Experimental Study Of The Mechanism Of Cardiac Shock Wave Therapy On Arteriogenesis Of Myocardium

Background:With high mortality and high morbidity,Coronary heart disease(CHD)has become a big challenge in the field of global public health,and existing routine therapies for CHD has many limitations.Extracorporal cardiac shock wave therapy(CSWT)is a noval noninvasive approach in the treatment of coronary heart disease(CHD),which can produce low-energy pulse waves.The low-energy pulsewaves can induce cavitation effect,which will exert a mechanical shear stress on the myocardial tissue and cells.Previous clinical studies of our group have verified the effectiveness and the safety of CSWT.According to the results from the previous studies of our group,the CSWT was able to promote angiogenesis in the ischemic myocardium of acute myocardial infarction pigs,and increase the expression of many growth factors related to angiogenesis in hman umbilical endothelial cells.But the molecular mechanism of the therapeutic effect of CSWT is still not clear.Since chinese traditional medcine is a effective strategy for CHD,whether the scutellarin(SCU)has positive cooperative effect to CSWT in improving arteriogenesis is worth discussing.Part ?:Experimental study of the effect of cardiac shock wave therapy on human cardiac microvascular endothelial cells and its signal conduction pathwayObjectives:to explore whether the CSWT can affect the expression of eNOS,VEGF,MCP-1 and ITGB1 in human cardiac microvascular endothelial cells,and to find whether integrin-focal adhesion kinase(FAK)pathway and calcium-sensitive potassium channel(KCa)were associated with the signaling pathways.We also want to explore whether the scutellarin(SCU)has positive cooperative effect to CSWT in improving arteriogenesis.Methods:1.HCMEC cells were recovered,and stable cell experimental platform was established.2.HCMEC cells were divided into the Control group,the CSWT group,the CSWT+Iberiotoxin group,and the CSWT+PF-573228 group,CSWT+SCUgroup.Each experimental group were added stimulant,and were cultured for 48h.,then recieved one low-energy extracorporeal shock wave therapy(0.09mJ/mm2,200Times).3.Being cultured for 24 hours after CSWT,the cells were collected and the sample was processed for analyzing the expression level of ITGB1,eNOS,VEGF,MCP1 by real-time quantitative PCR.Western blot assay was also performed to analyze the expression of ITGB1,eNOS,VEGF and MCP1.At the same time the expression of ITGB1,eNOS,VEGF and MCP1 was also analyzed by using immunohistochemistry assay.Results:1.In CSWT group,VEGF,eNOS,MCP-1 mRNA expression increased(4.61±0.05,3.99±0.01,1.57±0.09)compared with control group,but ITGB1 mRNA expression decreased(0.54 ±0.14)and the difference was statistically significant(P<0.05).VEGF?eNOS?MCP-1?ITGB1 protein expression increased(0.43 vs 0.35,0.63 vs 0.50,0.76 vs 0.54,0.68 vs 0.63).2.In CSWT+PF-57328 group,VEGF,eNOS,ITGB1 mRNA expression decreased(0.62±0.10,0.40±0.02,0.98±0.03)compared with CSWT group,but MCP-1 mRNA expression increased(17.1±0.01),and the difference was statistically significant(P<0.05).VEGF,eNOS protein expression decreased(0.29 vs 0.43,0.37 vs 0.63),but MCP-1,ITGB1 protein expression increased(1.05 vs 0.76,0.76 vs 0.63).3.In CSWT+Iberiotoxin group,VEGF,eNOS,ITGB1 mRNA expression decreased(0.53+1.02,0.64±0.02,0.35±0.01)compared with CSWT group,and the difference was statistically significant(P<0.05),and MCP-1 mRNA expression also decreased(0.82±0.02),but no statistic significance was found between this two groups(P>0.05).VEGF,eNOS,MCP-1,ITGB1 protein expression all decreased(0.30 vs 0.43,0.30 vs 0.63,0.48 vs 0.76,0.54 vs 0.63).4.In CSWT+SCU50um group,VEGF mRNA expression increased(5.98±0.05)compared with CSWT group,and the difference was statistically significant(P<0.05),but eNOS,MCP-I,ITGB1 mRNA expression decreased(0.29±0.02,1.39±0.03,0.39±0·.01,),but no statistic significance was found between this two groups(P>0.05).VEGF,eNOS protein expression increased(0.87 vs 0.43,0.65 vs 0.63),but MCP-1,ITGB1 protein expression decreased(0.33 vs 0.54,0.51 vs 0.63).5.In CS WT+SCU100um group,VEGF mRNA expression increased(5.29±0.31),compared with CSWT group,and the difference was statistically significant(P<0.05),but eNOS,MCP-1,ITGB1mRNA expression decreased(0.27±0.04,0.42±0.03,0.31 ±0.04),but no statistic significance was found between this two groups(P>0.05).VEGF,eNOS,MCP-1,ITGB1 protein expression all decreased(0.03 vs 0.35,0.13 vs 0.50,0.31 vs 0.54,0.18 vs 0.63)'.6.In CSWT+SCU200um group,VEGF mRNA expression increased(5.19±0.66)compared with CSWT group,and the difference was statistically significant(P<0.05),but eNOS,MCP-1,ITGB1 mRNA expression decreased(0.43±0.02,0.86±0.04,0.41 ±0.01),but no statistic significance was found between this two groups(P>0.05).VEGF,MCP-1,ITGB1 protein expression decreased(0.22 vs 0.35,0.38 vs 0.54,0.42 vs 0.63),but eNOS protein expression increased(0.87 vs 0.50).7.The expression of eNOS,ITGB,VEGF and MCP1 was detected by immunohistochemical testing,there were no statictic difference between each groups.Conclusions:1.CSWT can activate HCMEC,which will express eNOS,VEGF and MCP-1,and then facilitate the arteriogenesis.2.KCa and integrin-FAK pathway may participate in the signal conduction pathway of CSWT.KCa may act as the mechanoreceptor of HCMEC,and participate the singnal conduction pathway when CSWT increase the expression of MCP-1 in HCMEC.But the integrin-FAK pathway may not participate the process of activation of HCMEC by CSWT.Part II:Experimental study of the effect of cardiac shock wave therapy on acute myocardial infarction rats and its signal conduction pathwayObjectives:to explore whether the CSWT can affect the expression of acute myocardial infarction rats in the levels of eNOS,VEGF,MCP-1 and ITGB1,and to find whether adhesion kinase and calcium-sensitive potassium channel were associated with the signaling pathways.we also want to explore whether the scutellarin(SCU)has positive cooperative effect to CSWT in improving arteriogenesis in acute myocardial infarction rats.Methods:1.The SD rats' acute myocardial infarction models were built by ligation of the left anterior descending artery(LAD)in thoracotomy surgery.2.Three kind of different drugs were injected into rat model followed by the CSWT stimulation,and then the expression level of ITGB1,eNOS,VEGF,MCP1 by real-time quantitative PCR.Western blot assay was also performed to analyze the expression of ITGB1,eNOS,VECF and MCP1.3.SD rats were divided into the Control group,the CSWT group,the CSWT+Iberiotoxin group,and the CSWT+PF-573228 group,CSWT+SCUgroup.Each experimental group were added stimulant,and then recieved one low-energy extracorporeal shock wave therapy(0.09mJ/mm2,200Times).Results:1.VEGF,MCP-1 mRNA expression increaseed in CSWT group(1.62±0.26,1.18±0.06)compared with control group,eNOS mRNA expression decreaseed in CSWT group(0.96±0.03)compared with control group,but no statistic significance was found between this two groups(P>0.05).ITGB1 mRNA expression increaseed in CSWT group(2.58 ± 0.05),and the difference was statistically significant(P<0.001).VEGF,eNOS,ITGB1 protein expression decreased in CSWT group(1.66 vs 1.86,0.09 vs 0.37,0.50 vs 1.11),but MCP-1 protein expression increased in CSWT group(1.24 vs 0.30).2.VEGF,eNOS,ITGB1mRNA expression increaseed in CSWT+ PF-57328 group(2.09±0.06,2.50±0.07,2.28±0.02)compared with CSWT group,and MCP-1 mRNA expression decreaseed(0.71±0.04),but no statistic significance was found between this two groups(P>0.05).eNOS,MCP-1 protein expression increased in CSWT+PF-57328 group(0.70 vs 0.09,1.73 vs 1.24),butVEGF,ITGB1 protein expression decreased(0.11 vs 1.66,1.00 vs 1.11).3.In CSWT+ Iberiotoxin group,VEGF,MCP-1 mRNA expression decreaseed(1.58±0.11,0.449±0.02)compared with control group,and eNOS mRNA expression increased(1.17±0.02),but no statistic significance was found between this two groups(P>0.05).VEGF,ITGB1 protein expression decreased(0.77 vs 1.66,0.84 vs 1.11),but eNOS,MCP-1 protein expression increased in CSWT+PF-57328 group(0.27 vs 0.09,1.59 vs 0.24).4.In CSWT+ SCU10mg/kg group,VEGF,eNOS,MCP-1,ITGB1 mRNA expression increased(2.66±0.07,2.29±0.03,2.10±0.03,2.79±0.02)compared with CSWT group,but no statistic significance was found between this two groups(P>0.05).VEGF,eNOS protein expression increased(1.86 vs 1.66,0.37 vs 0.09),but MCP-1 protein expression decreased(0.30 vs 1.24),and there is no difference of the ITGB1 protein expression(0.30 vs 1.24)between this group and that of the control group.5.In CSWT+ SCU30mg/kg group,VEGF mRNA expression increased(3.03±0.13)compared with CSWT group,and eNOS,MCP-1 mRNA expression decreased(0.88±0.02,1.01 ±0.05),but no statistic significance was found between this two groups(P>0.05).ITGB1 mRNA expression increased(5.08 ±0.13),and the difference was statistically significant(P<0.001).VEGF,eNOS,MCP-1 protein expression increased(1.97 vs 1.66,0.87 vs 0.37,1.45 vs 1.24),ITGB1protein expression decreased(1.01 vs 1.11).6.In CSWT+ SCU90mg/kg group,VEGF,ITGB1mRNA expression increased(5.19±0.66,3.17±0.04)compared with CSWT group,and the difference was statistically significant(P<0.05),but eNOS,MCP-1 mRNA expression decreased(0.43±0.02,0.86±0.04),.VEGF,eNOS,MCP-1 protein expression increased(1.86 vs 1.66,0.37 vs 0.09),but MCP-1 protein expression decreased(2.02 vs 1.66,0.92 vs 0.09,1.70 vs 1.24),ITGBlprotein expression decreased(1.05 vs 1.11).Conclusions:1.CSWT can promote the expression of MCP-1 in acute myocardial infarction rats,but CSWT can not up-regulate the expression of eNOS and VEGF.2.It 's still unclear whether KCa and integrin-FAK are parts of the signal conduction pathway of CSWT4.The SCU has positive cooperative effect to CSWT in improving arteriogenesis in acute myocardial infarction rats.