夹脊电针对急性脊髓损伤大鼠脊髓组织微环境Rho-ROCK Ⅱ通路相关因子的影响
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摘要
目的:观察夹脊电针对脊髓损伤大鼠神经细胞轴突再生相关抑制因子重组人Ras同源物基因家族成员A(RhoA)、Rho蛋白激酶Ⅱ(ROCKⅡ)、肌球蛋白轻链(MLC)蛋白的影响,探讨夹脊电针治疗急性脊髓损伤(ASCI)的作用机制。方法:雌性Wistar大鼠随机分为假手术组、模型组、夹脊电针组、抑制剂组,每组12只。采用脊髓打击法制备大鼠ASCI模型。夹脊电针组于模型制备成功后3h进行夹脊电针治疗,每日1次,每次30 min,连续治疗14d、28d;抑制剂组于造模成功后立即给予腹腔注射盐酸法舒地尔(10mg/kg),每日1次,连续治疗14d、28d。采用BBB评分法评估大鼠后肢运动功能变化,免疫组化法检测各组大鼠脊髓组织中RhoA、ROCKⅡ、MLC蛋白的表达情况。结果:BBB评分结果显示:与假手术组比较,造模后14d、28d模型组大鼠运动功能评分显著降低(P<0.05);与模型组比较,造模后14d、28d夹脊电针组、抑制剂组大鼠运动功能评分均显著升高(P<0.05);夹脊电针组、抑制剂组造模后28d大鼠肢体运动功能评分较造模后14d显著升高(P<0.05)。免疫组化结果显示:与假手术组比较,造模后14d、28d模型组大鼠脊髓组织RhoA、ROCKⅡ、MLC阳性细胞数增加(P<0.05);与模型组相比,造模后14d、28d夹脊电针组、抑制剂组大鼠脊髓组织RhoA、ROCKⅡ、MLC阳性细胞数显著降低(P<0.05);夹脊电针组、抑制剂组造模后28d大鼠脊髓组织RhoA、ROCKⅡ、MLC阳性细胞数较造模后14d显著降低(P<0.05)。结论:夹脊电针能够显著改善ASCI大鼠肢体运动功能,其作用机制可能与抑制大鼠脊髓损伤组织微环境Rho-ROCKⅡ信号通路RhoA、ROCKⅡ、MLC抑制因子表达有关。
Objective To observe the effect of electroacupuncture(EA)of"Jiaji"(EX-B 2)on limb locomotor function and expression of Ras homolog gene family member A(RhoA),Rho-associated kinase Ⅱ(ROCK Ⅱ)and myosin light chain(MLC)proteins in the anterior horn of spinal cord in acute spinal cord injury(ASCI)rats,so as to explore its mechanisms underlying improvement of SCI-induced limb locomotor dysfunction.Methods Forty-eight female Wistar rats were randomly divided into sham operation(sham),ASCI model(model),EA EX-B 2(EA)and ROCK inhibitor(Fasudil)groups which were further divided into 14 dand 28 dsubgroups(n=6 in each).The ASCI model was made by using weight drop striking method.Three hours after modeling,EA(100 Hz,0.4,0.6 mA)was applied to EX-B 2(T 9,T 11)for 30 min,once daily for 14 dand 28 d,respectively.The ROCK inhibitor(hydrochloride Fasudil,10 mg/kg)was administrated by intraperitoneal injection immediately after modeling,once a day,continuously for 14 dor 28 d.The expression of RhoA,ROCK Ⅱ and MLC proteins in the spinal cord anterior horn tissue(T 10)was detected by immunohistochemistry.The rats' hindlimb locomotor function was assessed according to Basso,Beattie and Bresnahan(BBB)locomotor rating scale(21-points).Results After ASCI,the BBB scores were significantly lower in the model group than in the sham group on day 14 and 28(P<0.05),and obviously higher in the EA and inhibitor groups than in the model group(P<0.05),suggesting an improvement of the hindlimb locomotor function after EA intervention or suppression of ROCK.Immunohistochemical results indicated that the numbers of RhoA,ROCK Ⅱ and MLC immune-reaction positive cells in the anterior horn of spinal cord were significantly more in the model group than in the sham group(P<0.05),and remarkably decreased in both EA and inhibitor groups on day 14 and 28 relevant to the model group(P<0.05).The therapeutic effects of EA were markedly weaker than those of inhibitor Fasudil in up-regulating BBB score and down-regulating the number of RhoA,ROCKⅡ and MLC positive cells(P<0.05).Conclusion EA of EX-B 2 can improve the hindlimb locomotor function in ASCI rats,which may be associated with its effect in down-regulating the expression of RhoA,ROCK Ⅱ and MLC proteins(i.e.,inhibiting the RhoA/ROCK signaling pathway)in the anterior horn of spinal cord.
Objective To observe the effect of electroacupuncture(EA)of"Jiaji"(EX-B 2)on limb locomotor function and expression of Ras homolog gene family member A(RhoA),Rho-associated kinase Ⅱ(ROCK Ⅱ)and myosin light chain(MLC)proteins in the anterior horn of spinal cord in acute spinal cord injury(ASCI)rats,so as to explore its mechanisms underlying improvement of SCI-induced limb locomotor dysfunction.Methods Forty-eight female Wistar rats were randomly divided into sham operation(sham),ASCI model(model),EA EX-B 2(EA)and ROCK inhibitor(Fasudil)groups which were further divided into 14 dand 28 dsubgroups(n=6 in each).The ASCI model was made by using weight drop striking method.Three hours after modeling,EA(100 Hz,0.4,0.6 mA)was applied to EX-B 2(T 9,T 11)for 30 min,once daily for 14 dand 28 d,respectively.The ROCK inhibitor(hydrochloride Fasudil,10 mg/kg)was administrated by intraperitoneal injection immediately after modeling,once a day,continuously for 14 dor 28 d.The expression of RhoA,ROCK Ⅱ and MLC proteins in the spinal cord anterior horn tissue(T 10)was detected by immunohistochemistry.The rats' hindlimb locomotor function was assessed according to Basso,Beattie and Bresnahan(BBB)locomotor rating scale(21-points).Results After ASCI,the BBB scores were significantly lower in the model group than in the sham group on day 14 and 28(P<0.05),and obviously higher in the EA and inhibitor groups than in the model group(P<0.05),suggesting an improvement of the hindlimb locomotor function after EA intervention or suppression of ROCK.Immunohistochemical results indicated that the numbers of RhoA,ROCK Ⅱ and MLC immune-reaction positive cells in the anterior horn of spinal cord were significantly more in the model group than in the sham group(P<0.05),and remarkably decreased in both EA and inhibitor groups on day 14 and 28 relevant to the model group(P<0.05).The therapeutic effects of EA were markedly weaker than those of inhibitor Fasudil in up-regulating BBB score and down-regulating the number of RhoA,ROCKⅡ and MLC positive cells(P<0.05).Conclusion EA of EX-B 2 can improve the hindlimb locomotor function in ASCI rats,which may be associated with its effect in down-regulating the expression of RhoA,ROCK Ⅱ and MLC proteins(i.e.,inhibiting the RhoA/ROCK signaling pathway)in the anterior horn of spinal cord.
引文
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[2]吕威,李冰,景泉凯,等.电针“大椎”“命门”对脊髓损伤大鼠神经元细胞凋亡及JNK信号通路相关蛋白表达的影响[J].针刺研究,2017,42(1):14-19.
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[10]陈涛,张秀清,唐吉友.Rho/ROCK信号通路与中枢神经轴突再生[J].国际神经病学神经外科学杂志,2009,36(3):275-279.
[11]阚伯红,于建春,刘存志,等.Rho信号通路抑制中枢神经轴突再生的研究进展[J].中国老年学杂志,2009,29(20):2683-2685.
[12]DORAN J D,LIU X,TASIIMI P,et al.New insights into the structure-function relationships of Rho-associated kinase:a thermodynamic and hydrodynamic study of the dimmertomonomer transition and its kinetic implications[J].Biochem J,2004,384(2):255-262.
[13]VINSON M,RAUSCH O,MAYCOX P R,et al.Lipid rafts mediate the interaction between myelin-associated glycoprotein(MAG)on myelin and MAG-receptors on neurons[J].Molecul Cellul Neurosci,2003,22(3):344-352.
[14]SAMUEL F,HYNDS D L.RHO GTPase signaling for axon extension:is prenylation important[J].Molecul Neurobiol,2010,42(2):133-142.
[15]韩佳寅,易艳,梁爱华,等.Rho-ROCK信号通路研究进展[J].药学学报,2016,51(6):853-859.
[16]张辉,张海飞,李艳花,等.ROCK抑制剂在中枢神经系统再生和修复中的作用[J].中国免疫学杂志,2013,29(11):1227-1229.
[17]FEHLINGS M G,TATOR C H.The effect of direct current field polarity on recovery after acute experimental spinal cord injury[J].Brain Res,1992,579(1):32-42.
[18]孙连珠,马睿杰,张柳娟,等.夹脊电针干预脊髓损伤大鼠再髓鞘化的研究[J].浙江中医药大学学报,2014,38(5):626-630.
[19]李晓宁,吴磊,迟蕾,等.不同治疗周期夹脊电针对急性脊髓损伤大鼠运动功能及细胞凋亡的影响[J].针刺研究,2016,41(6):492-496.
[2]吕威,李冰,景泉凯,等.电针“大椎”“命门”对脊髓损伤大鼠神经元细胞凋亡及JNK信号通路相关蛋白表达的影响[J].针刺研究,2017,42(1):14-19.
[3]SCHWAB M E.Repairing the injured spinal cord[J].Science,2002,295(5557):1029-1031.
[4]朱枫,鲁秀敏,王永堂.中枢神经再生抑制因子及免疫治疗研究[J].现代生物医学进展,2008,8(9):1743-1745.
[5]CROWE M J,BRESNAHAN J C,SHUMAN S L,et al.Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys[J].Nat Med,1997,3(1):73-76.
[6]ANTEL J.Oligodendrocyte/myelin injury and repair as a function of the central nervous system environment[J].Clin Neurol Neurosurg,2006,108(3):245-249.
[7]FELDBLUM S,AMAUD S,SIMON M,et al.Efficacy of a new neuroprotective agent,gacyclidine,in a model of rat spinal cord injury[J].J Neurotraum,2000,17(11):1079-1093.
[8]FAWCETT J W,ASHER R A.The glial scar and central nervous system repair[J].Brain Res Bullet,1999,49(6):377-391.
[9]PLEMEL R J,KEOUGH B M,DUNCAN J G,et al.Remyelination after spinal cord injury:is it a target for repair[J].Progr Neurobiol,2014,117:54-72.
[10]陈涛,张秀清,唐吉友.Rho/ROCK信号通路与中枢神经轴突再生[J].国际神经病学神经外科学杂志,2009,36(3):275-279.
[11]阚伯红,于建春,刘存志,等.Rho信号通路抑制中枢神经轴突再生的研究进展[J].中国老年学杂志,2009,29(20):2683-2685.
[12]DORAN J D,LIU X,TASIIMI P,et al.New insights into the structure-function relationships of Rho-associated kinase:a thermodynamic and hydrodynamic study of the dimmertomonomer transition and its kinetic implications[J].Biochem J,2004,384(2):255-262.
[13]VINSON M,RAUSCH O,MAYCOX P R,et al.Lipid rafts mediate the interaction between myelin-associated glycoprotein(MAG)on myelin and MAG-receptors on neurons[J].Molecul Cellul Neurosci,2003,22(3):344-352.
[14]SAMUEL F,HYNDS D L.RHO GTPase signaling for axon extension:is prenylation important[J].Molecul Neurobiol,2010,42(2):133-142.
[15]韩佳寅,易艳,梁爱华,等.Rho-ROCK信号通路研究进展[J].药学学报,2016,51(6):853-859.
[16]张辉,张海飞,李艳花,等.ROCK抑制剂在中枢神经系统再生和修复中的作用[J].中国免疫学杂志,2013,29(11):1227-1229.
[17]FEHLINGS M G,TATOR C H.The effect of direct current field polarity on recovery after acute experimental spinal cord injury[J].Brain Res,1992,579(1):32-42.
[18]孙连珠,马睿杰,张柳娟,等.夹脊电针干预脊髓损伤大鼠再髓鞘化的研究[J].浙江中医药大学学报,2014,38(5):626-630.
[19]李晓宁,吴磊,迟蕾,等.不同治疗周期夹脊电针对急性脊髓损伤大鼠运动功能及细胞凋亡的影响[J].针刺研究,2016,41(6):492-496.