Abstract: Objective To investigate the activation and mechanism of microglia (MG) in Tourette syndrome (TS) rats induced by 3,3'-aminodipropionitrile (IDPN). Methods Thirty six male Sprague Dawley rats were randomly divided into a TS group and a Sham group (Sham group),with 18 rats in each group.The TS group received intraperitoneal injection of IDPN for 7 days,with the dose of 300mg/(kg·d),while the Sham group received intraperitoneal injection of physiological saline for 7 days,with the dose of 5mL/(kg·d).Double immunofluorescence staining was used to detected the expression of inducible nitric oxide synthase (iNOS),a marker protein of M1 type microglia,and its co-localization with Iba-1(a specific marker for microglial activation) in rat striatum,as well as the expression of arginase 1 (Arg-1) and its co-location with Iba-1 in M2 microglia.The expression levels of iNOS,Arg-1 in rat striatal tissues were quantified by enzyme-linked immunosorbent measurement (ELISA),real-time PCR (qRT-PCR),meanwhile the expression levels of inflammatory cytokines tumor necrosis factor α (TNF-α),interleukin-6(IL-6),and interleukin-10(IL-10) were detected. Results Immunofluorescence double staining showed that the positive expressions of Iba-1 and iNOS in striatum of TS group were enhanced and co-located compared with Sham group,while the positive expression of Iba-1 and Arg-1 in striatum of TS group decreased and showed co-localization.The ELISA results showed that compared with the Sham group,the TS group had a significant increase in the content of M1 type MG marker protein iNOS (t=5.796,P<0.001),a significant decrease in the content of M2 type MG marker protein Arg-1 (t=4.348,P<0.01),and a significant increase in inflammatory factors,including TNF-α,IL-6 and IL-10 (t=5.654,5.748,8.231, P<0.001).The qRT-PCR results showed that compared with the Sham group,the mRNA expression level of iNOS,a M1 type MG marker protein,was significantly increased in the TS group (t=9.914,P<0.001),while the mRNA expression level of Arg-1,a M2 type MG marker protein,was significantly reduced (t=4.390,P<0.01),and the mRNA expression levels of inflammatory factors in the TS group,including TNF-α,IL-6 and IL-10 were significantly increased (t=12.056,14.147,13.350,P<0.001). Conclusion TS rats induced by IDPN exhibit MG activation,with M1 type MG upregulated and M2 type MG downregulated,thereby mediating neuroinflammation.

Key words: Tourette syndrome, microglia, neuroimmunity, inflammatory factor

摘要： 目的 探讨小胶质细胞(MG)在3,3'-亚氨基二丙腈(IDPN)诱导的Tourette综合征(TS)大鼠中的活化及作用机制。 方法 雄性Sprague-Dawley大鼠36只,随机分为TS组和假用药组(Sham组),每组18只,TS组腹腔注射IDPN[300mg/(kg·d),7d],Sham组腹腔注射生理盐水[5mL/(kg·d),7d]。使用免疫荧光双染检测大鼠纹状体组织M1型MG标记蛋白诱导型一氧化氮合酶(iNOS)表达及与MG活化特异性标记物离子钙接头蛋白-1(Iba-1)共定位的情况,M2型MG标记蛋白精氨酸酶-1(Arg-1)表达及与Iba-1共定位的情况;使用酶联免疫吸附法(ELISA)、实时荧光定量PCR(qRT-PCR)对大鼠纹状体组织中iNOS、Arg-1的表达水平进行定量,同时检测炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)的表达水平。 结果 大鼠纹状体组织免疫荧光双染显示,相比Sham组,TS组大鼠纹状体组织中Iba-1、iNOS的阳性表达均有所增强且呈现共定位,Arg-1的阳性表达有所降低且与Iba-1呈现共定位。ELISA结果显示,相比Sham组,TS组M1型MG标记蛋白iNOS含量显著升高(t=5.796,P＜0.001),M2型MG标记蛋白Arg-1含量显著降低(t=4.348,P＜0.01),炎症因子TNF-α、IL-6、IL-10含量均显著升高(t=5.654、5.748、8.231,P＜0.001);qRT-PCR结果显示,与Sham组相比,TS组M1型MG标记蛋白iNOS的mRNA表达水平显著升高(t=9.914,P＜0.001),M2型MG标记蛋白Arg-1的mRNA表达水平显著降低(t=4.390,P＜0.01),炎症因子TNF-α、IL-6、IL-10的mRNA表达水平均显著升高(t=12.056、14.147、13.350,P＜0.001)。 结论 IDPN诱导的TS大鼠存在MG活化,且M1型MG上调,M2型MG下调,介导神经炎症。

关键词: Tourette综合征, 小胶质细胞, 神经免疫, 炎症因子