GLP-1R Activation Ameliorate Novel-object Recognition Function via Hippocampal AMPK/NF-κB Signaling Pathway in Neuropathic Pain Mice

Background: Growing evidences indicate that neuropathic pain is frequently accompanied with cognitive impairments, which aggravate the decrease in the quality of life of chronic pain patients. Furthermore, it has been shown that the activation of Glucagon-like-peptide-1receptor (GLP-1R) improved memory decit in multiple diseases such as Alzheimer’s disease (AD), stroke and so on. However, whether activating GLP-1R could improve memory impairment induced by neuropathic pain and the mechanism by which this improvement would occur remain unclear. Methods: The spared nerve injury (SNI) model was established as a kind of neuropathic pain. And novel-object recognition memory (hippocampus-dependent memory) was tested by the novel object recognition test (NORT).The expression levels of GLP-1,GLP-1R,adenosine monophosphate-activated protein kinase (AMPK),p-AMPKThr172, nuclear factor κ B p65 (NF-κB p65), interleukin-1β p17(IL-1β p17),and the synaptic associated proteins were tested in the murine hippocampus with memory decits caused by neuropathic pain. Then, exenatide acetate (Ex-4, a GLP-1R agonist), exendin(9-39)(Ex(9-39),a GLP-1R antagonist) and Compound C dihydrochloride ( CC, an AMPK inhibitor) were used to test the effects of activating GLP-1R in mice with neuropathic pain. Results: First, we uncovered that neuropathic pain could inhibit GLP-1/GLP-R axis, disturb inammatory signaling pathway, increase the expression of IL-1β and downregulate the synaptic associated proteins (postsynaptic density protein 95(PSD95) and Arc). Subsequently, we reported that Ex-4 treatment could improve recognition memory impairment and increase the ratio of p-AMPKThr172/AMPK, decrease the expression of NF-κB p65 and IL-1β p17, upregulate the levels of PSD95 and anti-PSD95 (1:1000; Cell Signaling Technology); rabbit anti-Arc (1:1000;ABclonal); rabbit anti-AMPK (1:1000; Cell Signaling Technology); rabbit anti-p-AMPKThr172 (1:1000; Cell Signaling Technology); rabbit anti-NF-κBp65 (1:1000; Proteintech), rabbit anti-pNF-κBp65 (1:1000; Cell Signaling Technology); rabbit anti-IL-1β p17 (1:1000; Anity). The protein bands tested (Pierce Western Blotting Substrate; Thermo Scientic) using a computerized image analysis system XRS+; BIO-RAD). with group). c training were no signicant differences in time spent on exploring two identical objects (familiar object) between the and SNI groups(n=10 mice per group). d In the NORT testing phase, sham mice spent more time exploring novel objects rather than familiar objects compared to the SNI group(n=10 mice per group). e DR represented recognition memory; the SNI mice demonstrated a signicant reduction in DR compared with the sham group(n=10 mice per group). f-g Neuropathic pain dramatically downregulated the protein expression level of GLP-1 and GLP-1R in the hippocampus. Data are presented as mean ± SEM,*P (cid:0) 0.05;**P (cid:0) 0.01;***P (cid:0) 0.001;****P (cid:0) 0.0001, compared with the sham group.


GLP-1R Activation
neuroin ammaton is a critical factor contributing to hippocampus-dependent memory impairment [9].Neuropathic pain can alos cause the release of pro-in ammatory cytokines in the hippocampus [3].
It has been reported that AMPK, as a downstream of GLP-1R, can be activated when activating GLP-1R [10][11][12].Furthermore, the activation of AMPK can inhibit the downstream protein p65 NF-κB, which is involved in controlling neuroin ammation [13,14]. Additionally, activation of the NF-κB could promote the secretion of multiple pro-in ammatory cytokines including IL-1β [14,15], which contribute to neuropathic pain-induced memory impairment [16,17], and IL-1β p17 is a mature fragment of IL-1β.In addition, the activation of GLP-1R can attenuate the increased levels of IL-1β in the brain [8,18].
However, little is known about the neuroprotection of GLP-1R activation during the chronic pain phase.
Also, whether the activation of GLP-1R could improve memory impairment induced by SNI, and the speci c mechanism by which this improvement would occur are still unclear. Here, we hypothesized that activating the GLP-1R/AMPK signaling pathway would attenuate memory de cits induced by neuropathic pain via the inhibition of neuroin ammation, raising the levels of synaptic associated proteins.

Animals
Male C57 BL/6(7-8 weeks) mice were purchased from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China. Mice were raised under controlled conditions (22-25°C, 12-hour alternate circadian rhythm, food, with food and water ad libitum). All animal studies were approved by the Animal Care and Use Committee of Tongji hospital.

Induction of neuropathic pain
The establishment of SNI models was according to Decosterd and Woolf [19].Brie y, under pentobarbital sodium anesthesia (50mg/kg, intraperitoneally), the left sciatic nerve (including the 3 branches: common peroneal , tibial, sural nerve) was exposured . The 6.0 silk thread (or suture) was tightly ligated with the two branches of the sciatic nerve (common peroneal nerve and tibial nerve). The distal end of the ligation site was cut off, and the distal nerve stump of 2-4 mm was removed. For the sham group, we only exposed the sciatic nerve without ligation.

Novel object recognition test
The NORT was conducted as previously described [9]. Brie y, the mice were handled for 1minute a day continuing 6 days before the test. Then, mice were put into the box and adapted to the condition for 5 minutes. In the training section, the mice were allowed to explore two identical rectangular blocks (familiar objects) for 5 minutes and rectangular blocks were placed on the same side of the box. Sni ng, licking, and climbing on objects were all regarded as the exploratory behaviors. In the testing phase, a rectangular block was superseded by a cylinder (novel object), and mice were allowed to explore the two objects for another 5 minutes in the box. The cognitive function was evaluated by the discrimination ratio (DR) which is represented by T C / (T R + T C ), where T C is the time spent exploring the novel object, T R is the time spent exploring the familiar object, and T R + T C is the total time spent exploring the two objects.

Experimental designs and Drugs treatment
There were four experiments in this study, as shown in (Fig 1A, 3A, 5A, 7A). First, we study whether neuropathic pain could induce recognition memory impairment. Then, to examine whether activating GLP-1R could alleviate memory impairment caused by neuropathic pain, Ex-4(0.4mg/kg, purchased from Selleck.cn) or Vehicle, was subcutaneously (s.c.) administrated into mice, starting from day 1 to day 21after surgery. The dosage of Ex-4 was selected according to previous research [21]. Ex(9-39)(0.1mg/kg, purchased from MedChemExpress) was intraperitoneally (i.p.) administered into mice 30 min before Ex-4 treatment from day 1 to day 21after operation. The selection of the dosage and administration route for Ex(9-39) was based on previous research [22].Finally, to test the neuroprotective mechanism of activating GLP-1R, CC(20mg/kg, purchased from MedChemExpress) was given to mice via intraperitoneal injection Page 6/26 30 min before Ex-4 ,from day 1 to 21 after nerve injury. The dosage of CC was selected depending on previous research [23].

Statistical analysis
All results are showed as mean ± SEM. When compared two groups, an unpaired Student's t test was used. When compared four groups, one-way ANOVA followed by Bonferroni post hoc test was used. Twoway ANOVA was used to analyze MPWT, NORT. GraphPad Prism 7.0 was used for statistical analysis, and P<0.05 as a statistically signi cant p value.

Results
SNI induced mechanical allodynia, hippocampus-dependent memory impairment and decreased GLP-1andGLP-1R in mice To evaluate development of mechanical allodynia after SNI surgery, the MPWT was tested on day 0(baseline),day 3,day 7,day 10,day 14,day 21 after the surgeries (Fig. 1B).The results showed that MPWT was signi cantly decreased in SNI mice ,compared to sham mice.
Next, we evaluated the novel-object recognition memory via the NORT. In the NORT, no signi cant difference was found in the total time spent on identical objects between the Sham and SNI groups during the training phase (Fig. 1C). In the testing stage, the mice spent more time on the novel object than on the familiar object in the Sham group (Fig. 1D).However, the time spent on the novel and familiar objects was similar in the SNI group. Furthermore, the discrimination ratio in the Sham group was higher than that in the SNI group (Fig. 1E).These results demonstrate that recognition memory was impaired in the mice with neuropathic pain.
In addition to this, we uncovered that the GLP-1/GLP-1R axis was inhibited in the murine hippocampus induced by neuropathic pain (Fig. 1F, G). West Blot showed that the levels of GLP-1 and GLP-1R were decreased in murine hippocampus.
SNI caused disturbance of in ammatory signaling, neuroin ammation, and decreased synaptic associated proteins in the murine hippocampus We found that the AMPK/NF-κB signaling was disturbed in the murine hippocampus with cognitive dysfunction. Neuropathic pain could inhibit the activation of AMPK by inhibiting its phosphorylation at the Thr172 site ( Fig. 2A) but activate its downstream NF-κB p65 by increasing the level of p-NF-κB p65 (Fig. 2B) compared with the Sham group. Furthermore, activating NF-κB p65 could induce neuroin ammation by up-regulating the expression of pro-in ammatory cytokines, including mature IL-1β (IL-1β p17) (Fig. 2C) in the hippocampus of mice. Also, over expressed IL-1β could decrease the level of synaptic associated proteins, such as PSD95 (Fig. 2D) and Arc (Fig. 2E), thus inducing memory impairment, compared with Sham mice.
Ex-4, a GLP-1R agonists, improved memory impairment caused by SNI in mice First, we evaluated the effects of Ex-4 on mechanical allodynia and memory de cit in mice with neuropathic pain after SNI. In the MPWT test, we found that both vehicle and Ex-4 application had no effect on allodynia in the SNI mice compared with that in the Sham + Veh and Sham + Ex-4 groups (Fig. 3B).
In the training phase of the NORT, no difference was found in the total time spent on identical objects among the four groups (Fig. 3C). In the testing stage, the mice spent more time on the novel object than on the familiar object in the Sham + Veh, Sham + Ex-4 and SNI + Ex-4groups.However, no difference was found in the time spent with novel and familiar objects in the SNI + Veh group (Fig. 3D). In addition, the DR in the SNI + Veh group was lower than that in the Sham + Veh group, but Ex-4 application could increase the DR in SNI mice compared with that in SNI + Veh mice (Fig. 3E).Taken together, these results suggested that Ex-4 can ameliorate de cits in the recognition memory caused by neuropathic pain.
Ex-4 improved in ammatory signaling, neuroin ammation, and upregulated synaptic proteins in mice hippocampus Next, we explored whether Ex-4 treatment could regulate the in ammatory signaling pathway and improve in ammation, the decreased synaptic associated-proteins. It was found that, in the murine hippocampus with memory de cits, Ex-4 could reverse the down-regulated levels of p-AMPKThr172 compared with Veh group (Fig. 4A), and could also inhibit the phosphorylation of NF-κB p65 downstream of AMPK (Fig. 4B).Furthermore, Ex-4 could attenuate neuroin ammation by down-regulating the secretion of IL-1β p17 (Fig. 4C).The results suggested that Ex-4 restored the decreased levels of PSD95 (Fig. 4D) and Arc (Fig. 4E) in the murine hippocampus, which was consistent with the memory protection of EX-4.
Ex(9-39),a GLP-1R antagonist, reversed the memory protection of Ex-4 in SNI mice We further veri ed the memory protective effect of activating GLP-1R in SNI mice via administration with Ex-4 and Ex(9-39).In the MPWT ,the results showed both Ex-(9-39) and Ex-4 + Ex(9-39) treatment did not affect the decrease of MPWT in SNI mice compared with that in the SNI + Veh group (Fig. 5B).In the NORT, the four groups spent a similar total time exploring two identical objects during the training phase (Fig. 5C). However, in the testing stage, the SNI + Ex-4 + Veh group spent more time on the novel object than on the familiar object compared with the SNI + Veh and SNI + Ex-4 + Ex(9-39) groups (Fig. 5D).It was found that Ex(9-39) could abrogate the increase in DR induced by Ex-4 in mice (Fig. 5E).
Ex(9-39) abolished the anti-in ammatory effect of Ex-4 in thehippocampus of SNI mice First, we reported that Ex-4 could activate AMPK by increasing the level of p-AMPKThr172, however, Ex(9-39) could inhibit the activation of AMPK induced by Ex-4 in SNI mice (Fig. 6A). Similarly, Ex(9-39) could also upregulate the decreased level of p-NF-κBp65 downstream of AMPK caused by Ex-4 in SNI mice (Fig. 6B).Moreover, Ex(9-39) could reverse the anti-in ammatory effect of Ex-4 by promoting the expression of IL-1β p17 (Fig. 6C).We also uncovered that Ex(9-39) could reversed the increased synaptic associated proteins PDS95 (Fig. 6D) and Arc (Fig. 6E) induced by Ex-4 in mice with memory impairment caused by neuropathic pain.

CC, an AMPK inhibitor, reversed improved recognition memory caused by Ex-4 in SNI mice
In order to further explore the mechanism of activating GLP-1R to improve recognition memory dysfunction caused by neuropathic pain, we applied Ex-4 and CC in SNI mice. We found that both CC and Ex-4 + CC did not in uence the MPWT in SNI mice as compared to that in the SNI + Veh mice (Fig. 7B). In the NORT, during the training phase, no signi cant difference was found in the total time spent on identical objects among the four groups (Fig. 7C).However, the SNI + Ex-4 group spent signi cantly more time on the novel object than on the familiar object compared to the SNI + Veh or SNI + Ex-4 + CC groups (Fig. 7D).About DR, the results demonstrated that application with CC could reverse the increased the DR induced by Ex-4 in SNI mice (Fig. 7E).

CC weakened the neuroprotection of Ex-4 in SNI mice
We used CC to verify whether the improved recognition memory effect via activation of GLP-1R was mediated by regulating the AMPK/NF-κB signaling pathway. We discovered that activating GLP-1R could inhibit the activation of NF-κB via AMPK. On the contrary, when an AMPK inhibitor was administered in mice, it was found that the protein level of p-NF-κB p65 in the SNI+Ex-4+CC group was signi cantly higher than that in the SNI+Ex-4+Veh group (Fig 8A).Furthermore, activation of GLP-1R by Ex-4 treatment in the mice with memory dysfunction induced by SNI could ameliorate neuroin ammation, but CC could abolish this effect (Fig 8B).We also tested synaptic-associated proteins, the results suggested that the combined application of Ex-4 and CC could downregulate the contents of PSD95( Fig 8C) and Arc (Fig 8D) proteins compared that of Ex-4 alone in SNI mice. The results suggested that activating GLP-1R might rescue the decreased level of synaptic associated proteins via improving neuroin ammation through regulation of AMPK/NF-κB signaling.

Discussion
The major ndings of this research were as follows: rst, neuropathic pain can impair recognition memory and the level of GLP-1R was decreased in the hippocampus of mice with memory impairment. Second, the activation of GLP-1R could improve memory impairments caused by neuropathic pain. Third, the function of Ex-4 of improving memory de cits induced by chronic pain of Ex-4 was reversed by GLP-1R antagonists. Fourth, AMPK inhibitors abrogated the neuroprotection of GLP-1R in mice with neuropathic pain. Taken together, it was likely that hippocampal GLP-1R played a critical role in protecting memory from neuropathic pain by downregulating the expression level of IL-1β by regulating the AMPK/NF-κB signaling pathway.
Previous studies have shown that the activation of GLP-1R could improve impaired learning and memory function [6,[24][25][26]. It was detected that GLP-1 and GLP-1R were both decreased in AD human brain and APP/PS1/Tau transgenic (3xTg) murine brains [26]. However, improving the level of GLP-1and GLP-1R expression by dipeptidyl peptidase-4 (DPP-4) inhibitors in the hippocampus and cortex of AD mice brains could improve impaired learning and memory, increase the O-Glycosylation and synaptic proteins, and decrease abnormal phosphorylation of tau and neuro laments (NFs) and intercellular β-amyloid (Aβ) accumulation [26]. In this study, we showed that the GLP-1/GLP-1R axis was repressed in mice with memory impairment induced by neuropathic pain. We also found that Ex-4 treatment (0.4 mg/kg,s.c.) did not ameliorate pain hypersensitivity induced by SNI. However, Gong et al. reported that intrathecal exenatide potently alleviated peripheral nerve injury (tight ligation of peripheral L5-L6 spinal nerves) induced hypersensitivity states by 60-90% in a dose and time-dependent antinociception way [27]. It is possible that the dose of Ex-4 in our experiment did not reach the concentration which could suppress pain hypersensitivity via activation of GLP-1R in the spinal cord.
AMPK is a critical sensor of energy status, which regulates cellular energy homeostasis [28]. Several studies have shown that activating AMPK could ameliorate in ammation by inhibiting the activation of NF-κB [13,14,29,30].Furthermore, it has been reported that AMPK could be activated as a downstream of GLP-1R,when GLP-1R is activated [10][11][12]31]. Stimulating GLP-1R using sitagliptin could facilitate axonal regeneration and locomotor functional repair after spinal cord injury via GLP-1R induced AMPK/PGC-1α signaling pathway activation [12]. A certain study has demonstrated that GLP-1 may suppress hepatic fat accumulation and in ammatory response induced by nutrients via the activation of the AMPK pathway [31].In our study, we demonstrated that AMPK was suppressed; however, NF-κB was hyperactive in mice with memory de cits caused by neuropathic pain. Nevertheless, the activation of GLP-1R could reverse the disturbance of AMPK/NF-κB signaling in the murine hippocampus induced by neuropathic pain.
It is well known that neuroin ammation is a common cause of memory de cits, and neuropathic pain often promotes the secretion of pro-in ammatory mediators in the murine hippocampus. The level of TNF-α in the bilateral hippocampus was increased, which could impair LTP in the hippocampus and reduce the dendrite lengths and spine densities in hippocampal CA1 pyramidal neurons [3]. However, it was found that the activation of GLP-1R could ameliorate neuroin ammation [7,8,18,32]. The activation of GLP-1R by Ex-4 could reduce the transcriptional expression level of pro-in ammatory cytokines (IL-1β, IL-6, IL-17, TNF-α) in the injured spinal cords of experimental autoimmune encephalomyelitis (EAE) mice [18]. Besides this, it was found that activating GLP-1R using Ex-4 could rescue surgery-induced spatial learning and memory de cits via inhibiting NF-κB signaling and reducing the expression of IL-1β [32]. In our study, we found that the activation of GLP-1R could reduce the secretion of mature IL-1β in murine hippocampus induced by neuropathic pain.
PSD95 is a member of the membrane-associated guanylate kinase (MAGUK) family, which can interact with certain neurotransmitter receptors, including N-methyl-D-aspartate (NMDA) receptors [33], and αamino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [34], and participate in synaptic plasticity and synaptic stabilization in LTP [35]. Arc is a protein that modulates the subsynaptic cytoskeleton and LTP [36], which contributes to memory consolidation and represents the activation of neurons during learning. Also, Arc participates in multiple synaptic plasticities mediated by the AMPA receptors [37].Furthermore, the overexpression of IL-1β might downregulate the expression of PSD95 [38] and Arc [36], which contributes to memory impairment. In this study, we found that neuropathic pain decreased the contents of PSD95 and Arc in the hippocampus of mice with memory impairment caused by neuropathic pain, but activating GLP-1R could upregulate the protein levels of PSD95 and Arc.

Conclusions
In short, the current study demonstrated that GLP-1R plays a critical role in improving neuropathic paininduced novel object recognition memory dysfunction. Importantly, we found that the expression of GLP-1R was downregulated in the murine hippocampus with recognition memory dysfunction, which exacerbated neuropathic pain-induced recognition memory de cits. Moreover, the activation of GLP-1R by Ex-4 could reverse recognition memory dysfunction by regulating the AMPK/NF-κB signaling pathway, which could ameliorate neuroin ammation and restore the decreased expression of synaptic proteins. Therefore, GLP-1R might as a potential target for improving memory impairment induced by neuropathic pain.     Administration with Ex-4 could improve the unbalanced AMPK/NF-κB signaling pathway, neuroin ammation and upregulate the level of synaptic proteins in mice with memory impairment. a-b neuropathic pain decreased the expression level of p-AMPKThr172 and increased p-NF-κB p65; however, application with Ex-4 could reverse the effects (n=4 mice per group). c Compared to the SNI+Veh group, Ex-4 treatment could signi cantly reduce the protein level of IL-1β p17 (n=4 mice per group). d-e Ex-4 treatment abolished the decrease of synaptic proteins in mice with memory dysfunction induced by neuropathic pain (n=4 mice per group). Data are presented as mean ± SEM, *P<0.05; **P<0.01; ****P<0.0001; #P<0.05; ##P<0.01:###P<0.001; #### P <0.0001.

Figure 5
Ex(9-39), a GLP-1R antagonist, reversed the improved recognition memory caused by Ex-4 treatment. a The protocol of application with Ex(9-39) or Ex-4,pain behavior test and NORT. Ex(9-39) was The neuroprotection of Ex-4 was abrogated by treatment with GLP-1R antagonist. a-b Ex(9-39) reversed the attenuated disturbed AMPK/NF-κB signaling compared with SNI+Ex-4+Veh group. c Ex-4 could attenuate neuroin ammation, however; the effect was inhibited with Ex(9-39) application before Ex-4. d-e Ex(9-39) treatment could abolish the upregulated synaptic associated proteins induced by Ex-4 in the SNI(n=12 mice per group). c In the NORT training phase, there were no signi cant differences in the time spent on exploring two identical objects (familiar object) between the four groups(n=10 mice per group).
d In the NORT testing section, a signi cant difference was found in the time exploring novel objects and familiar objects in the SNI+Ex-4+Veh group compared with the SNI+CC+Ex-4 group(n=10 mice per group).