针刺对局灶性脑缺血大鼠脑梗死灶周围皮质单羧酸转运体2表达的影响
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摘要
目的:观察针刺对局灶性脑缺血大鼠脑组织单羧酸转运体(MCT)2表达的影响,探讨针刺治疗缺血性脑血管病的作用机制。方法:Wistar大鼠随机分为正常组、假手术组、模型组和针刺组,每组20只。采用线栓法建立局灶性脑缺血大鼠模型。针刺组取"百会""风府""曲池""足三里","百会""风府"行平补平泻手法,"曲池""足三里"连接电针仪,20min/次,每天1次,共7d。采用Longa神经功能缺损评分评价大鼠神经功能缺损情况;酶化学法检测脑梗死灶周围皮质乳酸脱氢酶(LDH)、果糖-6-磷酸激酶(PFK)和丙酮酸激酶(PK)的活力;免疫荧光检测脑梗死灶周围皮质MCT2的表达和定位;实时荧光定量PCR和Western blot法检测脑梗死灶周围皮质MCT2mRNA和蛋白的相对表达量。结果:与正常组和假手术组比较,模型组大鼠神经功能缺损评分显著增高(P<0.01);脑梗死灶周围皮质LDH、PFK、PK的活力显著降低(P<0.01);MCT2阳性表达的荧光强度显著增加(P<0.01);MCT2蛋白的相对表达显著升高(P<0.01),MCT2mRNA的相对表达差异无统计学意义(P>0.05)。与模型组比较,针刺组大鼠神经功能缺损评分显著降低(P<0.01);LDH、PFK、PK的活力显著升高(P<0.01,P<0.05);MCT2阳性表达的荧光强度、MCT2蛋白和mRNA的相对表达量显著升高(P<0.01)。结论:针刺可上调MCT2在脑梗死灶周围皮质中的表达水平,进而改善大脑能量供应和代谢,促进神经功能修复。
Objective To observe the effect of manual acupuncture(MA)+ electroacupuncture(EA)on changes of neurological function and expression of monocarboxylate transporter 2(MCT2)in cerebral ischemia(CI)rats,so as to explore its mechanism underlying improvement of ischemic cerebrovascular disease.Methods Eighty male Wistar rats were equally randomized into four groups:normal control(normal),sham operation(sham),model and acupuncture.The CI model was induced by middle cerebral artery occlusion(MCAO)with a thread embolus.Manual acupuncture stimulation(mild twisting reinforcingreducing method)was applied to"Baihui"(GV20)and"Fengfu"(GV16)for 10 min.EA(1 mA,2 Hz/15 Hz)was respectively applied to bilateral"Quchi"(LI11)and"Zusanli"(ST36)for 20 min,once per day for 7 days.The neurological deficit severity was evaluated according to Zea Longa's methods.The activity of lactate dehydrogenase(LDH),fructose-6-phosphate kinase(PFK)and pyruvate kinase(PK)in the peri-ischemic cortex tissue was detected by enzymatic chemistry,and the expression of MCT2 detected by immunofluorescence histochemistry,Western blot and quantitative real-time PCR,separately.Results After CI and in comparison with the normal and sham groups,the Zea Longa's score,the fluorescence intensity and the expression level of MCT2 protein were significantly increased(P<0.01),and the activity of LDH,PFK and PK in the peri-ischemic cortex was significantly decreased in the model group(P<0.01).There was no significant change in the relative expression of MCT2 mRNA(P>0.05).Following the intervention and in comparison with the model group,the Zea Longa's score was considerably decreased in the acupuncture group(P<0.01),the activity of LDH,PFK and PK,and the expression levels of MCT2 protein and mRNA were considerably or further up-regulated in the acupuncture group(P<0.01,P<0.05).Conclusion Acupuncture intervention can improve neurological function in CI rats,which is possibly related with its effects in up-regulating the expression of MCT2 and promoting the utilization of lactate in peri-ischemic cortex.
Objective To observe the effect of manual acupuncture(MA)+ electroacupuncture(EA)on changes of neurological function and expression of monocarboxylate transporter 2(MCT2)in cerebral ischemia(CI)rats,so as to explore its mechanism underlying improvement of ischemic cerebrovascular disease.Methods Eighty male Wistar rats were equally randomized into four groups:normal control(normal),sham operation(sham),model and acupuncture.The CI model was induced by middle cerebral artery occlusion(MCAO)with a thread embolus.Manual acupuncture stimulation(mild twisting reinforcingreducing method)was applied to"Baihui"(GV20)and"Fengfu"(GV16)for 10 min.EA(1 mA,2 Hz/15 Hz)was respectively applied to bilateral"Quchi"(LI11)and"Zusanli"(ST36)for 20 min,once per day for 7 days.The neurological deficit severity was evaluated according to Zea Longa's methods.The activity of lactate dehydrogenase(LDH),fructose-6-phosphate kinase(PFK)and pyruvate kinase(PK)in the peri-ischemic cortex tissue was detected by enzymatic chemistry,and the expression of MCT2 detected by immunofluorescence histochemistry,Western blot and quantitative real-time PCR,separately.Results After CI and in comparison with the normal and sham groups,the Zea Longa's score,the fluorescence intensity and the expression level of MCT2 protein were significantly increased(P<0.01),and the activity of LDH,PFK and PK in the peri-ischemic cortex was significantly decreased in the model group(P<0.01).There was no significant change in the relative expression of MCT2 mRNA(P>0.05).Following the intervention and in comparison with the model group,the Zea Longa's score was considerably decreased in the acupuncture group(P<0.01),the activity of LDH,PFK and PK,and the expression levels of MCT2 protein and mRNA were considerably or further up-regulated in the acupuncture group(P<0.01,P<0.05).Conclusion Acupuncture intervention can improve neurological function in CI rats,which is possibly related with its effects in up-regulating the expression of MCT2 and promoting the utilization of lactate in peri-ischemic cortex.
引文
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[6]卢岩,赵海军,王媛,等.针刺对局灶性脑缺血大鼠血糖和胰岛素水平的影响[J].针刺研究,2013,38(6):435-440.
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[11]卢岩,赵海军,王媛,等.电针对局灶性脑缺血大鼠神经功能、脑血流及脑组织细胞色素P450表氧化酶基因表达的影响[J].针刺研究,2014,39(5):345-350.
[12]KIM W S,KIM I S,KIM S J,et al.Effect of electroacupuncture on motor recovery in a rat stroke model during the early recovery stage[J].Brain Res,2009,1248(13):176-183.
[13]GENC S,KURNAZ I A,OZILGEN M.Astrocyte-neuron lactate shuttle may boost more ATP supply to the neuron under hypoxic conditions:in silico study supported by in vitro expression data[J].BMC Syst Biol,2011,5:162.
[14]DALSGAARD M K,QUISTORFF B,DANIELSEN E R,et al.A reduced cerebral metabolic ratio in exercise reflects metabolism and not accumulation of lactate within the human brain[J].J Physiol(Lond),2004,554(Pt 2):571-578.
[15]CATER H L,CHANDRATHEVA A,BENHAM C D,et al.Lactate and glucose as energy substrates during,and after,oxygen deprivation in rat hippocampal acute and cultured slices[J].J Neurochem,2003,87(6):1381-1390.
[16]BLISS T M,IP M,CHENG E,et al.Dual-gene,dual-cell type therapy against an excitotoxic insult by bolstering neuroenergetics[J].J Neurosci,2004,24(27):6202-6208.
[17]OGAWA M,WATABE H,TERAMOTO N,et al.Understanding of cerebral energy metabolism by dynamic living brain slice imaging system with[18F]FDG[J].Neurosci Res,2005,52(4):357-361.
[18]MCKENNA M C,HOPKINS I B,CAREY A.Alpha-cyano-4-hydroxycinnamate decreases both glucose and lactate metabolism in neurons and astrocytes:implications for lactate as an energy substrate for neurons[J].J Neurosci Res,2001,66(5):747-754.
[19]WANIEWSKI R A,MARTIN D L.Astrocytes and synaptosomes transport and metabolize lactate and acetate differently[J].Neurochem Res,2004,29(1):209-217.
[20]BOUZIER-SORE A K,VOISIN P,CANIONI P,et al.Lactate is a preferential oxidative energy substrate over glucose for neurons in culture[J].J Cereb Blood Flow Metab,2003,23(11):1298-1306.
[21]VIJAY N,MORRIS M E.Role of monocarboxylate transporters in drug delivery to the brain[J].Curr Pharm Des,2014,20(10):1487-1498.
[22]KIRAT D,KATO S.Monocarboxylate transporter 1(MCT1)mediates transport of short-chain fatty acids in bovine caecum[J].Exp Physiol,2006,91(5):835-844.
[23]ERLICHMAN J S,HEWITT A,DAMON T L,et al.Inhibition of monocarboxylate transporter 2in the retrotrapezoid nucleus in rats:a test of the astrocyte-neuron lactate-shuttle hypothesis[J].J Neurosci,2008,28(19):4888-4896.
[24]HASUO H,AKASU T.Monocarboxylate transporters contribute to the adaptation of neuronal activity to repeated glucose deprivation in the rat lateral septal nucleus[J].Synapse,2003,49(2):97-105.
[2]WYSS M T,JOLIVET R,BUCK A,et al.In vivo evidence for lactate as a neuronal energy source[J].J Neurosci,2011,31(20):7477-7485.
[3]HERTZ L,DIENEL G A.Lactate transport and transporters:general principles and functional roles in brain cells[J].J Neurosci Res,2005,79(1-2):11-18.
[4]CORTES-CAMPOS C,ELIZONDO R,LLANOS P,et al.MCT expression and lactate influx/efflux in tanycytes involved in glia-neuron metabolic interaction[J].PLoS One,2011,6(1):e16411.
[5]LU Y,ZHAO H J,WANG Y,et al.Electro-acupuncture up-regulates astrocytic MCT1expression to improve neurological deficit in middle cerebral artery occlusion rats[J].Life Sci,2015,134:68-72.
[6]卢岩,赵海军,王媛,等.针刺对局灶性脑缺血大鼠血糖和胰岛素水平的影响[J].针刺研究,2013,38(6):435-440.
[7]LONGA E Z,WEINSTEIN P R,CARLSON S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[8]余曙光,徐斌.实验针灸学[M].2版.北京:人民卫生出版社,2016:287-289.
[9]殷颖,任硕,王世军.针康法对缺血性中风的研究现状分析[J].按摩与康复医学,2018,9(21):90-92.
[10]林忆诗,陈楚云,李丽霞.基于古籍数据挖掘分析中风后半身不遂针灸取穴规律研究[J].辽宁中医药大学学报,2018,20(4):151-153.
[11]卢岩,赵海军,王媛,等.电针对局灶性脑缺血大鼠神经功能、脑血流及脑组织细胞色素P450表氧化酶基因表达的影响[J].针刺研究,2014,39(5):345-350.
[12]KIM W S,KIM I S,KIM S J,et al.Effect of electroacupuncture on motor recovery in a rat stroke model during the early recovery stage[J].Brain Res,2009,1248(13):176-183.
[13]GENC S,KURNAZ I A,OZILGEN M.Astrocyte-neuron lactate shuttle may boost more ATP supply to the neuron under hypoxic conditions:in silico study supported by in vitro expression data[J].BMC Syst Biol,2011,5:162.
[14]DALSGAARD M K,QUISTORFF B,DANIELSEN E R,et al.A reduced cerebral metabolic ratio in exercise reflects metabolism and not accumulation of lactate within the human brain[J].J Physiol(Lond),2004,554(Pt 2):571-578.
[15]CATER H L,CHANDRATHEVA A,BENHAM C D,et al.Lactate and glucose as energy substrates during,and after,oxygen deprivation in rat hippocampal acute and cultured slices[J].J Neurochem,2003,87(6):1381-1390.
[16]BLISS T M,IP M,CHENG E,et al.Dual-gene,dual-cell type therapy against an excitotoxic insult by bolstering neuroenergetics[J].J Neurosci,2004,24(27):6202-6208.
[17]OGAWA M,WATABE H,TERAMOTO N,et al.Understanding of cerebral energy metabolism by dynamic living brain slice imaging system with[18F]FDG[J].Neurosci Res,2005,52(4):357-361.
[18]MCKENNA M C,HOPKINS I B,CAREY A.Alpha-cyano-4-hydroxycinnamate decreases both glucose and lactate metabolism in neurons and astrocytes:implications for lactate as an energy substrate for neurons[J].J Neurosci Res,2001,66(5):747-754.
[19]WANIEWSKI R A,MARTIN D L.Astrocytes and synaptosomes transport and metabolize lactate and acetate differently[J].Neurochem Res,2004,29(1):209-217.
[20]BOUZIER-SORE A K,VOISIN P,CANIONI P,et al.Lactate is a preferential oxidative energy substrate over glucose for neurons in culture[J].J Cereb Blood Flow Metab,2003,23(11):1298-1306.
[21]VIJAY N,MORRIS M E.Role of monocarboxylate transporters in drug delivery to the brain[J].Curr Pharm Des,2014,20(10):1487-1498.
[22]KIRAT D,KATO S.Monocarboxylate transporter 1(MCT1)mediates transport of short-chain fatty acids in bovine caecum[J].Exp Physiol,2006,91(5):835-844.
[23]ERLICHMAN J S,HEWITT A,DAMON T L,et al.Inhibition of monocarboxylate transporter 2in the retrotrapezoid nucleus in rats:a test of the astrocyte-neuron lactate-shuttle hypothesis[J].J Neurosci,2008,28(19):4888-4896.
[24]HASUO H,AKASU T.Monocarboxylate transporters contribute to the adaptation of neuronal activity to repeated glucose deprivation in the rat lateral septal nucleus[J].Synapse,2003,49(2):97-105.