EPO通过激活PI3K/Akt途径并上调HSP70的表达对低温下的心衰大鼠起保护作用
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摘要
目的：评价红细胞生成素(erythropoietin, EPO)对低温环境下心衰大鼠的影响并探讨其可能的作用机制。
方法：采用腹腔注射盐酸阿霉素(ADR)建立心衰大鼠模型,随机分为4组:①对照组(A组)8只；②心衰组(B组)20只：采用腹腔注射阿霉素制作大鼠心衰模型；③EPO组(C组)14只：造模后给予腹腔注射EPO,LY294002组(D组)18只：造模后尾静脉注射LY194002,30分钟后腹腔注射EPO后。观察10周后检测超声心动图的改变(?)并测定大鼠血清中SOD、MDA水平,Western blot测大鼠心肌组织中HSP70,Akt、pAkt的水平,RT-PCR测心肌组织中FasmRNA的表达、荧光定量PCR测心肌组织中PI3KmRNA的表达,Tunel法测心肌细胞凋亡。
结果：
(1)同对照组相比,阿霉素致心衰大鼠模型LVEDD:增大,EF下降(均p<0.01),符合慢性心衰模型的特征；
(2)心功能水平：心脏超声显示：和A组相比,B组LVIDd、LVIDs显著扩大,而C组明显低于B组,D组则介于B组和C组之间(P<0.01)；和A组相比,B组EF、FS值显著下降(p<0.01),而C组明显高于B组,D组则介于B组和C组之间。
(3)组织学特征：光镜下可见A组心肌细胞形态、胞质、间质及横(纵)纹均为正常,心衰组则存在心肌细胞横(纵)纹不清,部分区域肌质纤维溶解,胞浆空泡变性,不同程度的炎症细胞浸润和间质水肿病变表现。C组以上病变较B组有明显减轻,心肌细胞横(纵)纹存在；D组则介于心衰组和EPO组之间。
(4)氧化能力：与A组相比,B组SOD活力显著降低(p<0.01),MDA含量明显升高(p<0.01),而C组SOD活力显著高于B组(P<0.01),MDA含量明显低于A组(p<0.01), D组则介于B组和C组之间。
(5)蛋白免疫印迹(?)与A组相比,C组和D组大鼠心肌组织中pAkt、hsp70显著增加(p<0.01),B组与A组相较无明显变化,四组大鼠心肌组织中Akt的水平无显著差异。
(6)基因表达：与A组相比,B组Fas mRNA的表达明显增加(P<0.01),PI3KmRNA的表达下降(p<0.01),而C组Fas mRNA的表达较心衰组降低(p<0.01),PI3KmRNA的表达较B组明显增加(p<0.01),D组结果介于B、C组之间。
(7) TUNEL检测：与A组相比,B组TUNEL染色阳性细胞明显增多(p<0.01),C组TUNEL染色阳性细胞则显著低于B组(p<0.01), D组则介于B组和C组之间。结论：
1.阿霉素能够诱导血流动力学障碍、恶化心功能,且此作用可能与氧化应激有关。
2.低温会心衰大鼠的心功能进一步恶化并导致心肌细胞凋亡指数增加。PI3K/Akt信号通路是EPO发挥心肌保护作用的通路之一,其作用机制可能是EPO活化PI3K/Akt并上调内源性保护途径HSP70的表达,对低温下的心衰大鼠起心脏保护作用。
To investigated the effect of EPO on rats with heart failure in hypothermia and to explore Its possible mechanism
Method:Sprague-Dawley rats were administered adriamycin intraperitoneally injeetion All animals randomly divide into four groups:①control group (group A,n=10②heartfailure group (group B,n=16):Adriamycin was Intraperitoneally injected to induced HF model;③EPOgroup (group C,n=14):rats received erythropoietin(by Intraperitoneally injection) after operation,LY2940002 group(group D,n=18):rats received LY294002(by Tail vein injection) after operation and then received erythropoietin after thirty minutes. After 10 weeks of remedy,changs in echocardiography were observed, the oxidative stress including activities of superoxide dismutase (SOD) and malondialdehyde (MDA) concentrations were studied, the expression of HSP70,HSP27, pAkt and Akt in Myocardialwere tissues detected by western blot;Fas mRNA、PI3K mRNA were分别detected by reverse transcription and quantitative PCR and the apoptosis was assessed by using TUNEL method.
Results:
(1)Compared with normal group, LVIDd (P<0.01)was significantly increased and LVEF(P<0.01) reduced in adriamycin-induced cardiomyopathy rat models.(2)Cardiacfunction:①echocardiogrphy,compared with group A,group B LVIDd,LVIDs was significantly increased (P<0.01), and carvediloj group C decreased greatly (P<0.01);compared with group A, group B LVEF,FS was significantly decreased(p<0.01), while group C increased greatly(P<0.01), group D was between group B and group C.
(3)Cardiac histopathological characteristics:in group A the form of muscle cells, cytoplasm, interstitial and horizontal (vertical) lines were normal, and in group B horizontal (vertical) lines is unclear, some regional muscular fibers dissolved, cytoplasm vacuolation, inflammatory cells infiltration and the performance of interstitial edema lesions. compared with group B the lesions in group C were significantly reduced, myocardial cell horizontal (vertical) lines exist and crealy; group D was between group B and group C.
(4)Oxidative stress:Compared with group A, group B could significantly decrease the activity of SOD(p<0.01), and improve the contents of MDA greatly(p<0.01), while, in group C, the activity of SOD was enhanced significantly, (p<0.01), and the contents of MDA was decreased(p<0.05), group D was between group B and group C.
(5)Western blot:Compared with group A,the protein of pAkt、hsp70 expression were much higher in group C and D(p<0.01);but the expression of pAkt、hsp70 were no significant difference between group C and D,also between A and B;and the protein of Akt expression was no significant difference in all groups.
(6)Related genes:compared with group A,in group B,the mRNA of Fas expression were much higher(p<0.01), and the mRNA of PI3K expression were much lower than group A(p<0.01); while, in group C, the mRNA of Fas expression were much lower(p<0.01), and the mRNA of PI3K expression were much higher than group B(p<0.01), group D was between group B and group C.
(7)TUNEL:Compared with the group A, the positive cells in group B was increased significantly (p<0.01), the positive cells in grup C was significantly lower than group B(p<0.01), group D was between group B and group C.
Conclusion:
1.adriamycin can induced hemodynamic abnormality evidently, improve cardiac function, This mechanism may be concerned with its improvement of oxygen stress in cardiacmyocytes.
2. Cardiac function of rats with heart failure will worsen in hypothermia and the apoptosis index of Myocardial cells increased.
3.The pathway of PI3K/Akt may be one of the Cardioprotective ways of EPO.The possible mechanism is that EPO activates the PI3K/Akt pathway and upregulate the experssion of hsp70 (an endogenous Protective parthway) to show the cardioprotective in rats with heart failure in hypothermia.
方法：采用腹腔注射盐酸阿霉素(ADR)建立心衰大鼠模型,随机分为4组:①对照组(A组)8只；②心衰组(B组)20只：采用腹腔注射阿霉素制作大鼠心衰模型；③EPO组(C组)14只：造模后给予腹腔注射EPO,LY294002组(D组)18只：造模后尾静脉注射LY194002,30分钟后腹腔注射EPO后。观察10周后检测超声心动图的改变(?)并测定大鼠血清中SOD、MDA水平,Western blot测大鼠心肌组织中HSP70,Akt、pAkt的水平,RT-PCR测心肌组织中FasmRNA的表达、荧光定量PCR测心肌组织中PI3KmRNA的表达,Tunel法测心肌细胞凋亡。
结果：
(1)同对照组相比,阿霉素致心衰大鼠模型LVEDD:增大,EF下降(均p<0.01),符合慢性心衰模型的特征；
(2)心功能水平：心脏超声显示：和A组相比,B组LVIDd、LVIDs显著扩大,而C组明显低于B组,D组则介于B组和C组之间(P<0.01)；和A组相比,B组EF、FS值显著下降(p<0.01),而C组明显高于B组,D组则介于B组和C组之间。
(3)组织学特征：光镜下可见A组心肌细胞形态、胞质、间质及横(纵)纹均为正常,心衰组则存在心肌细胞横(纵)纹不清,部分区域肌质纤维溶解,胞浆空泡变性,不同程度的炎症细胞浸润和间质水肿病变表现。C组以上病变较B组有明显减轻,心肌细胞横(纵)纹存在；D组则介于心衰组和EPO组之间。
(4)氧化能力：与A组相比,B组SOD活力显著降低(p<0.01),MDA含量明显升高(p<0.01),而C组SOD活力显著高于B组(P<0.01),MDA含量明显低于A组(p<0.01), D组则介于B组和C组之间。
(5)蛋白免疫印迹(?)与A组相比,C组和D组大鼠心肌组织中pAkt、hsp70显著增加(p<0.01),B组与A组相较无明显变化,四组大鼠心肌组织中Akt的水平无显著差异。
(6)基因表达：与A组相比,B组Fas mRNA的表达明显增加(P<0.01),PI3KmRNA的表达下降(p<0.01),而C组Fas mRNA的表达较心衰组降低(p<0.01),PI3KmRNA的表达较B组明显增加(p<0.01),D组结果介于B、C组之间。
(7) TUNEL检测：与A组相比,B组TUNEL染色阳性细胞明显增多(p<0.01),C组TUNEL染色阳性细胞则显著低于B组(p<0.01), D组则介于B组和C组之间。结论：
1.阿霉素能够诱导血流动力学障碍、恶化心功能,且此作用可能与氧化应激有关。
2.低温会心衰大鼠的心功能进一步恶化并导致心肌细胞凋亡指数增加。PI3K/Akt信号通路是EPO发挥心肌保护作用的通路之一,其作用机制可能是EPO活化PI3K/Akt并上调内源性保护途径HSP70的表达,对低温下的心衰大鼠起心脏保护作用。
To investigated the effect of EPO on rats with heart failure in hypothermia and to explore Its possible mechanism
Method:Sprague-Dawley rats were administered adriamycin intraperitoneally injeetion All animals randomly divide into four groups:①control group (group A,n=10②heartfailure group (group B,n=16):Adriamycin was Intraperitoneally injected to induced HF model;③EPOgroup (group C,n=14):rats received erythropoietin(by Intraperitoneally injection) after operation,LY2940002 group(group D,n=18):rats received LY294002(by Tail vein injection) after operation and then received erythropoietin after thirty minutes. After 10 weeks of remedy,changs in echocardiography were observed, the oxidative stress including activities of superoxide dismutase (SOD) and malondialdehyde (MDA) concentrations were studied, the expression of HSP70,HSP27, pAkt and Akt in Myocardialwere tissues detected by western blot;Fas mRNA、PI3K mRNA were分别detected by reverse transcription and quantitative PCR and the apoptosis was assessed by using TUNEL method.
Results:
(1)Compared with normal group, LVIDd (P<0.01)was significantly increased and LVEF(P<0.01) reduced in adriamycin-induced cardiomyopathy rat models.(2)Cardiacfunction:①echocardiogrphy,compared with group A,group B LVIDd,LVIDs was significantly increased (P<0.01), and carvediloj group C decreased greatly (P<0.01);compared with group A, group B LVEF,FS was significantly decreased(p<0.01), while group C increased greatly(P<0.01), group D was between group B and group C.
(3)Cardiac histopathological characteristics:in group A the form of muscle cells, cytoplasm, interstitial and horizontal (vertical) lines were normal, and in group B horizontal (vertical) lines is unclear, some regional muscular fibers dissolved, cytoplasm vacuolation, inflammatory cells infiltration and the performance of interstitial edema lesions. compared with group B the lesions in group C were significantly reduced, myocardial cell horizontal (vertical) lines exist and crealy; group D was between group B and group C.
(4)Oxidative stress:Compared with group A, group B could significantly decrease the activity of SOD(p<0.01), and improve the contents of MDA greatly(p<0.01), while, in group C, the activity of SOD was enhanced significantly, (p<0.01), and the contents of MDA was decreased(p<0.05), group D was between group B and group C.
(5)Western blot:Compared with group A,the protein of pAkt、hsp70 expression were much higher in group C and D(p<0.01);but the expression of pAkt、hsp70 were no significant difference between group C and D,also between A and B;and the protein of Akt expression was no significant difference in all groups.
(6)Related genes:compared with group A,in group B,the mRNA of Fas expression were much higher(p<0.01), and the mRNA of PI3K expression were much lower than group A(p<0.01); while, in group C, the mRNA of Fas expression were much lower(p<0.01), and the mRNA of PI3K expression were much higher than group B(p<0.01), group D was between group B and group C.
(7)TUNEL:Compared with the group A, the positive cells in group B was increased significantly (p<0.01), the positive cells in grup C was significantly lower than group B(p<0.01), group D was between group B and group C.
Conclusion:
1.adriamycin can induced hemodynamic abnormality evidently, improve cardiac function, This mechanism may be concerned with its improvement of oxygen stress in cardiacmyocytes.
2. Cardiac function of rats with heart failure will worsen in hypothermia and the apoptosis index of Myocardial cells increased.
3.The pathway of PI3K/Akt may be one of the Cardioprotective ways of EPO.The possible mechanism is that EPO activates the PI3K/Akt pathway and upregulate the experssion of hsp70 (an endogenous Protective parthway) to show the cardioprotective in rats with heart failure in hypothermia.
引文
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[8]Liu L, Zhang XJ, Jiang SR, et al. Heat Shock Protein 27 Regulates Oxidative Stress-induced Apoptosis in Cardiomyocytes:Mechanisms via Reactive Oxygen Species Generation and Akt Activation[j]. Chin Med J(Engl),2007,120:(24):2271-2277.
[9]Iliodromitis EK, Karavclias GK, Botilis E, et al. Enhanced Protection of Heat Shock in Myocardial Infarction:Inhibition of Detrimental Effect of Systemic Hyperthermia[j].Cardiovasc Drugs Ther, 1999,13 (3):223-231.
[10]Baljinnyam E, Hasebe N, Morihira M, et al. Oral Pretreatment with Ebselen Enhances Heat Shock Protein 72 Expression and Reduces Myocardial Infarct Size[j]. Hypertens Res, 2006,29(11):905-913.
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[8]Liu L, Zhang XJ, Jiang SR, et al. Heat Shock Protein 27 Regulates Oxidative Stress-induced Apoptosis in Cardiomyocytes:Mechanisms via Reactive Oxygen Species Generation and Akt Activation[j]. Chin Med J(Engl),2007,120:(24):2271-2277.
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[22]Parra G, Quiroz Y, Salazar J, et al. Experimental Induction of Salt-Sensitive Hypertension is Associated with Lymphocyte Proliferative Response to HSP70[j]. Kidney Int Suppl, 2008,(111):S55-59.
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