LY294002+PD98059 group,the mRNA expression level of bcl-2 was significantly increased in HF+CSWT+LY29002+PD98059 group(0.43±0.05 vs 0.11±0.02,P<0.05),compared with HF+CSWT+LY294002 group,HF+CSWT+PD98059 group,The mRNA expression level of bcl-2 in HF+CSWT+LY29002+PD98059 group was significantly reduced(0.43±0.05 vs 0.62±0.04,0.43 ± 0.05 vs 0.71 ±0.03,P<0.05).The mRNA expression level of bcl-2 in HF+CSWT+LY294002 group was lower than that of HF+CSWT+PD98059 group(0.62±0.04 vs 0.71±0.03,P<0.05).The pro-apoptosis gene Bax,Caspase-3 and anti-apoptotic gene Bcl-2 mRNA expression levels were opposite in the above comparison.7.The results of immunohistochemical staining showed that the expression of p-AKT and P-ERK1/2 protein in HF group was significantly decreased compared with the control group(0.25±0.04 vs 1.00±0.02,0.22±0.04 vs 1.00±0.02,P<0.05).Compared with HF group,the expression of p-AKT and P-ERK1/2 protein in HF+CSWT group was significantly increased(0.79±0.07 vs 0.25±0.04,0.58±0.08 vs 0.22±0.04,P<0.05),Compare with HF+CSWT group,the expression of p-AKT protein was decreased in HF+CSWT+LY294002 group(0.45±0.04 vs 0.79±0.07 P<0.05),and the expression of P-ERK1/2 protein in HF+CSWT+PD98059 group was significantly decreased(0.22±0.03 vs 0.58±0.08,P<0.05).Anti-apoptotic protein Bcl-2 protein expression,compared with HF group,HF + CSWT group anti-apoptotic protein Bcl-2 expression was significantly increased(0.69±0.02 vs 0.28±0.04,P<0.05),HF + LY294002 group and HF+PD98059 group was significantly decreased(0.17±0.01 vs 0.28±0.04,0.21±0.02 vs 0.28±0.04,P<0.05),compare with HF+CSWT group,HF+CSWT+LY29002 group and CSWT+PD98059 group was significantly decreased(0.43±0.03 vs 0.69±0.02,0.56±0.02 vs 0.69±0.02,P<0.05),Anti-apoptotic protein Bcl-2 in HF+CSWT+LY29002 group was significantly lower than that of HF+CSWT+PD98059 group(0.43±0.03 vs 0.56±0.02,P<0.05).Compared with HF+CSWT+LY29002 group and HF+CSWT+PD98059 group,the expression of anti-apoptotic protein Bcl-2 in HF+CSWT+LY29002+PD98059 group was significantly decreased(0.28±0.02 vs 0.43±0.03,0.28±0.02 vs 0.56±0.02,P<0.05),The pro-apoptotic proteins Caspase-3,Bax and anti-apoptotic protein Bcl-2 showed opposite expression trends,and the anti-apoptotic protein Bcl-2 had the opposite result.Conclusion:1.High ligation of the thorax to the left anterior descending coronary artery can establish a parallel heart failure rat model and provide an ideal animal model for the cardiovascular field.2.The percentage of myocardial infarct size and cardiomyocyte apoptotic index,NT-BNP level,LVEF and other indexes of the SD rats after CSWT treatment were significantly lower than those of other groups,indicating that CSWT treatment can inhibit left ventricular remodeling,thus improving the HF rat's Cardiac Function.3.CSWT protects cardiac function by activating PI3K/Akt and ERK1/2 signaling pathways to inhibit cardiomyocyte apoptosis,and PI3K/Akt and ERK1/2 signaling pathways have a synergistic effect in the treatment of ischemic heart failure by CSWT and PI3K.The role of the/Akt signaling pathway in CSWT treated ischemic heart failure is better than that of the ERK1/2 signaling pathway.3.CSWT protects cardiac function by activating PI3K/Akt and ERK1/2 signaling pathways to inhibit cardiomyocyte apoptosis,and PI3K/Akt and ERK1/2 signaling pathways have a synergistic effect in the treatment of IHF with CSWT,and PI3K/Akt signaling pathway in CSWT treating IHF role is better than that of ERK1/2 signaling pathways.