Xiaoyaosan Exerts Antidepressant-like Effects by Regulating the Functions of Astrocytes and EAATs in the Prefrontal Cortex of Mice

Background: Mounting evidence indicates that the cerebral cortex is an important physiological system of emotional activity, and its dysfunction may be the main cause of stress. Glutamate is the primary excitatory neurotransmitter in the central nervous system (CNS), which initiates rapid signal transmission in the synapse before its reuptake into the surrounding glia, specifically astrocytes (ASTs). The astrocytic excitatory amino acid transporters 1 (EAAT1) and 2 (EAAT2) are the major transporters that take up synaptic glutamate to maintain optimal extracellular glutamic levels, thus preventing accumulation in the synaptic cleft and ensuing excitotoxicity. Growing evidence has shown that excitotoxicity is associated with depression. Therefore, we hypothesized that the underlying antidepressant-like mechanism of Xiaoyaosan (XYS), a Chinese herbal formula, may be related to the regulation of astrocytic EAATs. Therefore, we studied the antidepressant mechanism of XYS on the basis of EAAT dysfunction in ASTs.

Methods: Eighty adult C57BL/6 J mice were randomly divided into 4 groups: a control group, a chronic unpredictable mild stress (CUMS) group, a Xiaoyaosan (XYS) treatment group and a fluoxetine hydrochloride (Flu) treatment group. Except for the control group, mice in the other groups all received chronic unpredictable mild stress for 21 days. Mice in the control and CUMS groups received gavage administration with 0.5 mL of normal saline (NS) for 21 days, and mice in the XYS and Flu treatment groups were administered dosages of 0.25 g/kg/d and 2.6mg/kg/d by gavage. The effects of XYS on the depressive- like behavioral tests, including the open field test (OFT), forced swimming test (FST) and sucrose preference test (SPT), were examined. The glutamate (Glu) concentrations of the prefrontal cortex (PFC) were detected with colorimetry. The morphology of neurons in the PFC was observed by Nissl staining. The expression of glial fibrillary acidic protein (GFAP), NeuN, EAAT1 and EAAT2 proteins in the PFC of mice was detected by using Western blotting and immunohistochemistry. Quantitative real-time PCR (qPCR) was used to detect the expression of the GFAP, NeuN, EAAT1 and EAAT2 genes in the PFC of mice.

Results: The results of behavioral tests showed that CUMS-induced mice exhibited depressive-like behavior, which could be improved in some tests with XYS and Flu treatment. Immunohistochemistry and Western blot analysis showed that the protein levels of GFAP, NeuN, EAAT1 and EAAT2 in the PFC of CUMS mice were significantly lower than those in the control group, and these changes could be reversed by XYS and Flu. The results of qPCR analysis showed that the expression of GFAP, NeuN, EAAT1 and EAAT2 mRNAs in the PFC of CUMS mice was not significantly changed, with the exception of EAAT2, compared with that of the control group, while the expression of the above mRNAs was significantly higher in the XYS and Flu groups than that in the CUMS group.

Conclusion: XYS may exert antidepressant-like effects by improving the functions of AST and EAATs and attenuating glutamate-induced neuronal damage in the frontal cortex.