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

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.