Pharmacological activation of GPR55 improved cognitive impairment, neuroinammation, oxidative stress and apoptosis induced by lipopolysaccharide in mice

Neuroinammation, oxidative stress and apoptosis are implicated in the pathogenesis of Alzheimer’s disease (AD). The purpose of the present study was to investigate the neuroprotective effects and possible mechanism of G-protein coupled receptor 55 (GPR55) agonist, O-1602, on lipopolysaccharide (LPS)-induced cognitive decits in mice. of immunohistochemical The


Introduction
Neuroin ammation, oxidative stress and apoptosis are the major processes in the development of Alzheimer's disease (AD) [1][2][3]. Neurodegenerative diseases including AD are commonly characterized by cognitive dysfunction, which is probably associated with the abnormal activation of neuroin ammation [4]. Oxidative stress is a state in which oxidants produce overwhelming antioxidant defense, which is associated with the onset of AD [5]. Increased oxidative stress can cause damage to lipids, DNA, and proteins, leading to decreased neuronal function [6]. Oxidative stress is thought to upregulate the production of amyloid β(Aβ) peptide by inducing β-and γ-secretase activity [7]. Lipopolysaccharide (LPS) is an endotoxin isolated from bacteria, which activates the immune system and leads to behavioral and memory impairments, neuroin ammation, and oxidative brain damage [5,8,9]. Previous studies have shown that intracerebroventricular (i.c.v) injection of LPS in mice has been used to establish animal models of memory loss [10,11]. I.c.v administration of LPS in mice produces cognitive impairment by expression of pro-in ammatory cytokines and neuronal death [12]. Considerable evidence has shown that nuclear factor-κB (NF-κB) is a key transcription factor that regulates the expression of proin ammatory cytokines, and the levels of oxidative stress in neurons [13,14]. The neuroin ammatory process is characterized by the activation of glial cells such as microglia. Microglia, the brain's resident innate immune cells, is an essential component of neuroin ammatory response [15].
In addition, LPS induces neuroin ammation and oxidative stress-mediated neuronal apoptosis by the activation of caspase [16,17]. Caspase-3, has been identi ed as a key mediator of apoptosis in neuronal cells [18]. Bax, a pro-apoptotic molecule, can induce neuronal cell death while Bcl-2 possesses the opposite effect [19].
G-protein coupled receptor 55 (GPR55) is an orphan G-protein coupled receptor, which is activated by endocannabinoids and lipid transmitters [20,21].The expression of GPR55 in mouse tissue has been declared in the nervous system that includes the hippocampus, though its function remains unknown [22]. GPR55 agonists have been shown to have a neuroprotective effect in Parkinson's disease [23], anxiety [24], and pain perception [25]. It has been reported that activation of GPR55 can strongly protect from ER stress-induced apoptosis in pancreatic β-cells [26]. O-1602, a speci c GPR55 agonist, can decrease in ammation, improve glycemic control and energy expenditure [27].To date, the effects of GPR55 activation on LPS-induced cognitive de cits, neuroin ammation, oxidative stress and apoptosis have never been examined. In this study, we investigated the neuroprotective effects of O-1602 on LPSinduced cognitive de cits, neuroin ammation, oxidative stress and apoptosis in mice.

Animals
Male Institute of Cancer Research (ICR) mice, aged 6-week-old and weighing between 25 g and 28 g were obtained from the Center of Laboratory Animal of Anhui (Grade II). Mice maintained under standard conditions (12-   4.0 µg/mouse). ICV injections of LPS were given using the stereotaxic apparatus according to our previous report [28]. Mice were anesthetized using chloral hydrate(350 mg/kg, i.p.) and immobilized on a stereotacticframe. The injection volume of 5 µl of LPS solution or PBS (1 µl/min) was inserted into the mediolateral at a site 1.0 mm caudal to the bregma, 1.5 mm from the midline, and 2.0 mm below the dural surface. The micropipettes were left in place for 5 min to minimize back ux of liquid. After 3 days, PBS (5 µl) with or without O-1602 was infused into the same site. After 3 days of recovery, some of the mice were subjected to behavioral tests, and the other part of mice was subjected to biological tests.  [28,29]. The test was carried out in an open eld chamber (72 cm × 72 cm × 36 cm). Mice were placed in the centre of the open eld and allowed a 5-min acclimation period. For each trial, mice were placed in a corner of the device and allowed to explore for 5 min. The total distance, and the time spend in central squares were recorded by an overhead video camera.

Morris water maze (MWM) test
Spatial learning and memory function were assessed by MWM test according to our previous studies [30]. It was conducted for 6 days including a 5-days training sessions, and a probe trial on the sixth day. The MWM consisted of a large circular black pool (120 cm diameter and 60 cm height) lled to a depth of 30 cm with water at 24 ± 2 °C. The maze was divided into four quadrants, and a clear platform (9 cm in diameter) was placed inside. A mounted ag (height, 5 cm) was xed to the platform throughout the visible-platform trial (days 1-2). Each mouse was subjected to four training sessions every day in both visible platform and hidden-platform (days 3-5). Mice were released into the water facing the wall of the tank from one of four separate quadrants and were allowed to nd the platform within 90 s. If a mouse failed to nd the platform in time, the mouse would be guided to the platform. The mouse would be left on the platform to view spatial cues for 30 s, and then returned to the cage. In probe test (day 6, without ag attached), the mice were allowed to swim in the water tank for 90 s without the hidden-platform. The trend of the mouse to search for platform was measured by the time spent in the target quadrant, where the platform was previously located. The number of target platform location crossings was recorded by video tracking equipment and processed by a computer equipped with an analysis-management system (ANY-maze video tracking system, stoelting Company, China).

Novel object recognition (NOR) test
The recognition memory of mice was evaluated by NOR test as described previously [30,31]. Mice were placed in the center of the open eld chamber(72 cm × 72 cm × 36 cm). First, mice completed an acquisition trial that consisted of leaving the animals in the apparatus that contained two identical objects. Twenty-four hours later, recognition memory was assessed and a different pair of dissimilar objects (a familiar and a novel one, respectively) were presented. Behavior was recorded by a video camera mounted vertically above the test arena and analyzed using appropriate video-tracking software (Duoyi, Shanghai, China). Object exploration was de ned as when the mouse touched or sniffed the object from no more than 2 cm away. The time spent exploring the two objects were recorded for 5 min. The time spent on the two objects was recorded (T familiar and T novel). Results were expressed as discrimination index (DI), i.e. (T novel -T familiar)/ (T familiar + T novel).

Passive avoidance test
The passive avoidance test was carried out in a trough-shaped apparatus consisted of a white illuminated chamber and a dark chamber (20 cm × 12 cm × 60 cm, respectively). On day 1, mice had a right to explore both chambers for 4 min twice to acclimatize. Next day, the training trial was performed for 5 min after a 3-min adaption. When mice entered the dark chamber, an electric foot shock (40 V, 2 mA) was delivered. After 24 h, the consolidation trial was performed for 5 min in the same way as training and the tracking system was started once mice were placed into the light chamber. The step-through latency and error times to enter the dark chamber were recorded [32,33].

Western blot analyses
The method was performed as described previously [30].

Statistical Analysis
The data are shown as mean ± standard error of mean (SEM). Group differences were analyzed by a twoway repeated measure ANOVA with "days" as the within-subject factor and "group" as the betweensubject factor in the MWM test. All other data were analyzed by a one-way ANOVA followed by a Dunnett's post-hoc analysis for multiple comparisons. All analyses were carried out using SPSS v20.0. P < 0.05 was considered statistically signi cant.
To con rm 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 signi cant 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 nonspatial learning and memory. In the consolidation trial, LPS-induced cognitive de cits 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, speci cally a de cit in memory, which can be ameliorated by O-1602 treatment.

O-1602 reversed LPS-stimulated GPR55 expression downregulation
To con rm whether the protective effects of O-1602 on LPS-induced cognitive de cits were associated with GPR55, the level of GPR55 in the hippocampus was detected by western blot assay. One-way ANOVA revealed that LPS signi cantly 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 de cits induced by LPS in mice.

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).

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 signi cantly higher in the hippocampus of LPS-treated mice as compared with the control. However, one-way ANOVA showed that O-1602 treatment signi cantly 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).

5. O-1602 inhibits the hippocampal proin ammatory cytokines secretion induced by LPS
Because neuroin ammation is mainly due to the excessive secretion of proin ammatory 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

6. O-1602 prevents microglia activation in the hippocampus induced by LPS
Due to the important role that microglia activation plays in LPS-induced neuroin ammation, 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 signi cantly inhibited by O-1602 treatment(P < 0.05 or P < 0.01; Fig. 6B), suggesting that O-1602 could suppress LPSinduced microglia activation in the hippocampus of DG region in mice.

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 signi cantly 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)

Discussion
This study showed that O-1602 prevented LPS induced cognitive de cits, including de cits in spatial learning and memory. The behavioral tests were used as robust and reliable tests that re ect hippocampal dependent learning and memory. In the hidden platform test of MWM, the escape latency of these mice was obviously increased after LPS exposure. For the probe test, LPS-induced mouse model showed signi cant decrease in the time spent on the target quadrant and the number of target crossings. LPS treatment also caused a signi cant decline in DI. In the consolidation of the passive avoidance task, LPS-induced cognitive de cits were observed as shorter latency and more error times into the dark chamber than those of sham-operated mice. In addition, LPS treatment in mice led to memory de cit with neuroin ammation, oxidative stress and apoptosis as evidenced by increased nuclear translocation NF-κB p65, elevated pro-in ammatory cytokines, activated microglia cells, increased the MDA level, and decreased SOD activity, and increased TUNEL-positive cells, activation of caspase-3, decreased the ratio of Bcl-2/Bax. Importantly, we also found that LPS treatment decreased GPR55 expression in the hippocampus. However, O-1602 signi cantly inhibited such adverse cognitive and biochemical changes induced by the i.c.v. injection of LPS. Overall, these suggest that O-1602 may have potential to protect against LPS-induced cognition impairments in mice.
Increasing evidence indicates that neuroin ammation may play a crucial role in the development of memory de cits [36,37]. It has been reported that the ROS system is associated with the pathology that results from neuroin ammation [38]. LPS, a stressor produced by gut microbiota, impairs memory and induces in ammatory responses in the hippocampus [39]. I.c.v. administration of LPS in mice produces cognitive impairment by expression of pro-in ammatory cytokines and neuronal death. This method can be effectively used as an animal model for AD [29,35,40]. Expression of several in ammatory genes such as in ammatory cytokines, can be regulated by NF-κB activation. Importantly, in this study, O-1602 treatment prevented improved neuroin ammation by inhibition of in ammatory signaling molecule (NF-κB p65) and cytokines (TNF-α, IL-1β and IL-6) in the hippocampus of LPS-exposed mice. We speculate that the anti-in ammation effect of O-1602 might be mediated through NF-κB signaling pathway.
Microglia, the brains resident immune cells, are a key element in in ammatory processes and are mediating chronic neuroin ammation and aggravation of AD pathology [41,42]. Activated microglia are the major source of ROS in the brain [43]. To detect oxidative stress during neuroin ammation induced by LPS and to verify the antioxidative effect of O-1602, we measured the level of MDA and SOD activity in the hippocampus. In this study, treatment with O-1602 attenuated the LPS-induced increase in MDA and decrease in the activity of SOD. This result agrees with the evidence that suggests that ROS-induced oxidative stress is one of the most common toxic mechanisms. Furthermore, ROS has been shown to be capable of modulating gene expression. ROS may trigger the generation of cytokines and participate in in ammatory signaling. In addition, NF-κB is central to the acquisition of the pro-in ammatory phenotype, and it is particularly sensitive to ROS [34]. It has been reported that GPR55 expression was observed in microglia [44], and inhibition of NF-κB transcriptional activity in the microglia nucleus suppressed proin ammatory cytokine expression. Thus, our results demonstrated that O-1602 attenuated in ammatory oxidative stress.
Growing evidence suggests that the potential role of apoptosis in AD has become an area of intense research in recent years [45][46][47]. LPS-induced neuroin ammation enhances apoptotic neurodegeneration [48]. Early cell changes that occur during apoptosis are mediated by the Bcl-2 family of proteins, including the anti-apoptotic Bcl-2 and pro-apoptotic Bax [16]. In this study, the results are consistent with previous ndings. Our results showed that the protein level of the Bcl-2 was decreased; whereas, Bax was increased following LPS administration. Further, treatment with O-1602 downregulated the expression of Bax and upregulated the expression of Bcl-2 and therefore, mitigated caspase 3 activity and the number of apoptotic cells in LPS-treated mice.

Conclusion
In summary, our study indicates that O-1602 displays neuroprotective effects on LPS-induced memory impairment, neuroin ammation, oxidative stress and apoptosis in mice. Therefore, activation of GPR55 has potential therapeutic value for the treatment of neurodegenerative diseases such as AD. However, further investigations are warranted to support this notion and to better understand the exact protective mechanisms of GPR55 in the brain.