Chaihu-Shugan-San exerts an antidepressive effect by downregulating miR-124 and releasing inhibition of the MAPK14 and Gria3 signaling pathways

Abstract

Dysregulation of miR-124 has been reported to be involved in the pathophysiology of depression. Chaihu-Shugan-San, a traditional Chinese medicine, has antidepressive activity; however, the underlying mechanisms remain unclear. In this study, to generate a rodent model of depression, rats were subjected to a combination of solitary confinement and chronic unpredictable mild stress for 28 days. Rats were intragastrically administered Chaihu-Shugan-San (2.835 mL/kg/d) for 4 weeks, once a day. Real-time reverse-transcription quantitative polymerase chain reaction, miRNA microarray, western blot assay and transmission electron microscopy demonstrated that Chaihu-Shugan-San downregulated miR-124 expression and upregulated the mRNA and protein levels of mitogen-activated protein kinase 14 (MAPK14) and glutamate receptor subunit 3 (Gria3). Chaihu-Shugan-San also promoted synapse formation in the hippocampus. The open field test, sucrose consumption test and forced swimming test were used to assess depression-like behavior. After intragastric administration of Chaihu-Shugan-San, sucrose consumption increased, while the depressive behaviors were substantially reduced. Together, these findings suggest that Chaihu-Shugan-San exerts an antidepressant-like effect by downregulating miR-124 expression and by releasing the inhibition of the MAPK14 and Gria3 signaling pathways.

Keywords: Chaihu-Shugan-San; Gria3; MAPK14; depression; forced swimming test; miR-124; nerve regeneration; neural plasticity; neural regeneration; open-field test; sucrose consumption test; traditional Chinese medicine.

Figure 1

CHSGS exhibited an antidepressive effect on CUMS-induced depression in rats. (A) CHSGS treatment increased sucrose preference in the CUMS group. (B, C) CHSGS increased the time spent and the distance traveled (mm) by the CUMS rats in the central area of the open field. (D) The forced swimming time of CUMS rats was remarkably increased by treatment with CHSGS. The immobility time was decreased in the CHSGS group compared with the CUMS group. Data are presented as the mean ± SD (n = 3, one-way analysis of variance followed by least significant difference tests). *P < 0.05. CUMS: Chronic unpredictable mild stress; CHSGS: Chaihu-Shugan-San; s: second.

Figure 2

CHSGS promoted synapse formation in the hippocampus. Transmission electron microscopy was performed to measure the number of synapses, the formation of the synaptic cleft and the postsynaptic density (synapse junctions are indicated by arrows). (A) Control group (distilled water); (B) CUMS group (CUMS + distilled water); (C) fluoxetine group (CUMS + fluoxetine 1.8 mg/kg/day and distilled water); (D) CHSGS group (CUMS + CHSGS 2.835 g/kg/day and distilled water). The drugs were given once daily. (E) Relative synaptic density. Cross-sections of the hippocampus CA3 region were used for analysis of synapse number. Sections of hippocampus were randomly sampled. Synapses were counted in electron microscopic sections in a defined area (40 mm²) and normalized to the control group. At least 30 such samples from at least 10 sections were assessed for each animal. *P < 0.05. Data are presented as the mean ± SD from three independent replicates (n = 3, one-way analysis of variance followed by least significant difference tests). CUMS: Chronic unpredictable mild stress; CHSGS: Chaihu-Shugan-San.

Figure 3

CHSGS downregulated miR-124 expression. Total RNA was extracted from brain tissues, and real-time reverse transcription quantitative polymerase chain reaction was performed to evaluate miR-124 expression. *P < 0.05. Data are presented as the mean ± SD (n = 3; one-way analysis of variance followed by least significant difference tests). CUMS: Chronic unpredictable mild stress; CHSGS: Chaihu-Shugan-San; ns: not significant.

Figure 4

miR-124 target gene prediction analysis. (A) Venn diagram: The downstream targets of miR-124 predicted with the miRanda, miRDB and TargetScan databases. The candidate targets of miR-124 predicted by each database are shown, and approximately 190 genes are at the intersection of the three databases. (B) KEGG enrichment analysis by DAVID, showing the pathways that the predicted target genes are involved in. (C) Luciferase reporter assay: The binding between miR-124 with the 3′-UTR regions of MAPK14 and Gria3. Data are presented as the mean ± SD (n = 3, one-way analysis of variance followed by least significant difference tests). CHSGS: Chaihu-Shugan-San; WT: wild type; NC: negative control.

Figure 5

CHSGS increased the expression levels of MAPK14 and Gria3. (A, B) The mRNA levels of MAPK14 and Gria3 in the different groups (quantitative polymerase chain reaction). (C, D) Western blots for MAPK14 and Gria3, respectively. GAPDH was used as the endogenous control. (E, F) Quantification of protein levels of Gria3 and MAPK14 shown in C and D, respectively. Data are represented as the mean ± SD (n = 3; one-way analysis of variance followed by least significant difference tests). *P < 0.05, **P < 0.01, ***P < 0.001. CUMS: Chronic unpredictable mild stress; CHSGS: Chaihu-Shugan-San; MAPK14: mitogen-activated protein kinase 14; Gria3: glutamate receptor subunit 3.