Echinacoside exhibits antidepressant-like effects through AMPAR-Akt/ERK-mTOR pathway stimulation and BDNF expression in mice

Abstract

Background: Several natural products have been demonstrated to be effective in the treatment of depressive disorders. Echinacoside, a naturally occurring phenol extracted from Cistanche tubulosa, Echinacea angustifolia, and Cistanche spp, has a wide range of physiological effects, such as antioxidation, neuroprotection, anti-inflammatory, and immunoregulation, which are closely related to depression. In addition, echinacoside can activate protein kinase B (Akt), extracellular signal-regulated kinase (ERK), and brain-derived neurotrophic factor (BDNF) in the brain. A key downstream event of the Akt, ERK, and BDNF signaling pathways, namely mechanistic target of rapamycin (mTOR) signaling, plays a crucial role in generating an rapid antidepressant effect. Thus, echinacoside is a promising therapeutic agent for depression. However, research regarding the role of echinacoside in antidepressant effect and brain mTOR activation remains lacking.

Materials and methods: The forced swimming test and Western blot analysis in C57BL/6 mice was used to investigate the antidepressant-like activities of echinacoside and the underlying mechanism involved inα-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-Akt/ERK-mTOR pathway.

Results: We confirmed the suggestions by previous reports that echinacoside activates Akt/ERK signaling and further demonstrated that echinacoside could provide antidepressant-like effects in mice via the activation of AMPAR-Akt/ERK-mTOR pathway in the hippocampus.

Conclusions: To the best of our knowledge, our study is the first to reveal that echinacoside is a potential treatment for depressive disorders. Moreover, the present study suggests a mechanism for the neuroprotective effect of echinacoside.

Keywords: AMPAR; Akt; Antidepressant; ERK; Echinacoside; mTOR.

Fig. 1

Experimental mice treated with saline, desipramine (20 mg/kg, a tricyclic antidepressant as a positive control) and three different doses of echinacoside (20, 30 or 40 mg/kg) by intraperitoneally injection then detected behavior by forced swimming test (FST) (A) or open field test (OFT) (B). Mice were respectively administrated by normal saline, desipramine (20 mg/kg) and echinacoside (20, 30 or 40 mg/kg) 30 min before FST and 15-min pre-swimming were conducted at 24 h before the experiment (A). The percentage of immobility time significantly reduced in the groups of three different doses of echinacoside and desipramine-treated group (C) (ANOVA, F(4,45) = 32.454, p < 0.001, n = 10 per group). Mice were injected by normal saline, desipramine (20 mg/kg) and echinacoside (20, 30 or 40 mg/kg) 30 min before OFT (B). The total distance moved linked to the animals’ locomotor activity ability showed no difference between saline and all treating groups (D) (ANOVA, F(4,45) = 1.859, p> 0.05, n = 10 per group). (**p < 0.01, ***p < 0.001 compared with saline-treated group with Tukey post hoc analysis); values shown are mean ± SEM

Fig. 2

Effects of different echinacoside doses (20 mg/kg, 30 mg/kg or 40 mg/kg) and desipramine (20 mg/kg) intraperitoneally injected on the phosphorylation of mTOR, Akt, and ERK in the hippocampus of mice. Western blot analysis of phosphorylation of mTOR, Akt and ERK were conducted (A). The densitometry analyses of the blot (normalized to β-actin) verify the enhanced activity of pmTOR in group treated with echinacoside at 20 mg/kg and 30 mg/kg, not in groups treated with echinacoside at 40 mg/kg and desipramine (B). The increased pAkt in the mice hippocampus following treatment with echinacoside were showed in a dose-dependent manner (C). Only echinacoside at 30 mg/kg significantly increased pERK (D). Total levels of mTOR, Akt, and ERK were not different among the groups. (*p < 0.05 compared with saline-treated group by Mann-Whitney U test); n = 4 per group, values shown are mean ± SEM

Fig. 3

Effects of NBQX (30 mg/kg) or rapamycin (20 mg/kg) on the immobility duration in forced swim test (FST) and Western blotting of pmTOR, pAkt, and pERK from hippocampus of echinacoside (30 mg/kg)-treated mice. The timeline exhibits the experimental procedure under administration of drugs (A). Statistically analysis showed that the effect of reduction of immobility duration in FST resulted from echinacoside treatment is inhibited when the mice were pretreated with NBQX and rapamycin (B) (ANOVA, F(5.54) = 4.501, p < 0.01; n = 10 per group; *p < 0.05, **p < 0.01 compared with saline-treated group with Tukey post hoc analysis). Western blot analysis of phosphorylation of mTOR, Akt, and ERK was performed (C). The densitometry analyses of the blot (normalized to β-actin) confirmed the increased activity of pmTOR (D), pAkt (E), and pERK (E) in the echinacoside administrated group. The increased expression of pmTOR (D), pAkt (E), and pERK (F) resulted from echinacoside treatment being blocked when mice were pretreated with NBQX. The increased expression of pmTOR (D) resulted from echinacoside treatment is blocked when mice were pretreated with rapamycin and the increased expression of pAkt (E) and pERK (F) resulted from echinacoside treatment is not blocked; n = 4 each group; * p < 0.05, Mann–Whitney U test; Values shown are mean ± SEM

Fig. 4

Effects of SL327 (40 mg/kg) or MK2206 (60 mg/kg) on the antidepressant-like effect and Western blotting of pmTOR, pAkt, and pERK of echinacoside (30 mg/kg)-treated mice. The timeline exhibits the experimental procedure under administration of drugs (A). Statistically analysis showed that the effect of reduction of immobility duration in FST resulted from echinacoside treatment is inhibited when the mice were pretreated with SL327 and MK2206 (B) (ANOVA, F(5.54)=7.195, p < 0.001; n=10 per group; *p < 0.05, **p < 0.01 compared with saline-treated group with Tukey post hoc analysis). Western blot analysis of phosphorylation of mTOR, Akt, and ERK was performed (C). The densitometry analyses of the blot (normalized to β-actin) confirmed the increased activity of pmTOR (D), pAkt (E), and pERK (E) in the echinacoside administrated group. Western blotting shows the increased expression of pmTOR caused by echinacoside treatment is blocked when the mice were pretreated with SL327 and MK2206 (D). The enhanced expression of pAkt (E) is blocked by pretreatment of MK2206, not SL327. The enhanced expression of pERK (F) is blocked by pretreatment of SLE 327, not MK2206. Total levels of Akt, ERK and mTOR were not different among the four groups. n = 4 each group; * p < 0.05, Mann–Whitney U test; Values shown are mean ± SEM

Fig. 5

Representative Western blotting of pGluA1ser845 and pGluA1ser831 from hippocampus of mice treated with saline, desipramine (20 mg/kg) or echinacoside (20, 30 or 40 mg/kg) (A), after echinacoside (30 mg/kg) administration with pretreatment with NBQX (30 mg/kg) or rapamycin (20 mg/kg) (D) and after echinacoside (30 mg/kg) administration with pretreatment with SL327 (40 mg/kg) or MK2206 (60 mg/kg) (G). Echinacoside at 30 mg/kg treatment significantly increases the expression of pGluA1ser845 (B) and pGluA1ser831 (C), which were not observed in mice with other treatments. The densitometry analyses of the blot (normalized to β-actin) verify the enhanced activities of pGluA1ser845 (E), and pGluA1ser831 (F) in the echinacoside at 30 mg/kg administrated group and decreased activities in the NBQX, but, not in rapamycin pretreated groups (D–F). The up-regulations of pGluA1ser845 and pGluA1ser831 were not blocked by pretreatment of SL327 or MK2206 (G–I). n = 4 each group; *p < 0.05, Mann–Whitney U test; Values shown are mean ± SEM

Fig. 6

Representative Western blotting of the expression of BDNF from hippocampus of mice treated with saline, desipramine (20 mg/kg) or echinacoside (20, 30 or 40 mg/kg) (A), after echinacoside (30 mg/kg) administration with pretreatment with NBQX (30 mg/kg) or rapamycin (20 mg/kg) (B) and after echinacoside (30 mg/kg) administration with pretreatment with SL327 (40 mg/kg) or MK2206 (60 mg/kg) (C). Echinacoside at 20, 30 mg/kg treatment significantly increases the expression of BDNF (B), which was not observed in mice with other treatments. The densitometry analyses of the blot (normalized to β-actin) verify the enhanced expression of BDNF (B) in the echinacoside at 30 mg/kg administrated group and decreased activities in the NBQX and rapamycin pretreated groups (B). The up-regulation of BDNF was also blocked by pretreatment of SL327 or MK2206 (C). n = 4 each group; *p < 0.05, Mann–Whitney U test; Values shown are mean ± SEM