Engeletin alleviates depression-like phenotype by increasing synaptic plasticity via the BDNF-TrkB-mTORC1 signalling pathway

Abstract

Major depressive disorder (MDD) is a severe mental disorder associated with high rates of morbidity and mortality. Current first-line pharmacotherapies for MDD are based on enhancement of monoaminergic neurotransmission, but these antidepressants are still insufficient and produce significant side-effects. Consequently, the development of novel antidepressants and therapeutic targets is desired. Engeletin, a natural Smilax glabra rhizomilax derivative, is a compound with proven efficacy in treating ischemic stroke, yet its therapeutic effects and mechanisms for depression remain unexplored. The effects of engeletin were assessed in the forced swimming test (FST) and tail suspension test (TST) in mice. Engeletin was also investigated in the chronic restraint stress (CRS) mouse model of depression with fluoxetine (FLX) as the positive control. Changes in prefrontal cortex (PFC) spine density, synaptic plasticity-linked protein expressions and the brain-derived neurotrophic factor (BDNF)-tyrosine kinase B (TrkB)- mammalian target of rapamycin complex 1 (mTORC1) signalling pathway after chronic stress and engeletin treatment were then investigated. The TrkB and mTORC1 selective inhibitors, ANA-12 and rapamycin, respectively, were utilized to assess the engeletin's antidepressive mechanisms. Our data shows that engeletin exhibited antidepressant-like activity in the FST and TST in mice without affecting locomotor activity. Furthermore, it exhibited efficiency against the depression of CRS model. Moreover, it enhanced the BDNF-TrkB-mTORC1 pathway in the PFC during CRS and altered the reduction in dendritic spine density and levels of synaptic plasticity-linked protein induced by CRS. In conclusion, engeletin has antidepressant activity via activation of the BDNF-TrkB-mTORC1 signalling pathway and upregulation of PFC synaptic plasticity.

Keywords: BDNF-TrkB-mTORC1 signalling pathway; chronic restraint stress; engeletin; major depressive disorder; prefrontal cortex; synaptic plasticity.

FIGURE 1

The chemical structure of engeletin.

FIGURE 2

Engeletin produces an antidepressant‐like effect in the FST and TST. The mice received a single dose of saline (CON), FLX (10 mg/kg) or engeletin (2.5, 5, 10, 20 mg/kg). After 1 h of treatments, the behavioural tests were performed. (A) Engeletin dose‐dependently reduced the immobility time in the FST. (B) Engeletin dose‐dependently reduced the immobility time in TST. (C) Engeletin indicated no effect on spontaneous locomotor function in the OFT. The data are depicted as mean ± SEM (n = 12). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 substantially different from control; one‐way anova followed by post hoc LSD test.

FIGURE 3

Engeletin reverses CRS‐induced depressive‐like behaviours in mice. (A) Experimental protocol timeline. During the investigation, the mice's body weight was assessed every 7 days and behavioural tests, including TST, FST, SPT and OFT, were carried out on each mouse/group at the end of the experiment. (B) Influence of engeletin on the CRS mice's body weight alterations. (C) The sucrose preference rate in the SPT. (D) The FST immobility time. (E) The TST immobility time. (F) The total distance travelled in the OFT. The data are indicated as mean ± SEM (n = 11–12). *p < 0.05, **p < 0.01, ****p < 0.0001, two‐way anova followed by post hoc Bonferroni's test.

FIGURE 4

Engeletin reverses the change in PFC spine density and the decreased protein expression of PFC synaptic plasticity induced by CRS. (A) Representative photomicrograph of a Golgi‐stained PFC pyramidal neuron from each mouse/group. Scale bar = 10 mm. (B) The DSs/10 μm were quantified by Golgi staining. (C, D) Western blotting indicated that the engeletin regimen reversed the CRS‐mediated reduction of PFC's GLUR1 and PSD95 proteins. Data are depicted as mean ± SEM (n = 3). *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001. Two‐way anova followed by post hoc Bonferroni's test.

FIGURE 5

Chronic engeletin treatment increases BDNF signalling in the PFC of stressed animals. (A) RT‐PCR data indicated that 28 days of chronic engeletin treatment reversed the reduction of BDNF mRNA in CRS‐mediated PFC (n = 3). (B) Western blotting indicated that the engeletin regimen reversed the CRS‐mediated reduction of PFC's BDNF protein (n = 3). (C) Experimental protocol timeline. (D) In the CRS model, engeletin + ANA‐12 mice had reduced sucrose preference than engeletin mono‐treatment mice (n = 12). (E, F) In the CRS model, engeletin + ANA‐ 12 mice exhibited increased immobility time than engeletin mono‐treatment mice in the TST and FST (n = 12). (G) Neither ANA‐12 nor engeletin substantially affected the locomotor function of naive mice in the OFT (n = 12). Data are indicated as mean ± SEM. *p < 0.05, **p<0.01, ****p<0.0001. Two‐way anova followed by post hoc Bonferroni's test.

FIGURE 6

Rapamycin treatment prevents the effect of engeletin on depressant‐like depressant‐likes and synaptic plasticity in the PFC. (A, B) Western blotting indicated that the engeletin regimen reversed the CRS‐mediated reduction of PFC's p‐mTOR protein (n = 3). (C) Experimental protocol timeline. (D) In the CRS model, engeletin + rapamycin mice had reduced sucrose preference than engeletin mono‐treatment mice (n = 12). (E, F) In the CRS model, engeletin + rapamycin mice exhibited increased immobility time than engeletin mono‐treatment mice in the TST and FST (n = 12). (G) Neither rapamycin nor engeletin substantially affected the locomotor function of naive mice in the OFT (n = 12). (H, I) Golgi‐staining indicated that engeletin + rapamycin mice reversed the CRS‐mediated decrease of PFC DS density (n = 3). (J–L) Western blotting indicated that the engeletin + rapamycin reversed the engeletin‐mediated increase of PFCs' GLUR1 and PSD95 protein (n = 3). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two‐way anova followed by post hoc Bonferroni's test.