基于Toll样受体2/核转录因子kappa B通路探讨电针对脑缺血损伤大鼠的作用机制
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摘要
目的:探讨电针是否通过影响Toll样受体2/核转录因子kappa B(TLR2/NF-κB)信号通路以减轻脑缺血再灌注损伤。方法:SD大鼠随机分为空白组、假手术组、模型组、电针组和电针+抑制剂组,每组24只。参照Longa改良线栓法制备大脑中动脉栓塞(MCAO)模型。电针组以电针刺激大鼠"水沟""内关""三阴交""委中"穴,每次30 min,1次/d,共28d;电针+抑制剂组以NF-κB抑制剂PDTC腹腔注射后(120mg/kg)再行电针刺激,1次/d,共28d。治疗第3、7、14、28天后,分别对各组大鼠进行神经行为学评分,实时荧光定量PCR法检测缺血半暗带区脑组织中TLR2、白细胞介素-1受体相关激酶(IRAK)、NF-κB mRNA的表达。结果:治疗后4个时间点,模型组、电针组及电针+抑制剂组神经行为学评分均呈逐渐下降趋势。与模型组比较,电针组在第3、7、28天时神经行为学评分明显降低(P<0.05),电针+抑制剂组在治疗后4个时间点神经行为学评分均明显降低(P<0.05)。与电针组比较,电针+抑制剂组在治疗后4个观察时间点的神经行为学评分均降低,但差异无统计学意义(P>0.05)。与空白组比较,模型组TLR2mRNA表达在4个观察时间点均明显升高(P<0.05),IRAK mRNA表达在第3、7天时升高(P<0.05),NF-κB mRNA表达在第3、7、14天时明显升高(P<0.05)。与模型组比较,电针组TLR2mRNA、NF-κB mRNA表达在第3、7、14天时明显下降(P<0.05),IRAK mRNA表达虽在第3天时下降(P<0.05),但在第14、28天时表达上升(P<0.05);电针+抑制剂组TLR2 mRNA、NF-κB mRNA表达在4个观察点均明显下调(P<0.05),IRAK mRNA表达在第3天时下降(P<0.05)。与电针组比较,电针+抑制剂组TLR2mRNA表达在第28天时下降(P<0.05),IRAK mRNA表达在第3、14、28天时明显下降(P<0.05),NF-κB mRNA表达在第3、7、14天时明显下降(P<0.05)。结论:电针刺激能改善缺血再灌注损伤大鼠神经缺损症状,延迟IRAK表达高峰,其机制可能与电针抑制TLR2/NF-κB信号通路活性有关。
Objective To observe the effect of electroacupuncture(EA)on neurological behavior and activity of Tolllike receptor 2/nuclear factor kappa B(TLR2/NF-κB)signaling of the ischemic cerebral area in cerebral ischemia-reperfusion injury(CIRI)rats,so as to explore its mechanisms underlying improvement of CIRI.Methods A total of 120 male SD rats were randomly divided into blank control,sham operation,model,EA and EA+NF-κB inhibitor(Pyrrolidine Dithiocarbamate Hydrochloride,PDTC,EA+PDTC)groups which were further divided into 3,7,14 and 28 dsubgroups(n=6 in each subgroup).The CIRI model was established by occlusion of the middle cerebral artery for 90 min,followed by reperfusion.EA(1-20 Hz,6 V)was applied to"Shuigou"(GV26),"Neiguan"(PC6),"Sanyinjiao"(SP6)and"Weizhong"(BL40)for 30 min,once a day for 28 days.For rats of the EA+PDTC group,PDTC solution(120 mg/kg)was intraperitoneally injected on the 3 rd day after successful modeling and before EA intervention.The neurological deficit severity(Zea Longa score)was assessed 3,7,14 and 28 days after modeling.The expression levels of TLR2,Interleukin-1 receptor-associated kinase(IRAK)and NF-κB mRNAs in the ischemic penumbra region of brain tissue were detected by real-time fluorescence quantitative PCR.Results Following modeling,the neurological deficit scores were significantly increased from the 3 rd day on after CIRI(P<0.05),the expression levels of TLR2 mRNA on day 3,7,14 and 28,and IRAK mRNA on day 3 and 7,as well as NF-κB mRNA on day 3,7 and 14 were significantly up-regulated in the model group relevant to the blank control group(P<0.05).After EA intervention,the neurological deficit scores were significantly decreased in the EA group on day 3,7 and 28 and in the EA+PDTC group on day 3,7,14 and 28 in comparison with those of the model group(P<0.05).In addition,the expression levels of TLR2 mRNA and NF-κB mRNA on day3,7 and 14 in the EA group,and on day 3,7,14 and 28 in the EA+PDTC group,IRAK mRNA on day 3 in the EA and EA+PDTC group were significantly down-regulated(P<0.05),but those of IRAK mRNA on day 14 and 28 in the EA group were significantly up-regulated in comparison with those of the model group(P<0.05).The effect of the EA+PDTC was obviously superior to that of simple EA in down-regulating the expression of TLR2(on day 28),and IRAK(on day 3,14,28),and NF-κB(on day 3,7 and14)(all P<0.05).Conclusion EA stimulation can improve the symptoms of neurological deficits in CIRI rats,which may be related to its effect in suppressing the expression of TLR2,NF-κB and IRAK mRNAs of the ischemic cerebral tissue,i.e.,down-regulating the activity of TLR2/NF-κB signaling.
Objective To observe the effect of electroacupuncture(EA)on neurological behavior and activity of Tolllike receptor 2/nuclear factor kappa B(TLR2/NF-κB)signaling of the ischemic cerebral area in cerebral ischemia-reperfusion injury(CIRI)rats,so as to explore its mechanisms underlying improvement of CIRI.Methods A total of 120 male SD rats were randomly divided into blank control,sham operation,model,EA and EA+NF-κB inhibitor(Pyrrolidine Dithiocarbamate Hydrochloride,PDTC,EA+PDTC)groups which were further divided into 3,7,14 and 28 dsubgroups(n=6 in each subgroup).The CIRI model was established by occlusion of the middle cerebral artery for 90 min,followed by reperfusion.EA(1-20 Hz,6 V)was applied to"Shuigou"(GV26),"Neiguan"(PC6),"Sanyinjiao"(SP6)and"Weizhong"(BL40)for 30 min,once a day for 28 days.For rats of the EA+PDTC group,PDTC solution(120 mg/kg)was intraperitoneally injected on the 3 rd day after successful modeling and before EA intervention.The neurological deficit severity(Zea Longa score)was assessed 3,7,14 and 28 days after modeling.The expression levels of TLR2,Interleukin-1 receptor-associated kinase(IRAK)and NF-κB mRNAs in the ischemic penumbra region of brain tissue were detected by real-time fluorescence quantitative PCR.Results Following modeling,the neurological deficit scores were significantly increased from the 3 rd day on after CIRI(P<0.05),the expression levels of TLR2 mRNA on day 3,7,14 and 28,and IRAK mRNA on day 3 and 7,as well as NF-κB mRNA on day 3,7 and 14 were significantly up-regulated in the model group relevant to the blank control group(P<0.05).After EA intervention,the neurological deficit scores were significantly decreased in the EA group on day 3,7 and 28 and in the EA+PDTC group on day 3,7,14 and 28 in comparison with those of the model group(P<0.05).In addition,the expression levels of TLR2 mRNA and NF-κB mRNA on day3,7 and 14 in the EA group,and on day 3,7,14 and 28 in the EA+PDTC group,IRAK mRNA on day 3 in the EA and EA+PDTC group were significantly down-regulated(P<0.05),but those of IRAK mRNA on day 14 and 28 in the EA group were significantly up-regulated in comparison with those of the model group(P<0.05).The effect of the EA+PDTC was obviously superior to that of simple EA in down-regulating the expression of TLR2(on day 28),and IRAK(on day 3,14,28),and NF-κB(on day 3,7 and14)(all P<0.05).Conclusion EA stimulation can improve the symptoms of neurological deficits in CIRI rats,which may be related to its effect in suppressing the expression of TLR2,NF-κB and IRAK mRNAs of the ischemic cerebral tissue,i.e.,down-regulating the activity of TLR2/NF-κB signaling.
引文
[1]CHELLUBOINA B,KLOP-FENSTEIN J D,GUJRATIM,et al.Temporal regulation of apoptotic and anti-apoptotic molecules after middle cerebral artery occlusion followed by reperfusion[J].Mol Neurobiol,2014,49(1):50-65.
[2]WANG Q,TANG X N,YE-NARI M A.The inflammatory response in stroke[J].JNeuroimmune,2007,184(1-2):53-68.
[3]王萍,张密霞,庄朋伟,等.脑缺血再灌注损伤的炎症反应机制研究进展[J].天津中医药大学学报,2014,33(5):317-320.
[4]ELTZSCHIG H K,ECKLE T.Ischemia and reperfusion:from mechanism to translation[J].Nat Med,2011,17(11):1391-1401.
[5]YANAGISAWA K,TAGO K,HAYAKAWA M,et al.Anovel splice variant of mouse interleukin-1-receptor-associated kinase-1(IRAK-1)activates nuclear factor-kappaB(NF-kappaB)and c-Jun N-terminal kinase(JNK)[J].Biochem J,2003,370(1):159-166.
[6]冯晓红,王秀云.早期针灸介入综合治疗急性缺血性脑卒中的进展[J].陕西中医学院学报,2013,36(4):124-127.
[7]孙晶,刘喆.针灸抗脑缺血再灌注后炎症反应实验研究进展[J].浙江中西医结合杂志,2011,21(8):591-593.
[8]李璐,东贵荣,周艳丽,等.Toll样受体2与针刺穴位信息启动传导机制的作用研究进展[J].上海针灸杂志,2017,36(4):498-503.
[9]HUANG Y S,MISIOR A,LIL W.Novel role and regulation of the interleukin-1receptor associated kinase(IRAK)family proteins[J].Cell Mol Immunol,2005,2(1):36-39.
[10]LONGA E Z,WEINSTEIN P R,CARISON S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[11]王建波,曲怡,张立德.关于大鼠针刺、固定方法的研究与探讨[J].针灸临床杂志,2015,31(8):73-74.
[12]余曙光,郭义.实验针灸学[M].上海:上海科学技术出版社,2009:150-151.
[13]PFEILSCHIFTER W,CZECH B,HOFFMANNB P,et al.Pyrrolidine dithiocarbamate activates p38 MAPK and protects brain endothelial cells from apoptosis:a mechanism for the protective effect in stroke[J].Neurochem Res,2010,35(9):1391-1401.
[14]WANG Y J,CUI L Y,JI X M,et al.The China national stroke registry for patients with acute cerebrovascular events:design,rationale,and baseline patient characteristics[J].Int J Stroke,2011,6(4):355-361.
[15]李桂平,石磊,杜元灏,等.脑梗死早期侧支循环重建及电针干预效应研究[J].天津中医药大学学报,2011,30(2):102-104.
[16]CHELLUBOINA B,KLOPFENSTEINJ D,GUJRATI M,et al.Temporal regulation of apoptotic and anti-apoptotic molecules after middle cerebral artery occlusion followed by reperfusion[J].Mol Neurobiol,2014,49(1):50-65.
[17]冯吉杰,王珂,陈彤宇,等.针刺调控炎性反应在抗缺血再灌注损伤中的研究进展[J].针刺研究,2017,42(6):552-556.
[18]张晨蕾,李永峰,惠建荣,等.针刺调控不同信号通路防治缺血性脑血管病的研究进展[J].针刺研究,2018,43(8):531-536.
[19]CAMPANELLA M,SCIORATI C,TAROZZO G,et al.Flow cytometric analysis of inflammatory cells in ischemic rat brain[J].Stroke,2002,33(2):586-592.
[20]孙琳琳.电针对脑缺血再灌注模型大鼠炎性反应相关信号的影响及机制研究[D].北京:北京中医药大学,2013.
[21]GESUETE R,KOHAMAS G,STENZEL-POORE M P.Toll-like receptors and ischemic brain injury[J].J Neuropathol Exp Neurol,2014,73(5):378-386.
[22]ZIEGLER G,FREYER D,HARHAUSEN D,et al.Blocking TLR2in vivo protects against accumulation of inflammatory cells and neuronal injury in experimental stroke[J].J Cereb Blood Flow Metab,2011,31(2):757-766.
[23]张超男,黄学宽,骆言,等.电针对急性痛风性关节炎大鼠踝关节滑膜组织TLR/MYD88信号通路的影响[J].四川大学学报(医学版),2014,45(6):924-927.
[24]秦文熠,罗勇,余超.电针对局灶性脑缺血再灌注大鼠海马内白介素-1β及转录核因子κB抑制蛋白激酶的影响[J].针刺研究,2013,38(4):271-276.
[25]孔立红,孙国杰,刘胜洪.针刺对脑缺血再灌注大鼠海马组织核转录因子-κB表达及含量的影响[J].针刺研究,2006,31(3):140-144.
[26]兰岚.基于TLR4/NF-κB信号通路探讨电针治疗脑缺血再灌注损伤的抗炎机制[D].福州:福建中医药大学,2013.
[27]孙鹏,张清,韩继媛,等.体外模拟脑缺血再灌注条件下BV-2细胞TLR4信号途径对TLR2表达的影响[J].中国科学(C辑:生命科学),2009,39(11):1013-1018.
[28]尤晓欣.Toll样受体信号通路与缺血性中风炎症反应机制的研究进展[J].国际神经病学神经外科学杂志,2014,41(2):138-141.
[29]杨云峰,陈志,胡胜利,等.IRAK1/4抑制对大鼠神经元缺血缺氧性损伤的保护作用[C]∥中国医师协会神经外科医师分会第六届全国代表大会论文汇编,2011:302.
[30]WANG C,LIUX X,HUANG K B,et al.Preconditioning with recombinant high-mobility group box 1induces ischemic tolerance in a rat model of focal cerebral ischemia-reperfusion[J].J Neurochem,2016,137(4):576-588.
[2]WANG Q,TANG X N,YE-NARI M A.The inflammatory response in stroke[J].JNeuroimmune,2007,184(1-2):53-68.
[3]王萍,张密霞,庄朋伟,等.脑缺血再灌注损伤的炎症反应机制研究进展[J].天津中医药大学学报,2014,33(5):317-320.
[4]ELTZSCHIG H K,ECKLE T.Ischemia and reperfusion:from mechanism to translation[J].Nat Med,2011,17(11):1391-1401.
[5]YANAGISAWA K,TAGO K,HAYAKAWA M,et al.Anovel splice variant of mouse interleukin-1-receptor-associated kinase-1(IRAK-1)activates nuclear factor-kappaB(NF-kappaB)and c-Jun N-terminal kinase(JNK)[J].Biochem J,2003,370(1):159-166.
[6]冯晓红,王秀云.早期针灸介入综合治疗急性缺血性脑卒中的进展[J].陕西中医学院学报,2013,36(4):124-127.
[7]孙晶,刘喆.针灸抗脑缺血再灌注后炎症反应实验研究进展[J].浙江中西医结合杂志,2011,21(8):591-593.
[8]李璐,东贵荣,周艳丽,等.Toll样受体2与针刺穴位信息启动传导机制的作用研究进展[J].上海针灸杂志,2017,36(4):498-503.
[9]HUANG Y S,MISIOR A,LIL W.Novel role and regulation of the interleukin-1receptor associated kinase(IRAK)family proteins[J].Cell Mol Immunol,2005,2(1):36-39.
[10]LONGA E Z,WEINSTEIN P R,CARISON S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[11]王建波,曲怡,张立德.关于大鼠针刺、固定方法的研究与探讨[J].针灸临床杂志,2015,31(8):73-74.
[12]余曙光,郭义.实验针灸学[M].上海:上海科学技术出版社,2009:150-151.
[13]PFEILSCHIFTER W,CZECH B,HOFFMANNB P,et al.Pyrrolidine dithiocarbamate activates p38 MAPK and protects brain endothelial cells from apoptosis:a mechanism for the protective effect in stroke[J].Neurochem Res,2010,35(9):1391-1401.
[14]WANG Y J,CUI L Y,JI X M,et al.The China national stroke registry for patients with acute cerebrovascular events:design,rationale,and baseline patient characteristics[J].Int J Stroke,2011,6(4):355-361.
[15]李桂平,石磊,杜元灏,等.脑梗死早期侧支循环重建及电针干预效应研究[J].天津中医药大学学报,2011,30(2):102-104.
[16]CHELLUBOINA B,KLOPFENSTEINJ D,GUJRATI M,et al.Temporal regulation of apoptotic and anti-apoptotic molecules after middle cerebral artery occlusion followed by reperfusion[J].Mol Neurobiol,2014,49(1):50-65.
[17]冯吉杰,王珂,陈彤宇,等.针刺调控炎性反应在抗缺血再灌注损伤中的研究进展[J].针刺研究,2017,42(6):552-556.
[18]张晨蕾,李永峰,惠建荣,等.针刺调控不同信号通路防治缺血性脑血管病的研究进展[J].针刺研究,2018,43(8):531-536.
[19]CAMPANELLA M,SCIORATI C,TAROZZO G,et al.Flow cytometric analysis of inflammatory cells in ischemic rat brain[J].Stroke,2002,33(2):586-592.
[20]孙琳琳.电针对脑缺血再灌注模型大鼠炎性反应相关信号的影响及机制研究[D].北京:北京中医药大学,2013.
[21]GESUETE R,KOHAMAS G,STENZEL-POORE M P.Toll-like receptors and ischemic brain injury[J].J Neuropathol Exp Neurol,2014,73(5):378-386.
[22]ZIEGLER G,FREYER D,HARHAUSEN D,et al.Blocking TLR2in vivo protects against accumulation of inflammatory cells and neuronal injury in experimental stroke[J].J Cereb Blood Flow Metab,2011,31(2):757-766.
[23]张超男,黄学宽,骆言,等.电针对急性痛风性关节炎大鼠踝关节滑膜组织TLR/MYD88信号通路的影响[J].四川大学学报(医学版),2014,45(6):924-927.
[24]秦文熠,罗勇,余超.电针对局灶性脑缺血再灌注大鼠海马内白介素-1β及转录核因子κB抑制蛋白激酶的影响[J].针刺研究,2013,38(4):271-276.
[25]孔立红,孙国杰,刘胜洪.针刺对脑缺血再灌注大鼠海马组织核转录因子-κB表达及含量的影响[J].针刺研究,2006,31(3):140-144.
[26]兰岚.基于TLR4/NF-κB信号通路探讨电针治疗脑缺血再灌注损伤的抗炎机制[D].福州:福建中医药大学,2013.
[27]孙鹏,张清,韩继媛,等.体外模拟脑缺血再灌注条件下BV-2细胞TLR4信号途径对TLR2表达的影响[J].中国科学(C辑:生命科学),2009,39(11):1013-1018.
[28]尤晓欣.Toll样受体信号通路与缺血性中风炎症反应机制的研究进展[J].国际神经病学神经外科学杂志,2014,41(2):138-141.
[29]杨云峰,陈志,胡胜利,等.IRAK1/4抑制对大鼠神经元缺血缺氧性损伤的保护作用[C]∥中国医师协会神经外科医师分会第六届全国代表大会论文汇编,2011:302.
[30]WANG C,LIUX X,HUANG K B,et al.Preconditioning with recombinant high-mobility group box 1induces ischemic tolerance in a rat model of focal cerebral ischemia-reperfusion[J].J Neurochem,2016,137(4):576-588.