基于小胶质细胞-BDNF-神经元信号探讨电针对SNI大鼠的镇痛作用及机制

Analgesic effect and mechanism of electroacupuncture on SNI rats based on microglia-BDNF-neuron signal

目的:观察电针“环跳”“委中”对坐骨神经选择性神经损伤(SNI)大鼠脊髓背角胶质细胞活化、脑源性神经营养因子(BDNF)表达、神经元兴奋性及树突棘数量的影响,探讨电针对SNI的镇痛效应机制。方法:第一部分:60只SD大鼠随机分为假手术组、模型组、电针组和假电针组,每组15只。除假手术组外,其余组均制备SNI大鼠模型;假手术组仅做切开处理,不损伤神经。电针组于患侧“环跳”“委中”行电针干预,连续波,频率2 Hz,电流强度1 m A,每次30 min,每日1次,共14 d。假电针组于患侧“环跳”“委中”旁开0.5 cm处行电针干预,操作、电针参数及疗程同电针组。于造模前1 d,造模后3、7、14 d检测各组大鼠热缩足反射潜伏期和机械缩足反射阈值。造模后14 d,采用Western blot法检测各组大鼠脊髓背角离子钙接头蛋白1(Iba-1)、胶质纤维酸性蛋白(GFAP)、BDNF、即刻早期基因(c-Fos)蛋白表达;采用免疫荧光法检测各组大鼠脊髓背角Iba-1、c-Fos蛋白表达;采用免疫组化法检测各组大鼠脊髓背角GFAP蛋白表达;采用高尔基染色法检测各组大鼠脊髓背角神经元树突棘数量。第二部分:30只SD大鼠随机分为对照组、BDNF组、BDNF+anti-Trk B组,每组10只。对照组予10μL 0.9%氯化钠溶液和二甲基亚砜(DMSO)的1︰1混合液鞘内注射,BDNF组将10μg大鼠重组BDNF溶于10μL0.9%氯化钠溶液和DMSO的1︰1混合液鞘内注射,BDNF+anti-Trk B组将10μg大鼠重组BDNF和30μg酪氨酸激酶受体B(Trk B)抗体溶于10μL0.9%氯化钠溶液和DMSO的1︰1混合液鞘内注射。于鞘内注射前1 d,注射1、3、7 d后检测各组大鼠机械缩足反射阈值。注射7 d后,采用Western blot法和免疫荧光法检测各组大鼠脊髓背角c-Fos蛋白表达。结果:第一部分:造模后3、7、14 d,与假手术组比较,模型组大鼠热缩足反射潜伏期和机械缩足反射阈值降低(P<0.05);造模后7、14 d,与模型组比较,电针组大鼠热缩足反射潜伏期和机械缩足反射阈值增加(P<0.05)。模型组大鼠脊髓背角Iba-1、GFAP、BDNF、c-Fos蛋白表达及神经元树突棘数量较假手术组增加(P<0.05);电针组大鼠脊髓背角Iba-1、BDNF、c-Fos蛋白表达及神经元树突棘数量较模型组降低(P<0.05)。第二部分:鞘内注射3、7d后,BDNF组大鼠机械缩足反射阈值较对照组降低(P<0.05);BDNF+anti-Trk B组大鼠机械缩足反射阈值较BDNF组增加(P<0.05)。BDNF组大鼠脊髓背角c-Fos蛋白表达较对照组增加(P<0.05);BDNF+anti-Trk B组大鼠脊髓背角c-Fos蛋白表达较BDNF组降低(P<0.05)。结论:电针“环跳”“委中”对SNI大鼠产生镇痛作用,其机制可能是抑制脊髓背角小胶质细胞活化,从而阻断小胶质细胞-BDNF-神经元信号,最终降低神经元的兴奋性。

Objective To observe the effect of electroacupuncture(EA) at "Huantiao"(GB 30) and "Weizhong"(BL 40) on the activation of glial cells,the expression of brain-derived neurotrophic factor(BDNF),excitability and the number of dendritic spines of neurons in the spinal dorsal horn in rats with spared nerve injury(SNI) of sciatic nerve,and to explore the analgesic mechanism of EA on SNI.Methods PartⅠ:Sixty SD rats were randomly divided into a sham operation group,a model group,an EA group and a sham EA group,15 rats in each group.Except the sham operation group,the SNI rat model was established in the remaining groups.The rats in the sham operation group were only treated with incision without damaging the nerve.The rats in the EA group were treated with EA at "Huantiao"(GB 30) and "Weizhong"(BL 40) on the affected side,continuous wave,frequency of 2 Hz,current intensity of 1 m A,30 minutes each time,once a day,for 14 days.The rats in the sham EA group were treated with EA at points 0.5 cm next to "Huantiao"(GB 30) and "Weizhong"(BL 40) on the affected side;the manipulation,EA parameters and treatment course were the same as the EA group.The latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex were detected 1 day before modeling and 3,7 and 14 days after modeling.Fourteen days after modeling,Western blot was used to detect the protein expressions of ionized binding adapter junction protein 1(Iba-1),glial fibrillary acidic protein(GFAP),BDNF and c-Fos in the spinal dorsal horn;the expressions of Iba-1 and c-Fos proteins in the spinal dorsal horn were detected by immunofluorescence staining;immunohistochemical method was used to detect the expression of GFAP protein in the spinal dorsal horn;Golgi staining was used to detect the number of dendritic spines in spinal dorsal horn neurons.PartⅡ:Thirty SD rats were randomly divided into a control group,a BDNF group and a BDNF+anti-Trk B group,10 rats in each group.The control group was treated with intrathecal injection of 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and dimethyl sulfoxide(DMSO);the BDNF group was treated with intrathecal injection of 10 μg rat recombinant BDNF dissolved in 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and DMSO;the BDNF+anti-Trk B group was treated with intrathecal injection of 10 μg rat recombinant BDNF and 30 μg tyrosine kinase receptor B(Trk B) antibody dissolved in 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and DMSO.The threshold of mechanical foot retraction reflex was detected 1 day before intrathecal injection and 1,3 and 7 days after injection.Seven days after injection,the expression of c-Fos protein in the spinal dorsal horn was detected by Western blot and immunofluorescence staining.Results PartⅠ:Compared with the sham operation group,3,7 and 14 days after modeling,the latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex in the model group were decreased(P<0.05);7 and 14 days after modeling,compared with the model group,the latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex in the EA group were increased(P<0.05).The expressions of Iba-1,GFAP,BDNF,c-Fos proteins and the number of neuronal dendritic spines in the spinal dorsal horn in the model group were higher than those in the sham operation group(P<0.05);the expressions of Iba-1,BDNF,c-Fos proteins and the number of neuronal dendritic spines in the EA group were lower than those in the model group(P<0.05).PartⅡ:3 and 7 days after intrathecal injection,the threshold of mechanical foot retraction reflex in the BDNF group was lower than that in the control group(P<0.05);the threshold of mechanical foot retraction reflex in the BDNF+anti-Trk B group was higher than that in the BDNF group(P<0.05).The expression of c-Fos protein in spinal dorsal horn in the BDNF group was higher than that in the control group(P<0.05);the expression of c-Fos protein in spinal dorsal horn in the BDNF+anti-Trk B group was lower than that in the BDNF group(P<0.05).Conclusion The analgesic effect of EA at "Huantiao"(GB 30) and "Weizhong"(BL 40) on SNI rats may be related to inhibiting the activation of microglia in the dorsal horn of the spinal cord,thereby blocking the signal of microglia-BDNF-neuron,and finally reducing the excitability of neurons.