电针“夹脊”穴预处理对心肌缺血再灌注损伤大鼠细胞色素P450信号通路蛋白表达的影响
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摘要
目的:观察电针"夹脊"穴预处理对心肌缺血再灌注损伤(MIRI)大鼠的心肌保护作用及对细胞色素P450(CYP450)信号通路蛋白表达的影响。方法:Wistar大鼠随机分为假手术组、模型组、夹脊组、内关组、阳陵泉组、曲池组,每组10只。采用左冠状动脉结扎法复制MIRI大鼠模型。各电针预处理组电针相应穴位,每次30min,每日1次,连续针刺7d。HE染色法观察各组大鼠心肌组织病理形态,透射电镜下观察心肌细胞超微结构,Western blot法检测心肌CYP450相关分子CYP1A1、CYP2B2、CYP2C11、CYP4A2蛋白的表达水平。结果:模型组缺血区心肌纤维肿胀、紊乱,可见局灶性坏死区,心肌间质有大量炎性细胞浸润。夹脊组和内关组均可见心肌纤维轻度肿胀,散见部分坏死,间质出血,与模型组比较,炎性细胞浸润明显减轻。透射电镜下可见模型组肌原纤维排列紊乱,线粒体结构模糊,肿胀、空泡化。夹脊组和内关组心肌纤维结构较清晰,线粒体数量和结构无明显异常。与假手术组比较,模型组心肌CYP1A1、CYP2B2蛋白表达水平均显著增高(P<0.05)。与模型组比较,夹脊组、内关组、阳陵泉组心肌CYP1A1、CYP4A2蛋白表达水平均显著降低(P<0.01),夹脊组和内关组心肌CYP2B2、CYP2C11蛋白表达水平显著增高(P<0.01)。与夹脊组比较,曲池组心肌CYP1A1、CYP4A2蛋白表达水平显著升高(P<0.05),CYP2B2、CYP2C11蛋白表达水平显著降低(P<0.05)。结论:电针"夹脊"穴和"内关"穴预处理均能够上调CYP450表氧化酶蛋白表达水平,下调CYP450ω-羟化酶蛋白表达水平,起到心肌保护作用。
Objective To investigate the effect of"Jiaji"(EX-B2)-electroacupuncture(EA)preconditioning on structural changes of myocardium and expression of cytochrome P450(CYP450)enzymes in acute myocardial ischemia-reperfusion injury(MIRI)rats.Methods Sixty male Wistar rats were randomly divided into sham operation,model,EX-B2,Neiguan(PC6),Yanglingquan(GB34)and Quchi(LI11)groups(n=10 in each group).The MIRI model was established by occlusion of the descending anterior branch of the left coronary artery for 30 min,followed by reperfusion for 30 min.EA preconditioning(2 Hz/100 Hz,1 mA)was respectively applied to EX-B2,PC6,GB34,and LI11 for 30 min,once daily for 7 days.Morphological changes of myocardium were observed by H.E.staining.The ultrastructure of myocardial cells was observed by transmission electron microscope(TEM).The protein expression levels of myocardial CYP450(CYP1 A1,CYP2 B2,CYP2 C11 and CYP4 A2)were detected by Western blot.Results In MIRI rats,myocardial pathological changes as swollen,disorganized arrangement,and partially ruptured myocardial fibers with unclear limit and necrosis and infiltration of lots of inflammatory cells under microscope,and focal myofilament degeneration,dissolution and necrosis with interstitial edema,vague mitochondria with swelling and vacuolation,ruptured and disorganized arrangement of crests under TEM were found in the ischemic area,which was obviously milder in rats of both EX-B2 and PC6 groups.After modeling,the expression of myocardial CYP1 A1 and CYP2 B2 protein were significantly up-regulated in the model group relevant to the sham operation group(P<0.05).In EA preconditioning groups,the expression levels of CYP1 A1 and CYP4 A2 proteins in the EX-B2,PC6 and GB34 groups were obviously down-regulated(P<0.01),and those of CYP2 B2 and CYP2 C11 proteins in both EX-B2 and PC6 groups were markedly up-regulated relevant to the model group(P<0.01).Compared with the EX-B2 group,the expression levels of CYP1 A1,CYP4 A2 proteins in the LI11 group were significantly up-regulated(P < 0.05),and CYP2 B2,CYP2 C11 proteins were significantly down-regulated(P<0.05).No significant differences were found between the model and sham operation groups in the expression levels of myocardial CYP2 C11 and CYP4 A2 proteins,between the LI11 and model groups in the expression of CYP1 A1,CYP2 B2,CYP2 C11 and CYP4 A2,between the GB34 and model groups in the expression of CYP2 B2,CYP2 C11(all P>0.05).Conclusion EA preconditioning at EX-B2 and PC6 can suppress MIRI induced up-regulation of myocardial CYP450ω-hydroxylase CYP1 A1 and CYP4 A2 expression and further up-regulate the expression of myocardial epoxygenase CYP2 B2 and CYP2 C11 proteins in acute MIRI rats,thus playing a role in myocardial protection.
Objective To investigate the effect of"Jiaji"(EX-B2)-electroacupuncture(EA)preconditioning on structural changes of myocardium and expression of cytochrome P450(CYP450)enzymes in acute myocardial ischemia-reperfusion injury(MIRI)rats.Methods Sixty male Wistar rats were randomly divided into sham operation,model,EX-B2,Neiguan(PC6),Yanglingquan(GB34)and Quchi(LI11)groups(n=10 in each group).The MIRI model was established by occlusion of the descending anterior branch of the left coronary artery for 30 min,followed by reperfusion for 30 min.EA preconditioning(2 Hz/100 Hz,1 mA)was respectively applied to EX-B2,PC6,GB34,and LI11 for 30 min,once daily for 7 days.Morphological changes of myocardium were observed by H.E.staining.The ultrastructure of myocardial cells was observed by transmission electron microscope(TEM).The protein expression levels of myocardial CYP450(CYP1 A1,CYP2 B2,CYP2 C11 and CYP4 A2)were detected by Western blot.Results In MIRI rats,myocardial pathological changes as swollen,disorganized arrangement,and partially ruptured myocardial fibers with unclear limit and necrosis and infiltration of lots of inflammatory cells under microscope,and focal myofilament degeneration,dissolution and necrosis with interstitial edema,vague mitochondria with swelling and vacuolation,ruptured and disorganized arrangement of crests under TEM were found in the ischemic area,which was obviously milder in rats of both EX-B2 and PC6 groups.After modeling,the expression of myocardial CYP1 A1 and CYP2 B2 protein were significantly up-regulated in the model group relevant to the sham operation group(P<0.05).In EA preconditioning groups,the expression levels of CYP1 A1 and CYP4 A2 proteins in the EX-B2,PC6 and GB34 groups were obviously down-regulated(P<0.01),and those of CYP2 B2 and CYP2 C11 proteins in both EX-B2 and PC6 groups were markedly up-regulated relevant to the model group(P<0.01).Compared with the EX-B2 group,the expression levels of CYP1 A1,CYP4 A2 proteins in the LI11 group were significantly up-regulated(P < 0.05),and CYP2 B2,CYP2 C11 proteins were significantly down-regulated(P<0.05).No significant differences were found between the model and sham operation groups in the expression levels of myocardial CYP2 C11 and CYP4 A2 proteins,between the LI11 and model groups in the expression of CYP1 A1,CYP2 B2,CYP2 C11 and CYP4 A2,between the GB34 and model groups in the expression of CYP2 B2,CYP2 C11(all P>0.05).Conclusion EA preconditioning at EX-B2 and PC6 can suppress MIRI induced up-regulation of myocardial CYP450ω-hydroxylase CYP1 A1 and CYP4 A2 expression and further up-regulate the expression of myocardial epoxygenase CYP2 B2 and CYP2 C11 proteins in acute MIRI rats,thus playing a role in myocardial protection.
引文
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[3]李洋,邵明璐,于慧娟,等.电针“夹脊”穴预处理对心肌缺血再灌注大鼠细胞色素P450信号通路基因表达的影响[J].针刺研究,2017,42(1):25-30.
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[8]SEUBERT J,YANG B,BRADBURY J A,et al.Enhanced postischemic functional recovery in CYP2J2transgenic hearts involves mitochondrial ATP-sensitive K+channels and p42/p44 MAPK pathway[J].Circ Res,2004,95(5):506-514.
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[11]DHANASEKARAN A,GRUENLOH S K,BUONACCORSIJ N,et al.Multiple antiapoptotic targets of the PI3K/Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia[J].Am J Physiol Heart Circ Physiol,2008,294(2):724-735.
[12]ONI-ORISAN A,EDIN M L,LEE J A,et al.Cytochrome P450-derived epoxyeicosatrienoic acids and coronary artery disease in humans:a targeted metabolomics study[J].J Lipid Res,2016,57(1):109-119.
[13]GARCIA V,GILANI A,SHKOLNIK B,et al.20-HETEsignals through G protein-coupled receptor GPR75(Gq)to affect vascular function and trigger hypertension[J].Cir Res,2017,120(11):1776-1788.
[14]ISHIZUKA T,CHENG J,SINGH H,et al.20-hydroxyeicosatetraenoic acid stimulates nuclear factor-κB activation and the production of inflammatory cytokines in human endothelial cells[J].J Pharmacol Exp Ther,2008,324(1):103-110.
[15]BAO Y,WANG X,LI W,et al.20-hydroxyeicosatetraenoic acid induces apoptosis in neonatal rat cardiomyocytes through mitochondrial-dependent pathways[J].J Cardiovasc Pharmacol,2011,57(3):294-301.
[16]NUTT L K,PATAER A,PAHLER J,et al.Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+stores[J].J Biol Chem,2002,277(11):9219-9225.
[17]田岳凤,林亚平,严洁,等.电针内关穴对心肌缺血-再灌注损伤预防保护作用的实验研究(Ⅰ)-对CK、ET、MDA、NO、NOS等的影响[J].中国中西医结合急救杂志,2002,9(1):12-14.
[18]田岳凤,易受乡,严洁,等.电针内关穴对心肌缺血-再灌注损伤预防保护作用的实验研究(Ⅱ)-对超微结构及细胞凋亡的影响[J].中国中西医结合急救杂志,2002,9(1):15-18.
[19]林海波,严洁,常小荣,等.针灸内关穴预处理对兔缺血再灌注损伤心肌细胞死亡受体通路Fas/FasL蛋白表达的影响[J].中华中医药杂志,2013,28(5):1286-1290.
[20]王超,谢文娟,刘密,等.针灸预处理对不同时间心肌缺血再灌注损伤兔血浆内皮素、血清肌酸激酶和心肌组织热休克蛋白70表达的影响[J].针刺研究,2014,39(5):372-376.
[21]谭成富,王超,杜琳,等.电针、艾灸预处理对心肌缺血再灌注损伤大鼠心肌自噬相关蛋白LC 3、Beclin 1表达的影响[J].针刺研究,2018,43(1):1-7.
[22]白桦,卢圣锋,陈婉莹,等.麦粒灸预处理对大鼠心肌缺血再灌注损伤不同时期保护效应及Beclin 1表达的影响[J].针刺研究,2017,42(6):471-476.
[23]崔承斌,王京京,吴中朝.从背俞穴与夹脊穴的关系论背俞功能带[J].中国针灸,2005,25(7):483-486.
[2]REDINGTON K L,DISENHOUSE T,LI J,et al.Electroacupuncture reduces myocardial infarct size and improves postischemic recovery by invoking release of humoral,dialyzable,cardioprotective factors[J].J Physiol Sci,2013,63(3):219-223.
[3]李洋,邵明璐,于慧娟,等.电针“夹脊”穴预处理对心肌缺血再灌注大鼠细胞色素P450信号通路基因表达的影响[J].针刺研究,2017,42(1):25-30.
[4]李洋,邵明璐,杨琳,等.基因芯片筛选电针预处理心肌缺血再灌注大鼠细胞色素P450代谢通路相关差异表达基因[J].辽宁中医杂志,2017,44(4):691-694.
[5]KROETZ D L,ZELDIN D.Cytochrome P450 pathways of arachidonic acid metabolism[J].Curr Opin Lipidol,2002,13(3):273-283.
[6]袁成凌,姚建铭,余增亮.花生四烯酸及其代谢物的生物学作用[J].中国药物化学杂志,2000,10(1):75-78.
[7]ZELDIN D C.Epoxygenase pathways of arachidonic acid metabolism[J].J Biol Chem,2001,276(39):36059-36062.
[8]SEUBERT J,YANG B,BRADBURY J A,et al.Enhanced postischemic functional recovery in CYP2J2transgenic hearts involves mitochondrial ATP-sensitive K+channels and p42/p44 MAPK pathway[J].Circ Res,2004,95(5):506-514.
[9]NODE K,HUO Y,RUAN X,et al.Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids[J].Science,1999,285(5431):1276-1279.
[10]WANG D,HIRASE T,NITTO T,et al.Eicosapentaenoic acid increases cytochrome P450 2J2gene expression and epoxyeicosatrienoic acid production via peroxisome proliferator-activated receptor gamma in endothelial cells[J].J Cardiol,2009,54(3):368-374.
[11]DHANASEKARAN A,GRUENLOH S K,BUONACCORSIJ N,et al.Multiple antiapoptotic targets of the PI3K/Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia[J].Am J Physiol Heart Circ Physiol,2008,294(2):724-735.
[12]ONI-ORISAN A,EDIN M L,LEE J A,et al.Cytochrome P450-derived epoxyeicosatrienoic acids and coronary artery disease in humans:a targeted metabolomics study[J].J Lipid Res,2016,57(1):109-119.
[13]GARCIA V,GILANI A,SHKOLNIK B,et al.20-HETEsignals through G protein-coupled receptor GPR75(Gq)to affect vascular function and trigger hypertension[J].Cir Res,2017,120(11):1776-1788.
[14]ISHIZUKA T,CHENG J,SINGH H,et al.20-hydroxyeicosatetraenoic acid stimulates nuclear factor-κB activation and the production of inflammatory cytokines in human endothelial cells[J].J Pharmacol Exp Ther,2008,324(1):103-110.
[15]BAO Y,WANG X,LI W,et al.20-hydroxyeicosatetraenoic acid induces apoptosis in neonatal rat cardiomyocytes through mitochondrial-dependent pathways[J].J Cardiovasc Pharmacol,2011,57(3):294-301.
[16]NUTT L K,PATAER A,PAHLER J,et al.Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+stores[J].J Biol Chem,2002,277(11):9219-9225.
[17]田岳凤,林亚平,严洁,等.电针内关穴对心肌缺血-再灌注损伤预防保护作用的实验研究(Ⅰ)-对CK、ET、MDA、NO、NOS等的影响[J].中国中西医结合急救杂志,2002,9(1):12-14.
[18]田岳凤,易受乡,严洁,等.电针内关穴对心肌缺血-再灌注损伤预防保护作用的实验研究(Ⅱ)-对超微结构及细胞凋亡的影响[J].中国中西医结合急救杂志,2002,9(1):15-18.
[19]林海波,严洁,常小荣,等.针灸内关穴预处理对兔缺血再灌注损伤心肌细胞死亡受体通路Fas/FasL蛋白表达的影响[J].中华中医药杂志,2013,28(5):1286-1290.
[20]王超,谢文娟,刘密,等.针灸预处理对不同时间心肌缺血再灌注损伤兔血浆内皮素、血清肌酸激酶和心肌组织热休克蛋白70表达的影响[J].针刺研究,2014,39(5):372-376.
[21]谭成富,王超,杜琳,等.电针、艾灸预处理对心肌缺血再灌注损伤大鼠心肌自噬相关蛋白LC 3、Beclin 1表达的影响[J].针刺研究,2018,43(1):1-7.
[22]白桦,卢圣锋,陈婉莹,等.麦粒灸预处理对大鼠心肌缺血再灌注损伤不同时期保护效应及Beclin 1表达的影响[J].针刺研究,2017,42(6):471-476.
[23]崔承斌,王京京,吴中朝.从背俞穴与夹脊穴的关系论背俞功能带[J].中国针灸,2005,25(7):483-486.
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