电针诱导Beagle犬展神经M2型小胶质细胞极化的初步研究

Electroacupuncture-induced polarization of M2 microglia in beagle dogs

目的探讨电针诱导是否促进展神经小胶质细胞向M2型极化。方法选用健康6月龄Beagle犬24只,建立Beagle犬展神经损伤模型,按照随机数字表法随机分为假手术组、神经损伤组、电针处理组及AM1241组,每组6只。假手术组:仅暴露分离展神经,术中不进行神经损伤处理,缝合切口皮肤,在电针刺激时进行束缚;损伤组:制作展神经损伤模型,在电针刺激时进行束缚;电针处理组:Beagle犬展神经损伤模型建立后连续2周进行电针刺激; AM1241组:展神经损伤+大麻素2型受体激动剂AM1241,展神经损伤后连续2周给予溶剂3 mL/kg进行腹腔注射,在电针刺激时进行束缚。将4组实验动物干预2周,于第7天、第14天取材进行Western Blot检测展神经小胶质细胞M1特异性标记蛋白诱导型一氧化氮合酶(iNOS)和白细胞介素1β(IL-1β),M2特异性标记蛋白精氨酸酶-1(Arginase)和脑源性神经营养因子(BDNF)的表达水平,进一步观察免疫荧光分析Arginase表达的情况,4组之间比较采用Kruskal-Wallis H检验,组内两组间比较用Mann-Whitney U检验。结果 (1)术后第7天4组iNOS表达分别为1. 370、1. 610、0. 190、0. 105。4组iNOS表达比较差异具有统计学意义(χ~2=21. 64,P <0. 05),电针处理组和AM1241组iNOS的表达较神经损伤组均降低(Z=-2. 89、-2. 89,P均<0. 05);术后第14天4组iNOS表达分别为1. 430、1. 990、0. 165、0. 150。4组iNOS表达比较差异具有统计学意义(χ~2=20. 99,P <0. 05),电针处理组和AM1241组iNOS的表达较神经损伤组均降低(Z=-2. 89、-2. 94,P均<0. 05)。(2)术后第7天,4组IL-1β表达分别为1. 255、1. 575、0. 180、0. 160。4组IL-1β表达比较差异具有统计学意义(χ~2=21. 34,P <0. 05),电针处理组和AM1241组IL-1β的表达较神经损伤组均降低(Z=-2. 90、-2. 91,P均<0. 05)。术后第14天4组IL-1β表达分别为1. 245、1. 485、0. 255、0. 185。4组IL-1β表达比较差异具有统计学意义(χ~2=21. 69,P <0. 05),电针处理组和AM1241组IL-1β的表达较神经损伤组均降低(Z=-2. 91、-2. 93,P均<0. 05)。(3) 4组均检测到M2特异性标记蛋白(Arginase、BDNF)。其中术后第7天4组Arginase表达分别为0. 090、0. 420、1. 795、1. 820。4组Arginase表达比较差异具有统计学意义(χ~2=21. 44,P <0. 05),且电针处理组和AM1241组的Arginase表达均高于神经损伤组(Z=-2. 92、-2. 93,P均<0. 05)。术后第14天4组Arginase表达分别为0. 165、0. 545、1. 850、1. 930。4组Arginase表达比较差异具有统计学意义(χ~2=20. 52,P <0. 05),电针处理组和AM1241组的Arginase表达均高于神经损伤组(Z=-2. 90、-2. 91,P均<0. 05)。(4)术后第7天4组BDNF表达分别为0. 075、0. 385、1. 715、1. 770。4组BDNF表达比较差异具有统计学意义(χ~2=21. 69,P <0. 05),电针处理组和AM1241组的BDNF表达均高于神经损伤组(Z=-2. 91、-2. 92,P均<0. 05)。术后第14天4组BDNF表达分别为0. 140、0. 485、1. 810、1. 870。4组BDNF表达比较差异具有统计学意义(χ~2=21. 72,P <0. 05),电针处理组和AM1241组的BDNF表达均高于神经损伤组(Z=-2. 93、-2. 92,P均<0. 05)。结论电针能够减少Beagle犬展神经M1型小胶质细胞表达,诱导M2型小胶质细胞极化促进损伤神经修复。同时大麻素2型受体激动剂AM1241可以发挥良好的电针协同作用,但AM1241可能不影响小胶质细胞M2型标记蛋白表达的上调。

Objective To investigate whether electroacupuncture induction promotes polarized NGF to M2 type.Methods Twenty-four Beagle dogs aged 6 months according to random number table method were randomly divided into sham operation group, nerve injury group, electroacupuncture group and AM1241 group, with 6 dogs in each group. Sham operation group： only the abducent nerve was exposed and separated , the incision skin was sutured without nerve injury during operation, and the abducent nerve injury model was made, and the abducent nerve injury model was bound by electroacupuncture. Nerve injury group： abducent nerve injury model was made and restrained by electroacupuncture ; electroacupuncture group： electroacupuncture stimulation was lasted for 2 weeks after beagle dog abducent nerve injury model established . Abducent nerve injury ＋ CB2R agonist AM1241 was injected intraperitoneally with a solvent of 3 mL/kg for 2 weeks after abducent nerve injury and restrained by electroacupuncture. The expression levels of M1-specific marker proteins iNOS and IL-1 beta, M2-specific marker proteins Arginase and BDNF were detected by western blot on the 7th and 14th day after 2 weeks of intervention. The Kruskal-Wallis H test was used between the 4 groups, and Mann-Whitney U test was used to compare the two groups. Results （1） on the seventh day after operation, the median expression of iNOS in the 4 groups was 1.370, 1.610, 0.190 and 0.105 respectively. The expression of iNOS in the four groups had statistical significance （ χ^ 2=21.64, P 〈 0.05）. The expression of iNOS in the electro-acupuncture treatment group and AM1241 group was lower than that in the nerve injury group （ Z =-2.89, -2.89, all P 〈 0.05）. On the 14th day after operation, the expression of iNOS in the four groups was 1.430, 1.990, 0.165 and 0.150, respectively. The expression of iNOS in four groups had statistical significance （ χ ^2=20.99, P 〈0.05）. The expression of iNOS in electro-acupuncture treatment group and AM1241 group was lower than that in nerve injury group （ Z =-2.89, -2.94 , all P 〈 0.05）. （2） on the seventh day after operation, the median of IL-1βin the 4 groups was 1.255 , 1.575, 0.180 and 0.160 respectively. The expression of IL-1β in the four groups had statistical significanc e （ χ^ 2=21.34, P 〈0.05）. The expression of IL-1βin electro-acupuncture treatment group and AM1241 group was lower than that in nerve injury group （ Z=-2.90,-2.91, all P 〈0.05）. On the fourteenth day after operation , the expression of IL-1β in the 4 groups was 1.245, 1.485, 0.255 and 0.185 respectively. There was significant difference in the expression of IL-1β between the four groups （ χ ^2=21.69, P 〈0.05）. The expression of IL-1β in electroacupuncture group and AM1241 group was lower than that in nerve injury group （ Z =-2.91,-2.93,all P 〈0.05）. （3） M2 specific marker proteins （Arginase, BDNF） were detected in the 4 groups . Seventh days after operation, the expression of Arginase in 4 groups was 0.09 0, 0.420, 1.795 and 1.820 respectively. The expression of Arginase in the four groups had statistical significance （ χ ^2=21.44, P 〈 0.05）, and the expression of Arginase in the electro-acupuncture treatment group and AM1241 group was higher than that in the nerve injury group （ Z =-2.92, -2.93,all P 〈0.05）. On the fourteenth day after operation, the expression of Arginase in the 4 groups was 0.165, 0.545, 1.850 and 1.930 respectively. There was a significant difference in Arginase expression between the four groups （ χ ^2=20 .5 2, P 〈0.05）. The expression of Arginase in electro-acupuncture group and AM1241 group was higher than that in nerve injury group （ Z =-2.90,-2.91,all P 〈0.05）. （4） on the seventh day after operation , the expression of BDNF in the 4 groups was 0.075, 0.385, 1.715 and 1.770 respectively. The expression of BDNF in four groups had statistical significance （ χ ^2=21.69, P 〈0.05）. The expression of BDNF in electro-acupuncture treatment group and AM1241 group was higher than that in nerve injury group （ Z =-2.91 ,-2.92,all P 〈0. 05）. On the fourteenth day after operation, the expression of BDNF in the 4 group s was 0.140, 0.485 , 1.810 and 1.870 respectively. There was a significant difference in the expression of BDNF between the four groups （ χ ^2= 21.72, P 〈0.05）. The expression of BDNF in electro-acupuncture group and AM1241 group was higher than that in nerve injury group （ Z =-2.93, -2.92, all P 〈0.05）.Conclusion Electroacupuncture can induce the polarization of M2 microglial cells in Beagle dogs and promote nerve repair. At the same time, AM1241, a CB2R receptor agonist, can exert a good electroacupuncture synergistic effect, but AM1241 does not affect the upregulation of M2 type marker protein expression in microglia.