Electroacupuncture-induced polarization of M2 microglia in beagle dogs

Abstract

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 (χ2=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 (χ2=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 significance (χ2=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 (χ2=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 0.090, 0.420, 1.795 and 1.820 respectively. The expression of Arginase in the four groups had statistical significance (χ2=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 (χ2=20.52, 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 (χ2=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 groups 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 (χ2=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. Key words: Electroacupuncture; Abducens nerve injury; Microglia cell; Beagle dogs