| Objectives:In this study,it was verified that CSWT pretreatment could inhibit the apoptosis of H9C2 cells induced by Anoxia/reoxygenation,and further explore whether CSWT could inhibit the apoptosis of H9C2 cells by activating PI3K/Akt and ERK1/2 signaling pathway,and at the same time reveals the above two signal pathways in CSWT inhibiting myocardial apoptosis have synergistic effect.and provides theoretical basis for future research on the protective effects of CSWT pretreatment ischemia-reperfusion injury,and lays a foundation for further research on the molecular mechanism of CSWT for ischemic heart disease(IHD).Methods:H9C2 cells derived from rat heart tissue from embryonic stage were selected as the research object.Divided into normal control group(control),Anoxia/Reoxygenation group(A/R)、CSWT+Anoxia/Reoxygenation group(CSWT+A/R)、LY294002+Anoxia/Reoxygenation group(LY294002+A/R)、PD98059+Ano-xia/Reoxygenation group(PD98059+A/R group)、PD98059+ LY294002 +Anoxia/Reoxygenation group(LY294002+PD98059 +A/R)、LY294002+CSWT+Anoxia/Re-oxygenation group(LY294002+CSWT+A/R),PD980 59+CSWT+Anoxia/Reoxygen-ation group(PD98059+CSWT+A/R),LY294002 +PD9 8059+CSWT+Anoxia/Re-oxygenation group(LY294002+PD98059+CSWT+A/R),LY294002 and PD98059 are PI3K/Akt and ERK1/2 signaling pathway inhibitors respectively.then were pretr-eatmented with CSWT(each specimen received 500 shots at 0.09mJ/mm2),using the simulation of ischemia/reperfusion solution to build the myocardial cell Anoxia/Reoxygenation model,and then using CCK8 and the flow cytometry to test the proliferation and apoptosis of the cardiac cells,tested the mRNA transcription of the bcl-2,Bax,Caspase-3 in each cell cultures by RT-PCR,The protein expression levels of bcl-2,Bax,caspase-3,P-AKT and P-ERK1/2 in cell culture were detected by western-blot.In order to further study whether the pretreatment of CSWT was regulated by the PI3K/Akt signaling pathway and ERK1/2 signaling pathway to regulate the apoptosis of H9C2 cells induced by hypoxia/reoxygenation,the cells were treated with LY294002 and PD98059,respectively blocking the above two pathways,and again detecting the proliferation and apoptosis of myocardial cells in each group,and the mRNA transcription of Bcl-2,Bax,Caspase-3 in each cell culture,and the protein expression levels of Bcl-2,Bax,Caspase-3,P-AKT and P-ERK1/2.Results:1、CCK8 method was used to detect the survival rate of H9C2 cells in each group:the cell survival rate of A/R group was lower than the Control group(73.40 ±5.23 vs 100.00±3.34,P<0.05).Compared with the cell survival rate of the A/R group(73.40±5.23),the survival rate of the LY294002+A/R group(49.22±3.63)、PD98059+A/R group(60.97± 4.48)was lower(P<0.05),while the survival rate of the A/R+CSWT group(96.88±6.55)was increased(P<0.05).Compared with the A/R+CSWT group(96.88±6.55),the survival rate of the Ly294002+A/R+CSWT group(67.01±4.71)、PD98059+A/R+CSWT group(77.29±1.55)decreased(P<0.05).Compared with the LY294002+A/R group(49.22±3.63),the cell survival rate of Ly294002+ PD98059+A/R group(41.06±1.42)decreased,while the cell survival rate of PD98059+A/R group(60.97±4.48)increased(P<0.05).Compared with PD98059+A/R group(60.97±4.48),the cell survival rate of Ly294002+PD98059+A/R group(41.06±1.42)decreased(P<0.05).Compared with Ly294002+PD98059+A/R+CSWT group(55.95±4.82),the cell survival rate of Ly294002+A/R+CSWT group(67.01±4.71)and PD98059+A/R+CSWT group(77.29±1.55)increased(P<0.05),while the cell survival rate of Ly294002+PD98059+A/R group(41.06±1.42)decreased(P<0.05).Compared with Ly294002+A/R+CSWT group,the cell survival rate of PD98059+A/R+CSWT group increased(77.29±1.55 vs 67.01 ±4.71,P<0.05).2.Flow cytometry was used to detect the apoptosis rate of H9C2 cells in each group:the apoptosis rate of A/R group was higher than Control group(6.29±0.21 vs 3.08±0.26,P<0.05).Compared with the A/R group(6.29±0.21),the apoptosis rate of LY294002+A/R group(9.54±0.25),PD98059+A/R group(7.95±0.39)was increased(P<0.05),while the apoptosis rate of A/R+CSWT group(3.83±0.46)was decreased(P<0.05).Compared with A/R+CSWT group(3.83±0.46),the apoptosis rate of Ly294002+A/R+CSWT group(5.83±0.29),PD98059+A/R+CSWT group(4.72±0.26)was increased(P<0.05).The apoptosis rate of Ly294002+PD98059+A/R group(11.7±1.31)was increased compared with LY294002+A/R group(9.54±0.25),while the apoptosis rate was decreased in PD98059+A/R group(7.95±0.39)(P<0.05).Compared with PD98059+A/R(7.95±0.39),the apoptosis rate of Ly294002+PD98059+A/R group(11.7 ±1.31)was increased(P<0.05).Compared with Ly294002+PD98059+A/R+CSWT(6.82±0.47),the apoptosis rate of Ly49002+A/R+CSWT group(5.83±0.29),PD98059+A/R+CSWT group(4.72±0.26)were decreased(P<0.05),while Ly294002+PD98059+A/R group(11 ±1.31)was increased(P<0.05).Compared with Ly294002+A/R+CSWT group,the apoptosis rate of PD98059+A/R+CSWT group was reduced(4.72±0.26 vs.5.83±0.29,P<0.05).3.RT-PCR was used to detect the mRNA transcription of Bcl-2,Bax and Casepase-3 in each group:Compared with the Control group,the mRNA transcription level of Bcl-2 in A/R group was decreased(0.40±0.02 vs 1±0.00,P<0.05),and the mRNA transcription level of Bax and Caspase-3 was increased(3.00±0.24 vs 1±0.00,1.89±0.16 vs 1± 0.00,P<0.05).Compared with A/R group,the mRNA transcription level of Bcl-2 in LY294002+ A/R group.PD98059+A/R group decreased(0.25±0.02 vs 0.40±0.02,0.30±0.01 vs 0.40±0.02,P<0.05),while the mRNA transcription level of Bax and Caspase-3 was opposite to that of Bcl-2 Compared with the A/R group.Compared with the A/R group,the transcription level of Bcl-2 mRNA in A/R+CSWT group was increased(0.85±0.01 vs 0.40±0.02,P<0.05),and the mRNA transcription level of Bax and Caspase-3 decreased(1.11±0.07 vs 3.00±0.24,1.07±0.09 vs 1.89±0.16,P<0.05).Compared with the A/R+CSWT group,mRNA transcription level of Bcl-2 in the Ly294002+A/R+CSWT group、PD98059 +A/R +CSWT group was reduced(0.53±0.08 vs 0.85±0.01,0.64±0.03 vs 0.85±0.01,P<0.05),and the mRNA transcription level of Bax increased(2.06 ±0.24 vs 1.11±0.07,1.47±0.15 vs 1.11±0.07,P<0.05),The mRNA transcription level of Caspase-3 in Ly294002+A/R+CSWT group was also increased(1.74±0.16 vs 1.07±0.09,P<0.05).Compared with LY294002+A/R group,the mRNA transcription level of Bcl-2 in Ly294002+PD98059+A/R group was decreased(0.16±0.02 vs 0.25±0.02,P<0.05),and the mRNA transcription level of Bax and Caspase-3 was increased(7.86±0.16 vs 5.40±0.05,5.71 ±0.25 vs 4.36±0.04,P<0,05).The mRNA transcription level of Bcl-2 in PD98059+A/R group was increased(0.30±0.01 vs 0.25±0.02,P<0.05),and the mRNA transcription level of Bax and Caspase-3 decreased(3.84±0.17 vs 5.40±0.05,3.061±0.26 vs 4.36±0.04,P<0.05).Compared with PD98059+A/R,the mRNA transcription level of Bcl-2 in Ly294002+PD98059+A/R group decreased(0.16±0.02 vs 0.301±0.01,P<0.05),while the mRNA transcription level of Bax and Caspase-3 was increased(7.86±0.16 vs 3.84±0.17,5.71±0.25 vs 3.06±0.26,P<0.05).Compared with Ly294002+PD98059+A/R+CSWT,the mRNA transcription level of Bcl-2 in Ly294002+A/R+CSWT group、PD98059+A/R+CSWT group was increased(0.53士0.08 vs 0.38±0.01,0.64±0.03 vs 0.38±0.01,P<0.05),while the mRNA transcription level of Bax and Caspase-3 was compared with that of Bcl-2 in the above group.In comparison with Ly294002+PD98059+A/R group and Ly294002+PD98059+A/R+CSWT,the mRNA transcription level of Bcl-2 was reduced(0.16±0.02 vs 0.38±0.01,P<0.05),while the mRNA transcription level of Bax and Caspase-3 was increased(7.86±0.16 vs 2.97±0.40,5.71±0.25 vs 2.26±0.18,P<0.05).PD98059 + A/R + CSWT compared with Ly294002 + A/R + CSWT,the transcription level of Bcl-2 mRNA increase(0.64±0.03 vs 0.53±0.08,P<0.05),Bax and Caspase-3 mRNA transcription level decreased(1.47±0.15 vs 2.06±0.24,1.20±0.16 vs 1.74±0.16,P<0.05).4.The expression of Bcl-2,Bax and Casepase-3 in each group was detected by Western-blot:Compared with the Control group,the relative expression of Bcl-2 protein in A/R group was significantly decreased(0.56±0.04 vs 1.54±0.09,P<0.05),and the relative expression of Bax and Caspase-3 increased(0.54±0.04 vs 0.08±0.01,0.16±0.04 vs 0.06±0.01,P<0.05).Compared with A/R group,the relative expression of Bcl-2 in LY294002+A/R group、PD98059+A/R group decreased(0.15±0.01 vs 0.56±0.04,0.36±0.06 vs 0.56±0.04,P<0.05),and the relative expression of Bax increased(0.87±0.02 vs 0.54±0.04,0.68±0.03 vs 0.54±0.04,P<0.05).In addition,the relative expression of Caspase-3 in LY294002+ A/R group was also increased(0.27±0.03 vs 0.16±0.04,P<0.05),and the relative expression of Bcl-2 in A/R+CSWT group increased(0.84±0.06 vs 0.56±0.04,P<0.05),while the relative expression of Bax and Caspase-3 decreased(0.11±0.01 vs 0.54±0.04,0.07±0.01 vs 0.16±0.04,P<0.05).Compared with the A/R+CSWT group,the relative expression of Bcl-2 was significantly reduced in the Ly294002+A/R+CSWT group(0.68±0.06 vs 0.84±0.06,P<0.05),The relative expression levels of Bax and Caspase-3 were increased(0.21±0.03 vs 0.11 ±0.01,0.14±0.02 vs 0.07±0.01,P<0.05),The relative expression of Bax in PD98059+A/R+CSWT group was significantly increased(0.16±0.01 vs 0.11 ±0.01,P<0.05).Compared with LY294002+A/R group,the relative expression of Bcl-2 of Ly294002+PD98059+A/R group decreased(0.06±0.01 vs 0.15±0.01,P<0.05),while the relative expression levels of Bax and Caspase-3 were increased(1.11±0.02 vs 0.87 ±0.02,0.54±0.09 vs 0.27±0.03,P<0.05).The relative expression of Bcl-2 in PD98059 +A/R group was increased(0.361±0.06 vs 0.15±0.01,P<0.05),while the relative expression of Bax and Caspase-3 decreased(0.68±0.03 vs 0.87±0.02,0.20±0.02 vs 0.27±0.03,P<0.05).Compared with PD98059+A/R group,the relative expression of Bcl-2 in Ly294002+PD98059+A/R group decreased(0.06±0.01 vs 0.36±0.06,P<0.05),and the relative expression of Bax and Caspase-3 increased(1.11±0.02 vs 0.68±0.03,0.54±0.09 vs 0.20±0.02,P<0.05).Compared with Ly294002+PD98059 +A/R+ CSWT,the relative expression of Bcl-2 in PD98059+A/R+CSWT group was increased(0.85±0.05 vs 0.47±0.06,P<0.05),and the relative expression of Bax and Caspase-3 decreased(0.16±0.01 vs 0.37±0.01,0.08±0.01 vs 0.17±0.03,P<0.05).The relative expression levels of Bcl-2 in Ly294002+A/R+CSWT group were increased(0.68±0.06 vs 0.47±0.06,P<0.05),and the relative expression of Bax decreased(0.21 ±0.03 vs 0.37±0.01,P<0.05),The relative expression of Bcl-2 in Ly294002+ PD98059+A/R group decreased(0.06±0.01 vs.0.47±0.06,P<0.05),and the relative expression of Bax and Caspase-3 increased(1.11±0.02 vs 0.37±0.01,0.54±0.09 vs 0.17±0.03,P<0,05).Compared with Ly294002+ A/R+CSWT,the relative expression of Bcl-2 in PD98059+A/R+CSWT was increased(0.85±0.05 vs 0.68±0.06,P<0.05),Bax and Caspase-3 decreased relative expression(0.16±0.01 vs 0.21 ±0.03,0.08±0.01 vs 0.14+0.02,P<0.05).5.The expression of p-AKT and p-ERK protein in each group was detected by Western-blot:Compared with the Control group,the relative expression of p-AKT and p-ERK protein in A/R group was significantly decreased(0.69±0.05 vs 0.98±1.05,0.2710.03.vs 0.46±0.04,P<0.05),while the expression of p-AKT and p-ERK protein was significantly increased in group A/R+CSWT group compared with group A/R(0.97±1.12 vs 0.69±0.05,0.38±0.06 vs 0.27±0.03,P<0.05).the relative expression of p-AKT protein in LY294002 +A/R+CSWT group was significantly decreased compared with that of A/R+CSW 0.72±0.82 vs 0.97±1.12,P<0.05).the relative expression of p-ERK protein in PD98059 +A/R+CSWT group was significantly decreased compared with A/R+CSW(0.25±0.01 vs 0.38±0.06,P<0.05).Conclusions:1.Anoxia/reoxygenation lead to H9C2 cells apoptosis,and inhibit cells express the Bcl-2 factor,promote cell Bax,Caspase 3 factor expression,suggesting that Anoxia/reoxygenation may mediate the occurrence of H9C2 cell apoptosis through mitochondrial pathway.2.PI3K/Akt and ERK1/2 signaling pathway have a protective effect on the H9C2 cells,inhibit the H9C2 cells apoptosis induced by hypoxia/reoxygenation.3.CSWT pretreatment can regulate the expression of downstream apoptosis related factor by activating the PI3K/Akt signaling pathway and ERK1/2 signaling pathway,inhibit the apoptosis of H9C2 cells induced by Anoxia/reoxygenation.4.The PI3K/Akt and ERK1/2 signal pathway have synergistic effects in the CSWT pretreatment of the apoptosis of H9C2 cells inhibited by the inhibition of anoxia/reoxygenation,and the PI3K/Akt signal pathway is dominant. |
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