Essential oil from the roots of Paeonia lactiflora pall. has protective effect against corticosterone-induced depression in mice via modulation of PI3K/Akt signaling pathway

Abstract

For thousands of years, the roots of Paeonia lactiflora Pall (PLP) has been considered by traditional Chinese medicine as a drug that can improve mental or emotional disorders, including depression, anxiety and affective disorders. Unfortunately, the research on the mechanism of action and active ingredients of this beneficial drug is not comprehensive. This study focused on the activity of essential oil from PLP (EOP), systematically studied the antidepressant effect of EOP for the first time, and discussed the potential mechanism of its antidepressant effect. In this study, we used a mouse model of corticosterone (CORT)-induced depression, and found that EOP had a significant antidepressant effect on the symptoms of CORT-induced depression in mice, and significantly down-regulated the levels of CRH, ACTH and cortisol in the brain tissues of mice. In addition, we found that EOP treatment alleviated CORT-induced hippocampal neuron injury in mice In vitro experiments. It was also found that EOP could inhibit CORT-induced apoptosis and improve the proliferation ability and cell viability of PC12 cells. Further, with the help of network analysis, it was revealed that PI3K-Akt might be one of the main signaling pathways of EOP against CORT-induced hippocampal neuron apoptosis. In this study, we also found that EOP up-regulated the phosphorylation of PI3K and Akt in CORT-induced mouse hippocampal neurons and PC12 cells, and promoted the nuclear transcription of Nrf2 in CORT-induced PC12 cells. In conclusion, with the integrated approach, we demonstrated that EOP exerted anti-apoptotic effects on hippocampal neurons through PI3K/Akt/Nrf2 signaling pathway.

Keywords: PI3K-Akt pathway; apoptosis; depression; experimental techniques; neuroprotection; oxidative stress.

FIGURE 1

EOP improves basic condition and behavior in corticosterone (CORT)-induced mice (A) Changes in body weight, (B) changes in food intake, (C) changes in sucrose preference percentage (%), (D) Forced swimming test (FST) rest time, (D) immobility time in tail suspension test (TST), (F) motion track in the open-field test (OFT), (G) time in the central grid of OFT (s), (H) p percent of central grid crossing number in the OFT (%). Data expressed as Mean ± SD, n = 6. **p＜0.01, *p＜0.05 vs. CORT group.

FIGURE 2

Effects of EOP on the morphology and structure of hippocampal neurons in mice (A) Hippocampal HE staining (400X), (B) hippocampal Nissl degeneration staining (400X), (C) changes of neurotransmitters in mouse brain tissue. Data expressed as Mean ± SD, n = 3, **p＜0.01, *p＜0.05 vs. CORT group.

FIGURE 3

EOP reduced the effect of CORT on the viability of PC12 cells (A) Effects of different concentrations of EOP on the viability of PC12 cells. (B) Effects of different concentrations of CORT on the viability of PC12 cells (C) Effects of EOP on the viability of PC12 cells treated with CORT. (D) Morphological changes of PC12 cells treated with EOP and CORT. (E) AO-EB staining representative diagram of PC12 cells after EOP and CORT intervention. Data expressed as Mean ± SD, n = 3, **p＜0.01, *p＜0.05 vs. CORT group.

FIGURE 4

EOP inhibits CORT-induced apoptosis of PC12 cells (A) Effects of EOP on CORT-induced cell membrane potential collapse of PC12 cells. (B) Effects of EOP on CORT-induced apoptosis of PC12 cells. Data expressed as Mean ± SD, n = 3. **p＜0.01, *p＜0.05 vs. CORT group.

FIGURE 5

EOP inhibited CORT-induced oxidative stress in PC12 cells (A) Laser confocal observation of EOP improved ROS production in PC12 cells, (B) flow cytometry analysis of ROS changes in PC12 cells. Data expressed as Mean ± SD, n = 3. **p＜0.01, *p＜0.05 vs. CORT group.

FIGURE 6

Prediction of antidepressant effects of EOP by network pharmacology (A) Intersection of the depression target library and the EOP target library. (B) EOP-composition-depressive disorder network map (EOP-DD) (C) Interaction network diagram of intersection gene (PPI). (D) KEGG enrichment results of intersection genes.

FIGURE 7

EOP treatment induced PI3K phosphorylation in hippocampal neurons of CORT-induced depressed mice, n = 3 (A) The changes of PI3K phosphorylation in hippocampal CA1 neurons were observed by immunofluorescence (IF). (B) The changes of PI3K phosphorylation in hippocampal CA3 neurons were observed by IF. **p＜0.01, *p＜0.05 vs. CORT group.

FIGURE 8

EOP treatment induced AKT phosphorylation in hippocampal neurons of CORT-induced depressed mice, n = 3 (A) The changes of AKT phosphorylation in hippocampal CA1 neurons were observed by immunofluorescence (IF). (B) The changes of AKT phosphorylation in hippocampal CA3 neurons were observed by IF. **p＜0.01, *p＜0.05 vs. CORT group.

FIGURE 9

EOP promoted the phosphorylation of PI3K in CORT-induced PC12 cells (A) Immunofluorescence (IF) assay demonstrated that EOP promoted the phosphorylation of PI3K in CORT-induced PC12 cells (B) The statistical results of the fluorescence intensity of the IF experiment. (C) WB was used to detect the effect of EOP on the phosphorylation of PI3K in CORT-induced PC12 cells. (D) Statistical results of the WB experiment. Data expressed as Mean ± SD, n = 3. **p＜0.01, *p＜0.05 vs. CORT group.

FIGURE 10

EOP promoted the phosphorylation of AKT and nuclear transcription of Nrf2 in CORT-induced PC12 cells. (A) Immunofluorescence (IF) assay demonstrated that EOP promoted the phosphorylation of AKT in CORT-induced PC12 cells. (B,G) WB was used to detect the effect of EOP on the phosphorylation of AKT in CORT-induced PC12 cells. (C) Statistical results of the IF experiment (D) IF assay confirmed that EOP promoted the nuclear transcription of Nrf2 in CORT-induced PC12 cells. (E–I) WB was used to detect the effect of EOP on CORT-induced NRF2 expression in the nucleus and cytoplasm of PC12 cells. Data expressed as Mean ± SD, n = 3. * *p＜0.01, *p＜0.05 vs. CORT group.