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

Modified Xiaoyao San promoted M2 activation of microglia via PINK1/Parkin-mediated mitophagy in adolescent depressive rats

关键词:  百欢逍遥汤，青少年抑郁症，线粒体自噬，小胶质细胞，海马

Key words:  Baihuan Xiaoyao Decoction, adolescent depression, mitophagy, microglia, hippocampus.

摘要:

目的 探讨改良逍遥散(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构建青幼期抑郁模型大鼠。将3周龄雄性SD大鼠随机分为对照组、CUMS组、氟西汀组(2 mg/kg)、百欢逍遥汤低、中、高剂量组(5.36 g/kg、10.71 g/kg、21.42 g/kg)。蔗糖偏好实验(Sucrose preference test, SPT)、强迫游泳实验(Forced swim test, FST)、旷场实验(Open field test, OFT)、Morris水迷宫实验(Morris water maze, MWM)测定大鼠抑郁行为。免疫荧光检测(Immunofluorescence staining, IF)海马区M1型小胶质细胞活化标志物一氧化氮合酶(iNOS)、M2型小胶质细胞极化标志物巨噬细胞甘露糖受体(CD206)以及离子钙结合适配器分子1(Iba-1)和线粒体自噬标志物帕金森病相关蛋白(Parkin)的表达；蛋白免疫印迹法(Western blot, WB)检测海马iNOS、CD206、微管相关蛋白1轻链3B(LC3B)、泛素结合蛋白p62、PTEN诱导假定激酶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(Transmission electron microscopy, TEM)观察海马CA1区小胶质细胞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。}

Abstract:

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. 3-weeks aged Sprague-Dawley (SD) rats were randomly divided into control group, CUMS group, fluoxetine group (2 mg/kg), low-dose (5.36 g/kg), medium-dose (10.71 g/kg) and high-dose Chinese medicine group (21.42 g/kg). Sucrose preference test (SPT), Forced swim test (FST), Open field test (OFT) and Morris water maze (MWM) 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 mitophagy marker Parkin in hippocampus; Western blot was used to detect the expression of iNOS, CD206, microtubule-associated protein 1 light chain 3B (LC3B), 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 experiment 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 detection 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 level of CD206, PINK1, Parkin, LC3Ⅱ/Ⅰ levels increased significantly (P<0.01) ,with iNOS, p62 protein level 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.