| Objective:For the construction of heart failure model,SD rats were used as a model to cut the sternum for acute thoracotomy.After the left anterior descending artery was found,the rat model survived for 4 weeks,which were treated by CSWT(extracorporeal shock),PI3K/Akt signaling pathway specific blocker LY294002 treatment,and ERK1/2 signaling pathway specific blocker PD98059 treatment for 4 weeks.To detecte the expression level of NT-proBNP?heart weight,mRNA and protein,and masson staining?immunofluorescence will be used to determine the degree of myocardial fibrosis.To explore the effect of CSWT on myocardial fibrosis of rat models of heart failure,and to study the role of PI3K/Akt,ERK1/2 two signals Pathway on myocardial fibrosis treated by CSWT.Method:1.120 adult male Sprague-Dawley rats,220-250g,were randomly selected.110 rats underwent thoracotomy,and the anterior descending coronary artery was ligated at a high level to establish a model of ischemic heart failure in SD rats.According to the establishment of the model and the way of post-modeling treatment,the rats that survived for four weeks after surgery were divided into nine groups:A,B,C,D,E,F,G,H,I,A:control group(10 cases);B:heart failure group(10 cases in HF group),C:heart failure+extracorporeal shock group(9 cases in HF+CSWT group),D:heart failure+inhibitor group 1(LY294002)(9 cases in HF+LY294002 group),E:heart failure+inhibitor 2 group(PD98059)(9 cases of HF+PD98059 group),F:heart failure+inhibitor group 1(LY294002)+inhibitor 2 group(PD98059)(9 cases of HF+LY294002+PD98059 group)G:heart failure+cardiac shock+inhibitor group 1(LY294002)(9 cases of HF+CSWT+LY294002 group),H:heart failure+cardiac shock wave+inhibitor 2 group(PD98059)(9 cases of HF+CSWT+PD98059 group)),I:heart failure+cardiac shock+inhibitor group 1(LY294002)+inhibitor group 2(PD98059)(9 cases of HF+CSWT+LY294002+PD98059 group).In vitro cardiac shock therapy,shock wave energy 0.24mJ/mm2,200 pulses each time,frequency 60 times/min,three times per week,once a day for a total of 4 weeks.The inhibitor LY294002 was administered intraperitoneally at 100 mg/kg,and the inhibitor PD98059 was administered intravenously at 10 mg/kg once a week.2.Cardiac function was measured by cardiac ultrasound technique to detect the cardiac function of rat at three point,before and after 4 weeks?8 weeks after surgery.Serum NT-proBNP level were determined according to the morphology of rat heart and rat heart was weighed.The degree of myocardial infarction was stand by heart/weight.The expression level of CD34 and aSMA in myocardial tissue was determined by RT-PCR.The expression level of CD34,aSMA,p-AKT and P-ERK1/2 in myocardial tissue was detected by immunohistochemistry.Result:1.The model of acute myocardial infarction was established in 110 of 120 experimental SD rats,and 10 of them were sham-operated.In the model of acute myocardial infarction,96 of them survived to 4 weeks after surgery,and sham operated rat were survived.73 of them underwent echocardiography and showed myocardial infarction,that is say the incidence of myocardial infarction was 76.04%.The heart/body weight ratio reflects the degree of myocardial infarction in rat2.Heart/weight ratio response:myocardial infarction degree in rats(because after myocardial infarction,the ventricular wall becomes thinner and the heart weight decreases).Compared with the control group(1.16±0.01),the heart weight of the HF group(1.06 ± 0.01)is lower(P<0.05).While the HF+CSWT group(1.11±0.03)rebounded compared with the HF group(1.06±0.01),(P<0.05).HF+LY294002 group(0.95±0.02)and HF+PD98059 group.(1.02±0.01)compared with the HF group(1.06±0.01),there was a significantly decreased(P<0.05),and the heart-to-weight ratio of the HF+LY294002+PD98059 group(0.69±0.03),treated by two signal pathway inhibitors was increased-Adding either HF+LY294002 group(0.95±0.02)or HF+PD98059 group(1.02±0.01)was significantly decreased(P<0.05),and HF+LY294002 group(0.951±0.02)was significantly lower than HF+PD98059 group(1.02±0.01)(P<0.05),(1.03±0.01)Compared with HF+CSWT group(1.11±0.03),HF+CSWT+LY294002 group(0.98±0.01)and HF+CSWT+PD98059 group wer decreased(P<0.05),after treating with two inhibitors,HF+CSWT+LY294002+PD98059 group(0.85±0.01)compared with HF+CSWT+LY294002 group(0.98±0.01)and HF+CSWT+PD9805,the heart weight was significantly decreased(P<0.05),and the HF+CSWT+LY294002 group(0.98±0.01)was lower than the HF+CSWT+PD98059 group(1.03±0.01)(P<0.05).3.4 weeks after model establishment,the left ventricle enlargement,wall thinning,ventricular septal motion,left ventricular end-systolic diameter(LVEDV)and left ventricular end-diastolic diameter were observed in each group.Compared with preoperative,LVESV level of HF group(5.76±0.20)was increased.FS,LVEF were decreased(31.21±3.21,53.29±2.41)&(51.70±4.4,92.77±2.28)(P<0.05);8 weeks postoperative echocardiography showed that LVEDV and LVESV level were increased in HF+CSWT group compared with HF group(10.12±0.33,7.85±0.35)&(8.05±0.46,5.97±0.43),(P<0.05),LVEF,FS were significantly higher than HF group(35.36±2.38,58.13±2.91)&(24.31±2.39,40.52±5.22)(P<0.05);Compared with HF,the LVEDV,LVESV(8.05±0.46,5.97±0.43)in the+CSWT group,LVEDV and LVESV in the HF+CSWT+LY294002 group were expanded(8.29±0.35,6.01±0.35)(P<0.05).While the LVEF and FS(35.36±2.38,58.13±2.91)in the HF+CSWT group were higher than those in the HF+CSWT+LY294002 group(33.55±1.65,50.35±2.71)(P<0.05);Compared with HF+CSWT group(8.05±0.46,5.97±0.43),LVEDV and LVESV(7.34±0.21,5.04±0.27)in CSWT±PPD98059 group were significantly lower than those in HF+CSWT+PD98059 group(40.07±3.21,59.67±4.17)compared with HF+CSWT group(35.36±2.38,58.13±2.91),(P<0.05);compared with HF+CSWT+LY294002 group(8.29±0.35,6.01±0.35)and HF+The LVEDV and LVESV values of the CSWT+PD98059 group(7.34±0.21,5.04±0.27),the HF+CSWT+LY294002+PD98059 group(9.54±0.20,7.22±0.21)increased significantly,while the LVEF,FS values,HF+CSWT+LY294002 The group(33.55±1.65,50.35±2.71),HF+CSWT+PD98059 group(40.07±3.21,59.67±4.17)was significantly higher than the HF+CSWT+LY294002+PD98059 group(28.23±2.76,41.87±3.00),(P<0.05).4.Serum NT-proBNP levels were measured at three point,before and after 4 weeks?8 weeks after surgery.Compared with the preoperative control group(9.058 ± 0.4).The NT-proBNP level in SD rats was significantly increased after operation 4 weeks.(27.86 ± 0.70)(P<0.05).At 8 weeks postoperatively,serum NT-proBNP level were significantly lower than that of HF+CSWT group(18.69±1,16)compared with the HF group(34.29±2.07),HF+LY294002 group(51.48±1.41),HF+PD98059 group(42.36±1.91)NT-proBNP level was significantlyincreased(P<0.05),compared with HF+CSWT group(18.69±1.16),HF+CSWT+LY294002 group(31.88±0.74),HF+CSWT+PD98059 group(23.64±)1.59)NT-proBNP level were elevated(P<0.05);NT-proBNP values in the HF+LY294002 group(51.48±1.41)were lower than that of the HF+PD98059 group(42.36±1.91),(P<0.05),and HF+Compared with LY294002 group(51.48 ± 1.41)and HF+PD98059 group(42.36±1.91),NT-proBNP level(63.65±3.19)was significantly increased in HF+LY294002+PD98059 group(P<0.05),HF+LY294002+PD98059 Compared with the group(63.65±3.19),the level of NT-proBNP was significantly lower than in the HF+CSWT+LY29002+PD98059 group(35.54±1.13),(P<0.05),and HF+CSWT+LY294002 group(31.88±0.74),HF+Compared with CSWT+PD98059 group(23.64±1.59),serum NT-proBNP levels were significantly higher in HF+CSWT+LY29002+PD98059 group(35.54±1.13)(P<0.05).5.The expression level of CD34 and aSMA mRNA were detected by RT-PCR.RT-PCR.The results showed that the expression level of fibrosis-related factors CD34 and aSMA were significantly increased in the HF group compared with the control group,HF+CSWT.The level of CD34 and aSMA in the group(1.24±0.08,1.19±0.01)were significantly lower than those in the HF group(1.71±0.06,2.59±0.16),while the HF+LY294002 group(2.79±0.10,3.67±0.14)and HF+The levels of CD34 and aSMA in PD98059 group(1.90±0.14,3.05±0.16)were significantly higher than those in HF group,and the levels of CD34 and aSMA in HF+LY294002 group(2.79±0.10,3.67±0.14)were lower than HF+.PD98059 group(1.90±0.14,3.05±0.16),(P<0.05),compared with HF+LY294002 group(2.79±0.10,3.67±0.14)and HF+PD98059 group(1.90±0.14,3.05±0.16)CD34,aSMA levels,HF+LY294002+PD98059 group(3.51±0.03,4.57±0.047)were significantly increased(P<0.05);with HF+LY294002 group(2.79±0.10,3.67±0.14)and HF+PD98059 group Compared with CD34 and aSMA levels(1.90±0.14,3.05±0.16),HF+CSWT+LY294002 group(1.53±0.12,1.75=0.10),HF+CSWT+PD98059 group(0.86±0.05)The expression level of 0.05 and 1.49±0.01 was significantly decreased.Compared with HF+LY294002+PD98059 group(3.51±0.03,4.57±0.047),HF+CSWT+LY29002+PD98059 group(2.43±0.03,1.94±0.04)CD34,aSMA The mRNA expression level was significantly decreased(P<0.05),compared with HF+CSWT+LY294002 group(1.53±0.12,1.75±0.10),HF+CSWT+PD98059 group(0.86±0.05,1.49±0.01),HF+The mRNA expression levels of CD34 and aSMA in CSWT+LY29002+PD98059 group(2.43±0.03,1.94±0.04)were significantly increased(P<0.05).6.Immunohistochemical stainingwas used to study changes in protein expression.Level.Compared with the control group(6.76±0.58,5.07±0.30),CD34 and aSMA(17.13±1.47,13.90±0.85)in the HF group increased significantly compared with the control group.The expression levels of p-AKT and P-ERK1/2(1.13±0.03,1.35±0.26)were significantly decreased in HF group(0.28±0.04,0.29±0.06)(P<0.05);and HF group(17.13±1.47).Compared with 13.90±0.85),the expression levels of CD34 and aSMA(10.25±0.48,5.78±0.51)in HF+CSWT group were significantly decreased,while the expression levels of p-AKT and P-ERK1/2 protein in HF group(0.28±0.04,0.29).±0.06)was significantly lower than HF+CSWT group(0.89±0.08,0.78±0.11),(P<0.05),compared with CD34 and aSMA values of HF+CSWT group(10.25±0.48,5.78±0.51),HF+CSWT+LY294002 group(17.79±0.26,9.92±1.49),HF+CSWT+PD98059 group(14.06±1.58,11.86±1.38)all increased significantly(P<0.05),while HF+CSWT+LY294002 group p-AKT protein The expression level(0.51±0.05)was significantly lower than that of HF+CSWT group(0.89±0.08),and the expression of ERK1/2 protein in HF+CSWT+PD98059 group was 0.30±0.04 compared with HF+CSWT group(0.78±0.11)also decreased significantly(P<0.05);and the CD34 and aSMA values of the HF+CSWT+LY294002 group(17.79±0.26,9.92±1.49)were higher than those of the HF+CSWT+PD98059 group(14.0611.58).11.86±1.38);compared with HF+LY294002 group(23.62±0.85,25.22±1.44)and HF+PD98059 group(18.80±0.86,27.80±3.12),HF+LY294002+PD98059 group CD34,aSMA protein expression level(26.95±1.49,33.85±3.25)significantly increased(P<0.05),while the HF+LY294002 p-Akt level(0.13±0.02)was significantly higher than HF+LY294002+PD98059(0.08±0.02),HF The level of p-ERK1/2 in the PD98059 group(0.11±0.03)was higher than that in the HF+LY294002+PD98059 group(0.09±0.01);the expression level of CD34 and aSMA protein in the HF+LY294002+PD98059 group(26.95±1.49,Compared with 33.85±3.25),HF+CSWT+LY294002+PD98059 group(19.47±0.59,14.15±0.70)significantly decreased;while HF+LY294002+PD98059 group p-Akt and p-ERK1/2 expression level(0.08±0.02),0.09±0.01)is significantly lower than HF+CSWT+LY294002+PD98059 group(0.30±0.03,0.18±0.05),(P<0.05),CD34,aSMA protein expression level on HF+The CSWT+LY294002 group(17.79±0.26,9.92±1.49)and the HF+CSWT+PD98059 group(14.06±1.58,11.86±1.38)were significantly lower than the HF+CSWT+LY294002+PD98059 group(19.47±0.59,14.15±0.70).(P<0.05),but the expression levels of p-Akt(0.51±0.05)and HF+CSWT+PD98059 p-ERK1/2(0.30±0.04)in HF+CSWT+LY294002 group were higher than HF+CSWT.The expression levels of p-Akt(0.30±0.03)and p-ERK1/2(0.18±0.05)in the+LY294002+PD98059 group.(P<0.05).7.The degree of myocardial fibrosis was assessed by Masson staining:compared with the control group(0.07±0.05),the fibrosis level of the HF group(53.89±1.01)increased significantly(P<0.05),while the HF+CSWT group(23.63±0.54).Compared with the HF group(53.89±1.01),there was a significant decrease(P<0.05),and HF+CSWT+LY29002(32.25±3.03),HF+CSWT+PD98059(29.18±1.73)after the addition of the signaling pathway inhibitor.After 1.73),the fibrosis level was significantly higher than that of the HF+CSWT group(23.63±0.54),(P<0.05),and the degree of fibrosis in the HF+LY29002 group(57.63±3.19)and the HF+PD98059 group(63.99±2.00).Compared with the HF group(53.89±1.01),the level increased significantly(P<0.05),and the degree of fibrosis in the HF+LY29002+PD98059 group(69.66±4.72)compared with the HF+LY29002 group(57.63±3.19)and the HF+PD98059 group(63.99±2.00),also significantly increased(P<0.05),the degree of fibrosis in the HF+CSWT+LY294002+PD98059 group(45.09±2.12)compared to the HF+CSWT+LY294002 group(32.25±3.03)and HF+CSWT+PD98059 group(29.18±1.73),there was a significant increase(P<0.05);8.Detection of aSMA/CD34 and Procollage-I/CD34 expression by immunofluorescence double labeling:in the aSMA/CD34 level,compared with the control group(0.75±0.24,1.07±0.07),HF group(7.14±0.69,13.44±)The expression level of 1.21)was significantly increased(P<0.05).Compared with the HF group(7.14±0.69,13.44±1.21),the level of HF+CSWT(5.04±0.77,5.48±0.48)was significantly decreased.The HF+LY294002 group(24.21±1.47,16.37±2.13)showed a significant increase trend(P<0.05),and a double inhibitor was added compared with the HF+PD98059 group(8.23±0.45,15.55±0.62).The expression levels of CD34 and aSMA in the HF+LY294002+PD98059 group(35.31±1.59,19.10±1.64)were significantly increased(P<0.05),compared with HF+LY294002+PD98059(35.31±1.59,19.10±1.64).The expression levels of CD34 and aSMA in HF+CSWT+LY294002+PD98059 group(30.91 ± 1.98,9.16±1.47)after using extracorporeal shock wave treatment were significantly decreased(P<0.05),compared with HF+CSWT group(5.04±0.77,5.4810.48),HF+CSWT+LY294002 group(19.67±1.19,9.31±0.88)and HF+CSWT+PD98059 group(5.97±0.47,12.37±1.05)The expression level was significantly lower(P<0.05),and HF+CSWT with two inhibitors added to the HF+CSWT+LY294002 group(19.67±1.19)and the HF+CSWT+PD98059 group(5.97±0.47).The expression level of aSMA(30.91±1.98)in+LY294002+PD98059 group was significantly increased(P<0.05).In Procollage-I/CD34,compared with the control group(1.48±0.03,0.54±0.07),The expression level of HF group(3.45±1.02,1.93±0.47)was significantly increased(P<0.05),compared with HF group(3.45±1.02,1.93±0.47),HF+CSWT(0.41±0.16,1.43).The level of ±0.15)decreased significantly,while the HF+LY294002 group(37.81±4.99,15.01±3.21)showed a significant increase(P<0.05),compared with HF+LY294002+PD98059(39.65±).7.54,19.05±1.64),the expression levels of CD34 and aSMA in HF+CSWT+LY294002+PD98059 group(19.08±2.13,15.09±1.23)after using extracorporeal shock wave treatment were significantly decreased(P<0.05),compared with HF+.The expression levels of CSWT group(0.41±0.16,1.43±0.15)and HF+CSWT+LY294002 group(6.26±1.99,13.33±1.53)were significantly lower(P<0.05),compared with the HF+CSWT+LY294002 group(6.26±1.99),the expression level of Procollage-I(19.08±2.13)was significantly increased in the HF+CSWT+LY294002+PD98059 group with two inhibitors.(P<0.05),compared with HF+CSWT+PD98059 group(7.03±1.13,1.43±0.15),the expression level of HF+CSWT+LY294002+PD98059 group(19.08 ± 2.13,15.09±1.23)was significantly increased.(P<0.05).3.Conclusion:1.Surgical open thoracic high ligation of the left anterior descending coronary artery can establish a rat model of heart failure with good parallelism,which can provide an ideal animal model for the cardiovascular field.2.Compared with control group,shock wave treatment will emproved left ventricular ejection fraction,NT-proBNP levels,and expression level of CD34 and aSMA.and the degree of fibrosis,while the expression level of p-Akt and p-ERK were increased significantly.The above data indicate that CSWT has significant effects in the treatment of myocardial infarction,which can inhibit ventricular remodeling and reduce the degree of myocardial fibrosis,thereby improving heart failure.After rat heart function,improve prognosis.3.CSWT improves myocardial fibrosis by activating PI3K/Akt and ERK1/2 signaling pathways to protect heart function and improve heart failure,and PI3K/Akt and ERK1/2 signaling pathways have synergistic effects in the treatment of ischemic heart failure with CSWT,and The PI3K/Akt signaling pathway plays a better role in the treatment of IHF in CSWT than the ERK1/2 signaling pathway. |
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