Involvement of the glutamate/glutamine cycle and glutamate transporter GLT-1 in antidepressant-like effects of Xiao Yao san on chronically stressed mice

Abstract

Background: Xiao Yao San (XYS) is an herbal prescription which is used in the treatment of depression for thousands of years from Song dynasty in China (960-1127 A.D.), and is the bestselling and most popular herb formula for treating major depression. This study aimed to assess the chronic antidepressant effects of XYS and fluoxetine in depressed mice induced by chronic unpredictable mild stress (CUMS) and its association with alterations in glutamate/glutamine cycle and glutamate transporters.

Methods: Mice in the control and model group were given 0.5 ml physiological saline by intragastric administration. Mice in two treatment groups were given XYS (0.25 g/kg/d) and fluoxetine (2.6 mg/kg/d), respectively. The depressive-like behaviors such as forced swim test (FST), sucrose preference test (SPT) and novelty-suppressed feeding (NSF) test were measured after mice exposed to CUMS for 21 days. Body weight, contents of glutamate and glutamine, glutamine/glutamate ratio that is usually thought to reflect glutamate/glutamine cycle, and the protein and mRNA expressions of glutamate transporters (excitatory amino acid transporter 1-2,GLAST/EAAT1 and GLT-1/EAAT2) were measured. The immunoreactivities of GLAST and GLT-1 in the hippocampus were also investigated.

Results: After CUMS exposure, mice exhibited depressive-like behaviors, body weight loss, increased glutamate level, decreased glutamine level, elevated glutamine/glutamate ratio, decreased GLT-1 protein expression and mRNA level, and decreased average optical density (AOD) of GLT-1 in the CA1, CA3 and DG in the hippocampus. These abnormalities could be effectively reversed by XYS or fluoxetine treatment. In addition, the study also found that GLAST expression in the hippocampus could not be altered by 21-d CUMS.

Conclusion: The studies indicated that XYS may have therapeutic actions on depression -like behavior s induced by CUMS in mice possibly mediated by modulation of glutamate/glutamine cycle and glutamate transporter GLT-1 in the hippocampus.

Keywords: Animal research; Depression; Herbal medicine.

Fig. 1

Molecular structures of eight ingredients in XYS sample. a Palmitic acid. b Curcumin. c Paeoniflorin. d Liquiritin. e Atractylenolide II. f Saikosaponin D. g Saikosaponin B1. h Pachymic acid

Fig. 2

The chromatograms of eight ingredients in XYS sample. a Palmitic acid. b Curcumin. c Paeoniflorin. d Liquiritin. e Atractylenolide II. f Saikosaponin D + SaikosaponinB1. g Pachymic acid. References results are shown on the left side, and results on samples are shown on the right side

Fig. 3

Effects of XYS on body weight and behaviors in mice with CUMS. a Body weight was recorded once a week during 21d–CIS period. b The forced swimming test(FST) was conduct on day 21. c, d Sucrose preference test(SPT) was performed weekly. e, f The Novelty suppressed feeding (NSF) test was performed on day 21. Data were expressed as mean ± S.E.M, n = 15 per group. ^∗ P < 0.05, ^∗∗ p < 0.001 vs. control; ^△ p < 0.05, ^△△ p < 0.001 vs. model

Fig. 4

Alterations of contents of Glu and Gln, and the Gln/Glu ratio in the hippocampi of mice by CUMS as well as XYS and FLU treatment. a Chromatograms of Glu. b Chromatograms of Gln. Results on references are shown on the left side, and results on hippocampal samples are shown on the right side. c The contents of Glu and Gln in the hippocampi were detected by LC-MS/MS. d The glutamate/glutamine ratio was calculated. Data were expressed as mean ± S.E.M,n = 5 per group.^∗ P < 0.05,^△△ P < 0.01,^# P < 0.001 vs. control; ^△ P < 0.05, ^## P < 0.001 vs. model

Fig. 5

Alterations of protein and mRNA expressions of GLAST and GLT-1 in the hippocampi of mice by CUMS as well as XYS and FLU treatment. a, c The protein expressions of GLT-1 and GLAST were measured by Western blotting. b, d The mRNAs levels of GLT-1 and GLAST were measured by qRT-PCR. Data were expressed as mean ± S.E.M, n = 5 per group.^∗∗ p < 0.001 vs. control;^△ p < 0.05,^# p < 0.01 vs. model

Fig. 6

Alterations of GLAST and GLT-1 immunoreactivities in the hippocampi of mice by CUMS as well as XYS and FLU treatment. a, b The AOD of GLAST and GLT-1 in the hippocampal subregions CA1,CA3 and DG were determined by immunohistochemical staining. Abbreviations:CA1 and CA3: hippocampal subregions of ammons horn (cornu ammonis), DG:dentate gyrus. Data were expressed as mean ± S.E.M,n = 5 per group.^∗ P < 0.05 vs. control;^△ p < 0.05, ^# p < 0.01 vs. model