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    改良逍遥散通过线粒体自噬促进青幼期抑郁模型大鼠海马小胶质细胞M2型活化的机制研究

    Mechanism of Modified Xiaoyao San Promoted M2 Activation of Microglia via PINK1/Parkin-mediated Mitophagy in Adolescent Depressive Model Rats

      摘要
    • 摘要:
      目的  探索改良逍遥散(百欢逍遥汤)通过调节PINK1/Parkin介导的线粒体自噬促进小胶质细胞M2型活化，从而改善慢性不可预测温和应激(chronic unpredictable mild stress，CUMS)诱导的青幼期大鼠抑郁样行为的分子机制。
      方法 采用CUMS诱导3周龄雄性SD大鼠构建青幼期抑郁模型。将动物随机分为正常组，CUMS组，氟西汀组(2 mg·kg−1)，中药低、中、高剂量组(5.36、10.71、21.42 g·kg−1)。蔗糖偏好试验、强迫游泳试验、旷场试验、Morris水迷宫试验测定大鼠抑郁行为。免疫荧光标记海马区一氧化氮合酶(inducible nitric oxide synthase，iNOS)巨噬细胞甘露糖受体(cluster of differentiation 206，CD206)以及离子钙结合适配器分子1(ionized calcium binding adaptor molecule 1，Iba-1)和线粒体自噬标志物帕金森病相关蛋白(parkin RBR E3 ubiquitin protein ligase，Parkin)；Western blotting检测海马组织iNOS、CD206、微管相关蛋白1轻链3(microtubule-associated proteins light chain 3，LC3)、泛素结合蛋白p62(sequestosome 1，P62)、PTEN诱导假定激酶1(PTEN induced putative kinase 1，PINK1)、Parkin的表达。JC-1荧光探针检测大鼠海马线粒体膜电位(mitochondrial membrane potential，MMP)水平。透射电镜观察海马CA1区小胶质细胞内线粒体结构和线粒体自噬情况。
      结果 百欢逍遥汤干预后青幼期大鼠蔗糖偏好值明显升高(P<0.05)，不动时间显著缩短(P<0.01)，运动和探索行为明显增加(P<0.05)，学习和空间记忆能力显著提升(P<0.01)。百欢逍遥汤给药后海马组织中Iba1+iNOS+细胞减少，Iba1+CD206+细胞和Iba1+Parkin+细胞增加。百欢逍遥汤显著促进海马组织中CD206蛋白表达(P<0.01)，抑制iNOS蛋白表达(P<0.01)，并伴随PINK1、Parkin 、LC3Ⅱ/Ⅰ蛋白水平显著升高(P<0.01)以及p62蛋白水平降低(P<0.01)。百欢逍遥汤干预后小胶质细胞中损伤线粒体数量减少，线粒体自噬体增多，且MMP值显著升高(P<0.01)。
      结论 百欢逍遥汤可能通过促进PINK1/Parkin介导的线粒体自噬促进小胶质细胞M2型活化，从而改善CUMS诱导的青幼期大鼠抑郁样行为。

       

      Abstract:
      OBJECTIVE To investigate the effect of modified Xiaoyao San(Baihuan Xiaoyao Decoction, BHXYD) on the M2 activation of microglia by promoting PINK1/Parkin-mediated mitophagy in Chronic unpredictable mild stress(CUMS)-induced depressive adolescent rats.
      METHODS CUMS was used to establish a depression model in young rats. Three-weeks aged SD rats were randomly divided into normal group, CUMS group, fluoxetine group(2 mg·kg−1), low-dose(5.36 g·kg−1), medium-dose(10.71 g·kg−1) and high-dose(21.42 g·kg−1) Chinese medicine group. Sucrose preference test, forced swim test, open field test and morris water maze test were used to assess the depressive behavior. Immunofluorescence staining was employed to measure the expression of nitric oxide synthase(iNOS), macrophage mannose-receptor(CD206), ionic calcium binding adapter molecule 1(Iba-1) and Parkin in hippocampus; Western blotting was used to detect the expression of iNOS, CD206, microtubule-associated protein 1 light chain 3(LC3), ubiquitin-binding protein p62, PTEN-induced hypothesized kinase 1(PINK1) and Parkin in hippocampus. The level of mitochondrial membrane potential(MMP) in rat hippocampus was detected by JC-1 fluorescent probe. The mitochondrial structure and mitochondrial autophagy of microglia in CA1 region of hippocampus were observed by Transmission electron microscopy.
      RESULTS The results of behavioral experiments showed that the sucrose preference value was significantly increased(P<0.05), the immobility time was obviously shortened(P<0.01), the movement and exploration behaviors were dramatically increased(P<0.05), and the learning and spatial memory abilities were significantly improved(P<0.01) after the treatment of BHXYD. The results of IF showed that iNOS+ Iba1+ cells decreased, while Iba1+ CD206+ cells and Iba1+Parkin+ cells increased after administration of BHXYD. WB results showed that the protein levels of CD206, PINK1, Parkin, LC3Ⅱ/Ⅰ levels increased significantly(P<0.01), with iNOS, p62 protein levels decreased(P<0.01). TEM observation showed that the number of damaged mitochondria decreased and the number of mitochondria autophagosomes increased in BHXYD group. The results of JC-1 experiment showed that the MMP value after BHXYD treatment was significantly increased(P<0.01).
      CONCLUSION BHXYD may regulate the M2 microglial activation by promoting PINK1/Parkin-mediated mitophagy in CUMS-induced young rats.

       

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