Effect of DADS pre-treatment on LPS-induced depression-like behavior in mice
In order to determine whether DADS can prevent LPS-induced depression-like behavior, the mice were pretreated with DADS (40 or 80 mg/kg, i.p.) at 1 h before LPS injection (100 μg/kg, i.p., Fig. 1A). For TST, a two-way ANOVA revealed significant effects for LPS stimulation (F1,72 = 5.38, P < 0.05), drug pre-treatment (F3,72 = 4.69, P < 0.01), and LPS × drug interaction (F3,72 = 11.68, P < 0.001). For FST, the two-way ANOVA revealed significant effects for LPS stimulation (F1,72 = 6.94, P < 0.05), drug pre-treatment (F3,72 = 2.79, P < 0.05), and LPS × drug interaction (F3,72 = 3.08, P < 0.05). Post hoc analysis showed that DADS pre-treatment at the dose of 40 and 80 mg/kg markedly prevented the LPS-induced increase in immobility time in the TST and FST in mice. Similarly, imipramine pre-treatment (10 mg/kg) also prevented the LPS-induced in immobility time in the TST and FST in mice (Fig. 1B, C). The OFT results showed that both DADS and imipramine pre-treatment did not affect the locomotor activity in mice with or without LPS treatment (no significant effects for LPS stimulation (F1,72 = 0.34, P = 0.56), drug pre-treatment (F3,72 = 0.34, P = 0.79), and LPS × drug interaction (F3,72 = 0.19, P = 0.91), Fig. 1D). These results indicate that DADS has a prophylactic effect on LPS-induced depression-like behavior in mice.
Effect of DADS post-treatment on LPS-induced depression-like behavior in mice
We next evaluated the inhibitory effect of DADS on LPS-induced depression-like behavior in mice in a post-treatment model, in which the mice were administered with DADS (40 or 80 mg/kg, i.p.) at either 1.5 or 23.5 h after LPS treatment (100 μg/kg, i.p., Fig. 2A). For TST, a two-way ANOVA revealed significant effects for drug post-treatment (F3,72 = 4.76, P < 0.05) and LPS × drug interaction (F3,72 = 5.09, P < 0.001), but not for LPS stimulation (F1,72 = 0.34, P = 0.56). For FST, the two-way ANOVA revealed significant effects for LPS stimulation (F1,72 = 11.84, P < 0.001), drug post-treatment (F3,72 = 8.68, P < 0.001), and LPS × drug interaction (F3,72 = 11.45, P < 0.001). Post hoc analysis showed that DADS, administered at both doses (40 and 80 mg/kg) after LPS injection, markedly reversed the LPS-induced increase in immobility time in the TST and FST in mice (Fig. 2B, C). Similarly, imipramine administration at the dose of 10 mg/kg after LPS injection also suppressed the LPS-induced in immobility time in the TST and FST in mice (Fig. 2B, C). In the OFT, the locomotor activity of mice administered with LPS, DADS, or imipramine was not affected (no significant effects for LPS stimulation (F1,72 = 0.90, P = 0.34), drug post-treatment (F3,72 = 0.30, P = 0.83), and LPS × drug interaction (F3,72 = 0.18, P = 0.91), Fig. 2D). These results indicate that DADS post-treatment can reverse LPS-induced depression-like behavior in mice.
Effect of DADS pre- or post-treatment on LPS-induced increase in IL-1β and TNF-α levels in the hippocampus and prefrontal cortex
The systemic inflammation induced by LPS could lead to pro-inflammatory cytokine production in the CNS, among which IL-1β and TNF-α was closely related to the induction of depression-like behavior in mice. In this study, we evaluated the change of IL-1β and TNF-α levels in the hippocampus and prefrontal cortex in both pre- and post-treatment models. In the pre-treatment model, a one-way ANOVA for IL-1β (hippocampus: F3,36 = 8.72, P < 0.001; prefrontal cortex: F3, 36 = 15.74, P < 0.001) and TNF-α (hippocampus: F3,36 = 7.95, P < 0.001; prefrontal cortex: F3,36 = 5.09, P < 0.001), and post hoc analysis showed that both imipramine pre-treatment (10 mg/kg) and DADS pre-treatment at the dose of 40 and 80 mg/kg prevented the LPS (100 μg/kg)-induced increase in IL-1β (Fig. 3A, B) and TNF-α (Fig. 4A, B) levels in the hippocampus and prefrontal cortex. In the post-treatment model, the one-way ANOVA for IL-1β (hippocampus: F3,36 = 6.73, P < 0.01; prefrontal cortex: F3,36 = 4.52, P < 0.01) and TNF-α (hippocampus: F3,36 = 5.77, P < 0.01; prefrontal cortex: F3,36 = 5.80, P < 0.01), and post hoc analysis showed that both imipramine post-treatment (10 mg/kg) and DADS post-treatment (40 and 80 mg/kg) reversed the LPS (100 μg/kg)-induced increase in IL-1β (Fig. 3C, D) and TNF-α (Fig. 4C, D) levels in the hippocampus and prefrontal cortex. These results indicate that DADS pre- and post-treatment can reverse LPS-induced neuroinflammatory responses in the brain.
Effect of DADS pre- or post-treatment on LPS-induced increase in nitrite levels in the hippocampus and prefrontal cortex
Since NO is a crucial molecule which can bridge the neuroinflammatory response and oxidative stress together, we evaluated the change of nitrite levels in the hippocampus and prefrontal cortex in mice administered with LPS and/or DADS. In the pre-treatment model, a one-way ANOVA (hippocampus: F3,36 = 12.44, P < 0.001; prefrontal cortex: F3,36 = 10.72, P < 0.001) and post hoc analysis for nitrite levels showed that both imipramine pre-treatment (10 mg/kg) and DADS pre-treatment (40 and 80 mg/kg) prevented the LPS (100 μg/kg)-induced increase in nitrite levels in the hippocampus (Fig. 5A) and prefrontal cortex (Fig. 5B). In the post-treatment model, the one-way ANOVA (hippocampus: F3,36 = 15.91, P < 0.001; prefrontal cortex: F3,36 = 10.30, P < 0.001) and post hoc analysis for nitrite levels showed that both imipramine post-treatment (10 mg/kg) and DADS post-treatment at the dose of 40 and 80 mg/kg reversed the LPS (100 μg/kg)-induced increase in nitrite levels in the hippocampus (Fig. 5C) and prefrontal cortex (Fig. 5D).
Effect of DADS pre- or post-treatment on LPS-induced alterations in markers reflecting the oxidative and anti-oxidative status of the hippocampus and prefrontal cortex
Finally, we measured the change of MDA and GSH, two markers reflecting the levels of oxidative and anti-oxidative status, in the hippocampus and prefrontal cortex in mice administered with LPS and/or DADS. In the pre-treatment model, a one-way ANOVA and post hoc analysis for MDA (hippocampus: F3,36 = 6.97, P < 0.001; prefrontal cortex: F3,36 = 7.77, P < 0.001) and GSH (hippocampus: F3,36 = 4.32, P < 0.05; prefrontal cortex: F3,36 = 4.66, P < 0.01) levels showed that both imipramine pre-treatment (10 mg/kg) and DADS pre-treatment at the dose of 40 and 80 mg/kg prevented the LPS (100 μg/kg)-induced increase in MDA levels (Fig. 6A, B) as well as the LPS-induced decrease in GSH levels (Fig. 7A, B) in the hippocampus and prefrontal cortex. In the post-treatment model, the one-way ANOVA and post hoc analysis for MDA (hippocampus: F3,36 = 7.79, P < 0.001; prefrontal cortex: F3,36 = 6.02, P < 0.01) and GSH (hippocampus: F3,36 = 6.52, P < 0.01; prefrontal cortex: F3,36 = 4.43, P < 0.01) levels showed that both imipramine post-treatment (10 mg/kg) and DADS post-treatment (40 and 80 mg/kg) reversed the LPS (100 μg/kg)-induced increase in MDA levels (Fig. 6C, D) as well as the LPS-induced decrease in GSH levels (Fig. 7C, D) in the hippocampus and prefrontal cortex. These results indicate that both DADS pre- and post-treatment are capable of inhibiting high levels of oxidative stress in the brain.