Scopoletin ameliorates anxiety-like behaviors in complete Freund's adjuvant-induced mouse model

Abstract

Anxiety disorder is highly prevalent worldwide and represents a chronic and functionally disabling condition, with high levels of psychological stress characterized by cognitive and physiological symptoms. Scopoletin (SP), a main active compound in Angelica dahurica, is traditionally used for the treatment of headache, rhinitis, pain, and other conditions. Here, we evaluated the effects of SP in a mouse model of complete Freund's adjuvant (CFA)-induced chronic inflammation anxiety. SP (2.0, 10.0, 50.0 mg/kg) administration for 2 weeks dose-dependently ameliorated CFA-induced anxiety-like behaviors in the open field test and elevated plus maze test. Moreover, we found that SP treatment inhibited microglia activation and decreased both peripheral and central IL-1β, IL-6, and TNF-α levels in a dose-dependent manner. Additionally, the imbalance in excitatory/inhibitory receptors and neurotransmitters in the basolateral nucleus after CFA injection was also modulated by SP administration. Our findings indicate that the inhibition of the nuclear factor-kappa B and mitogen-activated protein kinase signaling pathways involving anti-inflammatory activities and regulation of the excitatory/inhibitory balance can be attributed to the anxiolytic effects of SP. Moreover, our molecular docking analyses show that SP also has good affinity for gamma-aminobutyric acid (GABA) transaminase and GABA_A receptors. Therefore, these results suggest that SP could be a candidate compound for anxiolytic therapy and for use as a structural base for developing new drugs.

Keywords: Amygdala; Anxiety; GABA; Glutamate; Inflammation; Scopoletin.

Fig. 1

SP relieved CFA-induced anxiety-like behaviors in mice. a Representative traces of locomotor activity in the OFT. b-d SP administration effectively reversed the reduction in the time spent (b) and the distance traveled (c) in the central area in the OFT after CFA injection, while the total distance traveled showed no significant difference in each group (d). e-g SP treatment obviously increased the percentage of time spent in (e) and the number of entries into (f) the open arms, and decreased the percentage of time spent in (g) the closed arms in the EPM test. n = 12 mice per group; **p < 0.01 versus control group; ^#p < 0.05, ^##p < 0.01 versus vehicle group

Fig. 2

SP suppressed pro-inflammatory cytokine levels in the serum and BLA of CFA-injected mice. a-c SP treatment significantly reduced the elevated levels of IL-1β (a), IL-6 (b), and TNF-α (c) in the serum, as shown by ELISA. d Representative western blot analysis of IL-1β, IL-6, and TNF-α expression. Administration of SP reversed the increased expression of IL-1β (e), IL-6 (f), and TNF-α (g) normalized to β-actin. n = 6 mice from three independent experiments; **p < 0.01 versus control group; ^#p < 0.05, ^##p < 0.01 versus vehicle group

Fig. 3

SP reduced microglial activation in the BLA of CFA-injected mice. a Slices were immunostained using the microglial marker Iba-1 antibody (red), and nuclei were stained with Hoechst 33258 (blue). Scale bar = 50 μm. b SP inhibited the activation of microglia in the BLA after CFA injection and had a dose-dependent effect. n = 6 mice from three independent experiments; *p < 0.05 versus control group; ^##p < 0.01 versus vehicle group

Fig. 4

SP improved changes in glutamate and GABA_A receptor expression in the BLA of CFA-injected mice. a Representative western blot analysis of GluA1, GluN2A, GluN2B, and PSD95 expression. SP treatment reversed the increased expression of GluA1 (b) and PSD95 (e), but had no apparent effect on GluN2A (c) and GluN2B (d) normalized to β-actin. f Representative western blot analysis of GABA_A α2 and GABA_A γ2 expression. SP treatment significantly reversed the decreased expression of GABA_A α2 (g) and GABA_A γ2 (h) normalized to β-actin. n = 6 mice from three independent experiments; **p < 0.01 versus control group; ^#p < 0.05, ^##p < 0.01 versus vehicle group

Fig. 5

Effect of SP on glutamate and GABA levels in the BLA of CFA-injected mice. SP treatment reversed the increase in glutamate (a) and reduced GABA (b). Data are from three independent experiments; *p < 0.05 versus control group; ^#p < 0.05 versus vehicle group

Fig. 6

SP inhibited GABA-T and NF-κB and MAPK signaling pathways in CFA-induced mice. a Representative western blot analysis of GABA-T, p-p38, p38, p-JNK, JNK, and p65 expression. SP treatment obviously reduced the CFA-induced upregulations of GABA-T (b), p-p38/p38 (c), p-JNK/JNK (d), and p65 (e) normalized to β-actin. n = 6 mice from three independent experiments; **p < 0.01 versus control group; ^#p < 0.05, ^##p < 0.01 versus vehicle group

Fig. 7

Binding interactions of SP with GABA-T, GABA_AR, NMDAR, and AMPAR. a-h Superimposition of SP (gray) with the co-crystallized ligands (light blue) vigabatrin (a), diazepam (c), ifenprodil (e), and NBQX (g) against GABA-T (PDB code: 1OHW), GABA_AR (PDB code: 6HUP), NMDAR (PDB code: 4PE5), and AMPAR (PDB code: 6QKC); the yellow and light blue dashes represent hydrogen bonds and π-π stacking, respectively. The 2D interaction diagram shows the major binding sites between SP and GABA-T (b), GABA_AR (d), NMDAR (f), and AMPAR (h); the purple arrow and the green line represent hydrogen bonds and π-π stacking, respectively

Fig. 8

SP alleviated CFA-induced anxiety behaviors by activating the GABA_A receptor. a Representative traces of locomotor activity in the OFT. b, c SP (50 mg/kg) administration effectively reversed the reduction in the time spent (b) and distance traveled (c) in the central area in the OFT, while this effect was reduced by co-administration of flumazenil (FLU, 10 mg/kg). d, e Mice showed a significant increase in the percentage of time spent in (d) and number of entries into (e) the open arms in the EPM test after SP (50 mg/kg) treatment, while this effect of SP was also reduced in the presence of FLU (10 mg/kg). n = 12 mice per group; **p < 0.01 versus control group; ^#p < 0.05, ^##p < 0.01 versus vehicle group; ^&p < 0.05, ^&&p < 0.01 versus SP group