督脉电针对急性脊髓损伤大鼠脊髓损伤区NR2B表达的影响

Influence of electroacupuncture of “Dazhui” (EX-B2) and “Mingmen” (GV4) on NR2B expression in anterior horns of spinal cord in rats with acute spinal cord injury

目的:观察督脉电针"大椎""命门"对急性脊髓损伤(SCI)大鼠脊髓损伤区N-甲基-D-天门冬氨酸受体(NMDAR)亚基NR2B表达的影响,探讨督脉电针促进急性SCI脊髓前角神经修复的分子机制。方法:SD大鼠随机分为假手术组、模型组、电针组及药物组,每组24只。采用脊髓打击器打击复制急性SCI模型。电针组于造模成功后电针大鼠"大椎""命门"穴,每次治疗30min,共治疗3次。药物组先予尾静脉注射甲基强的松龙(MP)30mg/kg冲击治疗,然后予5.4mg·kg^(-1)·h-1 MP维持,共24h。采用BBB行为学评分观察大鼠造模前及造模后6、24、48h运动功能的变化。采用实时荧光定量PCR法检测脊髓损伤区NR2B mRNA表达水平,采用蛋白免疫印迹法和免疫荧光技术检测脊髓损伤区NR2B蛋白表达水平。结果:与同时段假手术组相比,模型组BBB行为学评分于造模后6、24、48h均显著降低(P<0.001)。与同时段模型组相比,电针组和药物组的BBB评分于造模后各时点均未见明显改变(P>0.05)。与假手术组相比,模型组脊髓损伤区病理学改变明显,NR2B阳性神经元数量显著增加(P<0.001),NR2BmRNA和蛋白表达水平均显著升高(P<0.05)。与模型组相比,电针组和药物组脊髓损伤区病理学改变明显减轻,NR2B阳性神经元数量显著减少(P<0.001),NR2BmRNA和蛋白表达水平显著降低(P<0.05)。电针组与药物组比较,各指标变化的差异均无统计学意义(P>0.05)。结论:督脉电针"大椎""命门"穴可能通过抑制脊髓损伤区NR2B的表达,从而促进脊髓前角损伤神经的修复。

Objective To observe the influence of eletroacupuncture(EA)at"Dazhui"(EX-B2)and"Mingmen"(GV4)on expression of NR2 Bsubunit of N-methyl-D-aspartate receptor(NMDA)in the injured anterior horn(AH)area of rats with acute spinal cord injury(SCI),so as to explore its mechanisms underlying improvement of neural repair.Methods A total of 96 male Sprague-Dawley(SD)rats were randomly and equally divided into four groups:sham operation(sham),model,medication(Methyl-prednisone,MP)and EA(n=24 in each group).The acute SCI model was established by using a MASCIS spinal cord impactor.EA(2 Hz,0.5 mA)was applied to EX-B2 and GV4 for 30 min,once at 0.5 h,12 and 24 hafter SCI.Rats of the medication group were treated by tail intravenous injection of MP 30 mg/kg within 15 min(impact therapy)and 5.4 mg·kg^-1·h^-1(maintaining treatment)45 min thereafter for 23 h.The Basso,Beattie and Bresnahan(BBB)rating scale(0 to 21 points)was used to assess changes of locomotor function 6,24 and 48 hafter SCI.Histopathological changes of the injured spinal cord AH region were observed after sectioning and hematoxylin-eosin(H.E.)staining,and the expression levels of NR2 BmRNA and protein of AH were measured by quantitative real-time PCR,Western blot and immunofluorescence,respectively.Results After SCI,the BBB scores at 6,24 and 48 hwere significantly decreased in the model group compared with those of the sham group(P<0.001),but had no remarkable changes in both medication and EA groups after the intervention in comparison with those of the model group(P>0.05).After modeling,the histopathological changes(blurred border of the grey-white matter,cellular karyopyknosis,deepening of the cytoplasmic red stain,and rupture,dissolution and disordered arrangement of myelinated nerve fibers)in the injury area of the spinal cord in the model group were apparent,the number of NR2 Bpositive neurons and the relative expression levels of NR2 BmRNA and protein were significantly increased in the model group relevant to the sham group(P<0.05).In contrast to the model group,the injured severity of the spinal cord AH region was relatively milder,and the expression levels of NR2 BmRNA and protein were considerably down-regulated in both EA and MP groups(P<0.05).However,there were no significant differences between the EA and MP groups in the expression levels of NR2 BmRNA and protein(P>0.05).Conclusion EA at EX-B2 and GV4 may inhibit the expression of NR2 BmRNA and protein in acute SCI rats,which may contribute to its action in promoting nerve repair of the injured ventricolumna area of the thoracic spinal cord.