3.1. O-1602 ameliorates LPS-induced cognitive deficits in mice
To investigate the effects of O-1602 on cognitive performance, mice were tested in the open field, NOR, MWM, and passive avoidance tests. Firstly, results showed that there was no significant difference among the groups in the total distance traveled and the time spend at the center (F[3, 44] = 0.32, P = 0.81; Fig. 1A), and the time spend at the center (F[3, 44] = 0.24, P = 0.87; Fig. 1B), suggesting that the impaired performance was not due to a decrease in spontaneous locomotor activity.
Next, we assessed the performance of mice in the visible-platform variant (days 1–2) of the MWM test. Mice in each group exhibited similar escape latency (4 trials/mouse/day for 2 days, effect of day, F [3, 428] = 11.07, P < 0.05; effect of group, F [3, 428] = 1.05, P > 0.05; effect of group-by-day interaction, F [3, 428] = 0.07, P > 0.05; Fig. 1C). We then tested the mice in the spatial hidden-platform variant (days 3–5), and the data appeared that LPS treatment increased escape latency compared to the control, these were reversed by O-1602(4 trials/d for 3 d, effect of day, F [3, 620] = 13.01, P > 0.05; effect of group, F [3, 620] = 2.12, P > 0.05; effect of group-by-day interaction, F [3, 620] = 0.22, P > 0.05; Fig. 1D). In the probe trial (1 trial/mouse for day 6), the data appeared that the mice in the LPS + Veh group displayed a significant decrease in the percentage of time spent in the target quadrant (F[3, 44] = 5.47, P < 0.01; Fig. 1E), and the number of platform location crossings (F[3, 44] = 6.38, P < 0.01; Fig. 1F) compared to the control, suggesting memory impairment in the LPS-treated mice. However, mice in the LPS plus O-1602 group showed significant increases in both indices compared to LPS + Veh group (Fig. 1E and 1F).
To confirm the results observed in the MWM test, we also carried out the NOR and passive avoidance tests. The discrimination index (DI) was calculated as mentioned earlier and shown in Fig. 1G reveals that the group that received LPS alone showed negative DI values, indicating non-spatial memory impairment where the mice spent more time exploring the familiar object than the novel object(F [3, 44] = 4.15, P < 0.01; Fig. 1G). Notably, treatment withO-1602 showed a significant increase in DI compared with the LPS + Veh group (P < 0.05; Fig. 1G).The passive avoidance task is a fear-aggravated test used to evaluate non-spatial learning and memory. In the consolidation trial, LPS-induced cognitive deficits were observed as shorter latency and more error times into the dark chamber than those of sham-operated mice(P < 0.05 or P < 0.01; Fig. 1H and I). Compared with LPS-injected group, O-1602 treatments prolonged the latency, and reversed the increase of error times. Together, these results suggest that LPS induced cognitive impairment, specifically a deficit in memory, which can be ameliorated by O-1602 treatment.
3.2 O-1602 reversed LPS-stimulated GPR55 expression downregulation
To confirm whether the protective effects of O-1602 on LPS-induced cognitive deficits were associated with GPR55, the level of GPR55 in the hippocampus was detected by western blot assay. One-way ANOVA revealed that LPS significantly reduced GPR55 expression in the hippocampus, which was reversed by O-1602 treatment (F [3, 12] = 6.78, P < 0.01; Fig. 2A and B). These results suggest that GPR55 might be involved in cognitive deficits induced by LPS in mice.
3.3. O-1602 inhibits LPS-induced oxidative stress
MDA is a well-established indicator of lipid peroxidation, and SOD acts as an endogenous scavenger of ROS[34]. Our data indicated that the MDA level was increased (F [3, 12] = 6.78, P < 0.01; Fig. 3A) and that SOD activity (F [3, 12] = 6.78, P < 0.01; Fig. 3A) was decreased in the LPS group compared to control group. However, treatment with O-1602 decreased the changes in MDA (P < 0.05) and SOD (P < 0.05).
3.4. O-1602 suppresses LPS-activated NF-κB signaling in the hippocampus
LPS causes memory impairment by the activation of the NF-κB signaling pathway[35]. In this study, nuclear translocation of NF-κB p65 was significantly higher in the hippocampus of LPS-treated mice as compared with the control. However, one-way ANOVA showed that O-1602 treatment significantly prevented the LPS-induced increased nuclear translocation of NF-κB p65 in the hippocampus(F [3, 12] = 13.36, P < 0.01; Fig. 4A and B).
3. 5. O-1602 inhibits the hippocampal proinflammatory cytokines secretion induced by LPS
Because neuroinflammation is mainly due to the excessive secretion of proinflammatory factors, the levels of TNF-α, IL-1β, and IL-6 were detected by ELISA. One-way ANOVA revealed that the levels of TNF-α, IL-1β, and IL-6 were much higher in the LPS group compared with the control group, which was significantly suppressed by O-1602 treatment (F [3, 12] = 13.05, P < 0.01 for TNF-α; Fig. 5A; F [3, 12] = 14.46, P < 0.01 for IL-1β; Fig. 5B; F [3, 12] = 9.20, P < 0.01 for IL-6; Fig. 5C). These results suggest that O-1602 inhibited LPS-induced accumulation of proinflammatory cytokines in hippocampus.
3. 6. O-1602 prevents microglia activation in the hippocampus induced by LPS
Due to the important role that microglia activation plays in LPS-induced neuroinflammation, we used IHC to investigate microglia activation. The results showed that LPS caused obvious microglia activation in the mouse hippocampal DG region(F [3, 12] = 5.92, P < 0.01, Fig. 6A and B). This effect was significantly inhibited by O-1602 treatment(P < 0.05 or P < 0.01; Fig. 6B), suggesting that O-1602 could suppress LPS-induced microglia activation in the hippocampus of DG region in mice.
3. 7. O-1602 Decreased Lps-induced Neuronal Apoptosis In The Hippocampus
To determine whether O-1602 affected neuronal apoptosis, we performed TUNEL assay in the hippocampal DG region. One-way ANOVA revealed that the TUNEL-positive cells numbers of the hippocampal DG region in the LPS + Veh group was significantly higher than the control group (F [3, 12] = 9.39, P < 0.01, Fig. 7B). However, O-1602 treatment decreased the number of apoptotic cells compared with LPS + Veh group(P < 0.05 or P < 0.01, Fig. 7B). The anti-apoptotic effects of O-1602 were further investigated using western blot. The results showed that the expression of active caspase-3 was decreased by O-1602 treatment. The expression of Bcl-2 protein was significantly decreased in the LPS-treated mice whereas O-1602 treatment was able to restore Bcl-2 protein content to that comparable to the Veh + Veh group. The Bax expression was significantly less than that of LPS + Veh group, and the ratio of Bcl-2/Bax was markedly increased after O-1602 treatment (Caspase-3:F [3, 12] = 6.64, P < 0.01; Fig. 7D; Bcl-2/Bax: F [3, 12] = 7.94, P < 0.01; Fig. 7E). These data suggested that O-1602 may ameliorate the learning and memory ability by reducing LPS-induced hippocampal neuron apoptosis.