促红细胞生成素通过AMPK-KLF2信号通路调节脑缺血后血管新生的分子机制
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摘要
目的:探讨促红细胞生成素(EPO)通过AMPK-KLF2信号通路调大鼠脑缺血后血管新生的分子机制。方法:雄性SD大鼠92只,16只用于假手术组,剩余76只构建大脑中动脉栓塞(MCAo)模型;将造模成功的64只随机均分为脑缺血组、Compound C组、EPO组及EPO+Compound C组,建立大脑中动脉栓塞模型后及干预7 d时,镜下观察顶叶缺血周围区新生血管并计数全部新生血管数目,采用RT-PCR检测Krueppel样因子2(KLF2)、内皮一氧化氮合酶(e NOS)、血栓调节蛋白(TM)及血管内皮生长因子(VEGF) mRNA的转录水平,采用免疫印迹检测脑缺血区域的AMPK及KLF2蛋白表达。结果:干预7 d时,EPO组新生血管数量较脑缺血组增多,Compound C组和EPO+Compound C组新生血管数目少于脑缺血组,EPO+Compound C组新生血管数目多于Compound C组、少于脑缺血组(P<0.05);RT-PCR结果显示,干预7 d时,EPO组各mRNA的表达较脑缺血组升高,Compound C组各mRNA的表达量低于脑缺血组,EPO+Compound C组各mRNA的表达量高于Compound C组,差异有统计学意义(P<0.05);免疫组化检测发现,干预7 d时,Compound C组AMPK、KLF2表达量低于脑缺血组,EPO治疗组AMPK、KLF2蛋白的表达量与脑缺血组相比增加,差异有统计学意义(P<0.05)。结论:EPO可通过上调KLF2、eNOS、TM及VEGF mRNA的转录水平进而促进AMPK蛋白的表达从而发挥对脑缺血后血管新生的调控作用。
Objective:To explore the molecular mechanism of angiogenesis after cerebral ischemia by erythropoietin(EPO) through AMPK-KLF2 signaling pathway.Methods:A total of 92 male SD rats of clean grade for 24 weeks were selected and 16 were used in the sham operation group.The remaining 76 models were constructed for middle cerebral artery embolization(MCAo).11 models failed to be modeled,and 1 model was discarded.A total of 12 models were used for 64 follow-up experiments.The rats were randomly divided into 4 groups:The cerebral ischemia group,Compound C intervention group,EPO-treated group,the EPO + Compound C intervention group,with 16 rats in each group.After the establishment of middle cerebral artery embolization models,the number of neovascularization in parietal lobe was observed and counted under the microscope.Rot-pcr was used to test the mRNA transcription level of Krueppel-like factor 2(KLF2) endothelial nitric oxide(TM) Thrombomodulin(TM)vascular endothelial growth factor(VEGF).Western blot(WB) was used to detect AMPK and KLF2 protein expression in ischemic areas.Results:The number of new blood vessels in the EPO-treated group increased as compared with the cerebral ischemia group(P<0.05).The number of new blood vessels in the Compound C intervention group and the EPO + Compound C intervention group decreased as compared with the cerebral ischemia group(P<0.05).The number of new blood vessels in the EPO + Compound C intervention group increased as compared with the Compound C intervention group,which was lower than that in the cerebral ischemia group,but there was no significant difference(P>0.05).Then the RT-PCR experiments showed that the expression of mRNA in the EPOtreated group increased more than that in the cerebral ischemia group.The expression of mRNA in the Compound C intervention group decreased compared with the cerebral ischemia group(P<0.05).The expression of mRNA in the EPO + Compound C intervention group was higher than that in the Compound C intervention group,and was less than that in the cerebral ischemia group.There was no significant difference in the cerebral ischemic group(P>0.05).After that the immunohistochemical detection revealed.the expression of AMPK,KLF2 in the Compound C intervention group was lower than that in the cerebral ischemia group(P<0.05).The expression of AMPK and KLF2 protein in the EPO-treated group was higher than that in the cerebral ischemia group(P<0.05).The expression of AMPK and KLF2 protein was higher in the EPO + Compound C group than in the Compound C group,which was lower than that in the cerebral ischemia group.Conclusion:EPO can upregulate the expression of KLF2,eNOS,TM,VEGF mRNA and promote the expression of AMPK protein to regulate angiogenesis after cerebral ischemia.
Objective:To explore the molecular mechanism of angiogenesis after cerebral ischemia by erythropoietin(EPO) through AMPK-KLF2 signaling pathway.Methods:A total of 92 male SD rats of clean grade for 24 weeks were selected and 16 were used in the sham operation group.The remaining 76 models were constructed for middle cerebral artery embolization(MCAo).11 models failed to be modeled,and 1 model was discarded.A total of 12 models were used for 64 follow-up experiments.The rats were randomly divided into 4 groups:The cerebral ischemia group,Compound C intervention group,EPO-treated group,the EPO + Compound C intervention group,with 16 rats in each group.After the establishment of middle cerebral artery embolization models,the number of neovascularization in parietal lobe was observed and counted under the microscope.Rot-pcr was used to test the mRNA transcription level of Krueppel-like factor 2(KLF2) endothelial nitric oxide(TM) Thrombomodulin(TM)vascular endothelial growth factor(VEGF).Western blot(WB) was used to detect AMPK and KLF2 protein expression in ischemic areas.Results:The number of new blood vessels in the EPO-treated group increased as compared with the cerebral ischemia group(P<0.05).The number of new blood vessels in the Compound C intervention group and the EPO + Compound C intervention group decreased as compared with the cerebral ischemia group(P<0.05).The number of new blood vessels in the EPO + Compound C intervention group increased as compared with the Compound C intervention group,which was lower than that in the cerebral ischemia group,but there was no significant difference(P>0.05).Then the RT-PCR experiments showed that the expression of mRNA in the EPOtreated group increased more than that in the cerebral ischemia group.The expression of mRNA in the Compound C intervention group decreased compared with the cerebral ischemia group(P<0.05).The expression of mRNA in the EPO + Compound C intervention group was higher than that in the Compound C intervention group,and was less than that in the cerebral ischemia group.There was no significant difference in the cerebral ischemic group(P>0.05).After that the immunohistochemical detection revealed.the expression of AMPK,KLF2 in the Compound C intervention group was lower than that in the cerebral ischemia group(P<0.05).The expression of AMPK and KLF2 protein in the EPO-treated group was higher than that in the cerebral ischemia group(P<0.05).The expression of AMPK and KLF2 protein was higher in the EPO + Compound C group than in the Compound C group,which was lower than that in the cerebral ischemia group.Conclusion:EPO can upregulate the expression of KLF2,eNOS,TM,VEGF mRNA and promote the expression of AMPK protein to regulate angiogenesis after cerebral ischemia.
引文
[1]杜同信,王自正,傅雷,立彦,张学忠.促红细胞生成素、叶酸、维生素B_(12)、铁蛋白检测在缺铁性贫血诊疗中的应用分析[J].标记免疫分析与临床,2008,(1):20-22.
[2]章锁,李国玲,张明轩,赵志平,刘钟明.慢性肾衰贫血患者尿促红细胞生成素浓度的变化及意义[J].河南医学研究,1992,(2):140-143.
[3]尹秀东.益气补血法联合EPO改善恶性肿瘤相关性贫血(气血亏虚型)的疗效观察[D].长沙:湖南中医药大学,2017.
[4]EIBEL B,MARKOSKI M M,RODRIGUES C G,et al.VEGF gene therapy cooperatively recruits molecules from the immune system and stimulates cell homing and angiogenesis in refractory angina[J]. Cytokine,2017,91(1):44-50.
[5]郭婷.过表达膜联蛋白A2受体对新生血管形成的作用研究[D].第二军医大学,2017.
[6]梁有峰,王爱玲.促红细胞生成素与心肌缺血性疾病的研究进展[J].心血管病学进展,2009,30(1):118-121.
[7]EPO衍生肽HBSP抑制大鼠心肌微血管内皮细胞缺血/再灌注损伤的作用研究[D].第四军医大学,2015.
[8] ZHANG Q,CHEN Z W,ZHAO Y H,ET A L. Bone marrow stromal cells combined with sodium ferulate and n-butylidenephthalide promote the effect of therapeutic angiogenesis via advancing astrocyte-derived trophic factors after ischemic stroke[J]. Cell Transplantation,2016,26(2):229-242.
[9]ZHAO G C,YUAN Y L,CHAI F R,et al. Effect of Melilotus officinalis extract on the apoptosis of brain tissues by altering cerebral thrombosis and inflammatory mediators in acute cerebral ischemia[J]. Biomed Pharmacother. 2017,89:1346-1352.
[10]HABIB P,STAMM A S,ZEYEN T,et al. EPO regulates neuroprotective Transmembrane BAX Inhibitor-1Motif-containing(TMBIM)family members GRINA and FAIM2 after cerebral ischemia-reperfusion injury[J].Exp Neurol,2019,15:112978.
[11]YAN F,CHAO J,YAN M,et al. Erythropoietin improves hypoxic-ischemic encephalopathy in neonatal rats after short-term anoxia by enhancing angiogenesis[J].Brain Research,2016,1651(1):104-113.
[12]LEIGH R,CHRISTENSEN S,CAMPBELL B C V,et al. Pretreatment blood-brain barrier disruption and postendovascular intracranial hemorrhage[J]. Neurology,2016,87(3):263.
[13]MERNTIE M,RISSANEN R,LOTTONEN-RAIKASLEHTO L,et al. Doxycycline modulates vegf-a expression:failure of doxycycline-inducible lentivirus shrna vector to knockdown vegf-a expression in transgenic mice[J].Plo S One,2018,13(1):e0190981.
[14]SHI Y X,YANG R F,BO Y,et al. Recombinant human erythropoietin promotes healing of incisions and anemia in the postoperated patients with gout[J]. Chinese Journal of New Drugs,2016,25(14):1617-1620.
[15]YAN Z,DENG Y,JIAO F,et al. Lipopolysaccharide downregulates kruppel-like factor 2(klf2)via inducing DNMT1-mediated hypermethylation in endothelial cells[J]. Inflammation,2017,25(1):1-10.
[16]NA L I,JUAN-JUAN H E,WANG Q,et al. Effect and mechanism of phosphorylated ampk on hypoxia preconditioning with its special inhibitor-compound c[J]. Journal of Sun Yat-sen University(Medical Sciences),2016,37(2):168-174.
[17]NEMECZ M,ALEXANDRU N,TANKO G,et al. Role of microrna in endothelial dysfunction and hypertension[J]. Current Hypertension Reports,2016,18(12):87.
[18]PURROY C,FAIRCHILD R L,TANAKA T,et al.Erythropoietin receptor-mediated molecular crosstalk promotes t cell immunoregulation and transplant survival[J]. Journal of the American Society of Nephrology Jasn,2017,28(8):2377-2392.
[19]WANG D,SONG Y,ZHANG J,et al. AMPK-KLF2 signaling pathway mediates the proangiogenic effect of erythropoietin in endothelial colony forming cells[J]. American Journal of Physiology:Cell Physiology,2017,313(6):674-685.
[20]REDDY M,VASIR J K,HEGDE G V,et al. Erythropoietin induces excessive neointima formation:a study in a rat carotid artery model of vascular injury[J]. J Cardiovasc Pharmacol Ther,2007,12(3):237-247.
[21]WANG H. Anti-VEGF therapy in the management of retinopathy of prematurity:what we learn from representative animal models of oxygen-induced retinopathy[J]. Eye&Brain,2016,8(1):81-90.
[22]KOLUSARI A,OKYAY A G,KOKAYA E A. The effect of erythropoietin in preventing ischemia-reperfusion injury in ovarian tissue transplantation[J]. Reproductive Sciences,2017,25(6):406-413.
[2]章锁,李国玲,张明轩,赵志平,刘钟明.慢性肾衰贫血患者尿促红细胞生成素浓度的变化及意义[J].河南医学研究,1992,(2):140-143.
[3]尹秀东.益气补血法联合EPO改善恶性肿瘤相关性贫血(气血亏虚型)的疗效观察[D].长沙:湖南中医药大学,2017.
[4]EIBEL B,MARKOSKI M M,RODRIGUES C G,et al.VEGF gene therapy cooperatively recruits molecules from the immune system and stimulates cell homing and angiogenesis in refractory angina[J]. Cytokine,2017,91(1):44-50.
[5]郭婷.过表达膜联蛋白A2受体对新生血管形成的作用研究[D].第二军医大学,2017.
[6]梁有峰,王爱玲.促红细胞生成素与心肌缺血性疾病的研究进展[J].心血管病学进展,2009,30(1):118-121.
[7]EPO衍生肽HBSP抑制大鼠心肌微血管内皮细胞缺血/再灌注损伤的作用研究[D].第四军医大学,2015.
[8] ZHANG Q,CHEN Z W,ZHAO Y H,ET A L. Bone marrow stromal cells combined with sodium ferulate and n-butylidenephthalide promote the effect of therapeutic angiogenesis via advancing astrocyte-derived trophic factors after ischemic stroke[J]. Cell Transplantation,2016,26(2):229-242.
[9]ZHAO G C,YUAN Y L,CHAI F R,et al. Effect of Melilotus officinalis extract on the apoptosis of brain tissues by altering cerebral thrombosis and inflammatory mediators in acute cerebral ischemia[J]. Biomed Pharmacother. 2017,89:1346-1352.
[10]HABIB P,STAMM A S,ZEYEN T,et al. EPO regulates neuroprotective Transmembrane BAX Inhibitor-1Motif-containing(TMBIM)family members GRINA and FAIM2 after cerebral ischemia-reperfusion injury[J].Exp Neurol,2019,15:112978.
[11]YAN F,CHAO J,YAN M,et al. Erythropoietin improves hypoxic-ischemic encephalopathy in neonatal rats after short-term anoxia by enhancing angiogenesis[J].Brain Research,2016,1651(1):104-113.
[12]LEIGH R,CHRISTENSEN S,CAMPBELL B C V,et al. Pretreatment blood-brain barrier disruption and postendovascular intracranial hemorrhage[J]. Neurology,2016,87(3):263.
[13]MERNTIE M,RISSANEN R,LOTTONEN-RAIKASLEHTO L,et al. Doxycycline modulates vegf-a expression:failure of doxycycline-inducible lentivirus shrna vector to knockdown vegf-a expression in transgenic mice[J].Plo S One,2018,13(1):e0190981.
[14]SHI Y X,YANG R F,BO Y,et al. Recombinant human erythropoietin promotes healing of incisions and anemia in the postoperated patients with gout[J]. Chinese Journal of New Drugs,2016,25(14):1617-1620.
[15]YAN Z,DENG Y,JIAO F,et al. Lipopolysaccharide downregulates kruppel-like factor 2(klf2)via inducing DNMT1-mediated hypermethylation in endothelial cells[J]. Inflammation,2017,25(1):1-10.
[16]NA L I,JUAN-JUAN H E,WANG Q,et al. Effect and mechanism of phosphorylated ampk on hypoxia preconditioning with its special inhibitor-compound c[J]. Journal of Sun Yat-sen University(Medical Sciences),2016,37(2):168-174.
[17]NEMECZ M,ALEXANDRU N,TANKO G,et al. Role of microrna in endothelial dysfunction and hypertension[J]. Current Hypertension Reports,2016,18(12):87.
[18]PURROY C,FAIRCHILD R L,TANAKA T,et al.Erythropoietin receptor-mediated molecular crosstalk promotes t cell immunoregulation and transplant survival[J]. Journal of the American Society of Nephrology Jasn,2017,28(8):2377-2392.
[19]WANG D,SONG Y,ZHANG J,et al. AMPK-KLF2 signaling pathway mediates the proangiogenic effect of erythropoietin in endothelial colony forming cells[J]. American Journal of Physiology:Cell Physiology,2017,313(6):674-685.
[20]REDDY M,VASIR J K,HEGDE G V,et al. Erythropoietin induces excessive neointima formation:a study in a rat carotid artery model of vascular injury[J]. J Cardiovasc Pharmacol Ther,2007,12(3):237-247.
[21]WANG H. Anti-VEGF therapy in the management of retinopathy of prematurity:what we learn from representative animal models of oxygen-induced retinopathy[J]. Eye&Brain,2016,8(1):81-90.
[22]KOLUSARI A,OKYAY A G,KOKAYA E A. The effect of erythropoietin in preventing ischemia-reperfusion injury in ovarian tissue transplantation[J]. Reproductive Sciences,2017,25(6):406-413.
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