Protective effects of VMY-2-95 on corticosterone-induced injuries in mice and cellular models

Abstract

Nicotinic α4β2 receptor antagonists have drawn increasing attention in the development of new antidepressants. In this study, we aimed to investigate the protective effect of VMY-2-95, the new selective antagonist of α4β2 nicotinic acetylcholine receptor (nAChR) on corticosterone (CORT) injured mice and cellular models. Fluoxetine was applied as a positive control, and the effects of VMY-2-95 were investigated with three different doses or concentrations (1, 3, 10 mg/kg in mice, and 0.003, 0.03, 0.1 μmol/L in cells). As a result, VMY-2-95 showed significant antidepressant-like effects in the CORT injured mice by improving neuromorphic function, promoting hippocampal nerve proliferation, and regulating the contents of monoamine transmitters. Meanwhile, VMY-2-95 exhibited protective effects on cell viability, cell oxidant, cell apoptosis, and mitochondrial energy metabolism on corticosterone-impaired SH-SY5Y cells. Also, the PKA-CREB-BDNF signaling pathway was up-regulated by VMY-2-95 both in vitro and in vivo, and pathway blockers were also combined with VMY-2-95 to verify the effects furtherly. Therefore, we preliminarily proved that VMY-2-95 had protective effects in depressed mice and SH-SY5Y cells against injuries induced by corticosterone. This work indicated that the application of VMY-2-95 is a potential pharmacological solution for depression. This study also supported the development of α4β2 nAChR antagonists towards neuropsychiatric dysfunctions.

Keywords: Cholinergic–adrenergic theory; Corticosterone; Depression; PKA-CREB-BDNF signaling pathway; SH-SY5Y cells; VMY-2-95; α4β2 nAChR antagonist.

Graphical abstract

Figure 1

The structure of VMY-2-95·2HCl.

Figure 2

Behavior improving effects of VMY-2-95 in mice induced by corticosterone (CORT). (A) Changes in body weight, (B) immobility time in the tail suspension test (TST), (C) immobility time in the forced swimming test (FST), (D) changes in percent of sucrose preference, (E₁) motion track in the open-field test (OFT), (E₂) total crossing number in the OFT, (E₃) percent of central grid crossing number in the OFT, and (E₄) central grid crossing time in the OFT. Data are presented as mean ± SEM, n = 12. ∗∗P < 0.01, ∗∗∗P < 0.001 versus control group in A; and ^#P < 0.05, ^###P < 0.001 versus control group, and ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus model group in B–E.

Figure 3

The effects of VMY-2-95 on neuron morphology and proliferation. (A) Nissl denaturation staining in the hippocampus (400×, scale bar indicates 100 μm), (B₁) immunohistochemistry pictures of Ki67 in different groups of mice (400×, scale bar indicates 100 μm), (B₂) the mean optical density of Ki67 in hippocampus, (C₁) immunohistochemistry pictures of c-FOS in different groups of mice (400×, scale bar indicates 100 μm), (C₂) the mean optical density of c-FOS in hippocampus, (D) the effect of different concentrations of corticosterone (CORT) on the viability of cells, (E) the effect of different concentrations of VMY-2-95 on the viability of cells, (F) the effect of VMY-2-95 on the cell morphology (200×, scale bar indicates 100 μm), (G) the protect effect of VMY-2-95 on the cell viability, and (H) the protect effect of VMY-2-95 on the cytotoxicity. Data are presented as mean ± SEM, n = 3 in mice; and data are represented as the mean ± SD, n = 3 in SH-SY5Y cells. ∗P < 0.05, ∗∗∗P < 0.001 versus control cells in D and E; and ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus control group, and ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus model group in the others.

Figure 4

The effects of VMY-2-95 on contents of transmitter and expression of the receptors. The contents of acetylcholine (ACh, A) and norepinephrine (NE, B) in the plasma supernatant of mice, the contents of dopamine (DA, C) and 5-hydroxytryptamine (5-HT, D), and the mean optical density of dopamine 2 receptor (D2R, E₁) in dentate gyrus region of hippocampus, (E₂) immunohistochemistry pictures of D2R in different groups of mice (400×, scale bar indicates 100 μm), (F₁) the mean optical density of 5-hydroxytryptamine 1 receptor (5-HT1AR) in dentate gyrus region of hippocampus, (F₂) immunohistochemistry pictures of 5-HT1AR in different groups of mice (400×, scale bar indicates 100 μm), contents of corticotropin releasing hormone (CRH, G₁), adreno-cortico-tropic-hormone (ACTH, G₂), and corticosterone (CORT, G₃). Data are presented as mean ± SEM, n = 3 in E and F, and n = 10–12 in others. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus control group, and ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus model group.

Figure 5

The neuroprotective mechanisms of VMY-2-95 on SH-SY5Y cells against injury induced by corticosterone (CORT). The effect of VMY-2-95 on the contents of nitric oxide (NO, A), glutathione peroxidase (GPs, B), malondialdehyde (MDA, C), superoxide dismutase (SOD, D), and total antioxidant capacity of cells (E); flowcytometry analysis of the cell apoptosis results (F₁); the effect of VMY-2-95 on cell apoptosis ratio (F₂), cytochrome c (Cyt-c) level (G), adenosine triphosphate (ATP) level (H), and the activity of mitochondrial complex I (I). Data are represented as mean ± SD, n = 3. ^#P < 0.05, ^###P < 0.001 versus control cells, and ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus model cells.

Figure 6

Effects of VMY-2-95 on the PKA–CREB–BDNF signaling pathways. The protein expressions of (A) P-PKA and PKA, (B) P-CREB and CREB, and (C) BDNF in hippocampus; (D₁) immunohistochemistry pictures of BDNF in different groups of mice (400×, scale bar indicates 100 μm); (D₂) the mean OD of BDNF in hippocampus; the protein expressions of (E) P-PKA and PKA, (F) P-CREB and CREB, and (G) BDNF in SH-SY5Y cells; (H) cell viability when VMY-2-95 combined with H89 or J147; and (I) NO content when VMY-2-95 combined with H89 or J147. Data are presented as mean ± SEM, n = 12 in A–C; and data are presented as mean ± SD, n = 3 in others. ^##P < 0.01, ^###P < 0.001 versus control group, and ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus model group.