Inhibition of the P38 MAPK/NLRP3 Pathway Mitigates Cognitive Dysfunction and Mood Alterations in Aged Mice after Abdominal Surgery Plus Sevoflurane

doi:10.21203/rs.3.rs-3803551/v1

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Inhibition of the P38 MAPK/NLRP3 Pathway Mitigates Cognitive Dysfunction and Mood Alterations in Aged Mice after Abdominal Surgery Plus Sevoflurane

https://doi.org/10.21203/rs.3.rs-3803551/v1

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Background

Cognitive dysfunction, encompassing perioperative psychological distress and cognitive impairment, is a prevalent postoperative complication within the elderly population, and in severe cases, it may lead to dementia. Building upon our prior research that unveiled a connection between postoperative mood fluctuations and cognitive dysfunction with the phosphorylation of P38, this present investigation aims to delve deeper into the involvement of the P38 MAPK/NLRP3 pathway in perioperative neurocognitive disorders (PND) in an abdominal exploratory laparotomy (AEL) aged mice model.

Methods

C57BL/6 mice (male, 18-month-old) underwent AEL with 3% anesthesia. Then, inhibitors targeting P38 MAPK (SB202190, 1 mg/kg) and GSK3β (TWS119, 10 mg/kg) were administered multiple times daily for 7 days post-surgery. The NLRP3-cKO AEL and WT AEL groups only underwent the AEL procedure. Behavioral assessments, including the open field test (OFT), novel object recognition (NOR), force swimming test (FST) and fear conditioning (FC), were initiated on postoperative day 14. Additionally, mice designated for neuroelectrophysiological monitoring had electrodes implanted on day 14 before surgery and underwent novel object recognition while their local field potential (LFP) was concurrently recorded on postoperative day 14. Lastly, after they were euthanasized, pathological analysis and western blot were performed.

Results

SB202190, TWS119, and astrocyte-conditional knockout NLRP3 all ameliorated the cognitive impairment behaviors induced by AEL in mice and increased mean theta power during novel location exploration. However, it is worth noting that SB202190 may exacerbate postoperative depressive and anxiety-like behaviors in mice, while TWS119 may induce impulsive behaviors.

Conclusions

Our study suggests that anesthesia and surgical procedures induce alterations in mood and cognition, which may be intricately linked to the P38 MAPK/NLRP3 pathway.

cognitive dysfunction

perioperative stress

aging

P38 MAPK

NLRP3

GSK3β

Perioperative neurocognitive impairment (PND) remains a significant concern in the aging population. PND is a global issue with substantial implications for both communities and families, and among elderly individuals, it is a prevalent central nervous system (CNS) disorder, with reported incidence rates reaching up to 52%^{[1, 2]}. As the elderly population continues to grow worldwide, the incidence of PND is expected to also rise. PND not only leads to prolonged hospital stays but also incurs higher healthcare costs, imposing a considerable economic burden on families and society at large. Nonetheless, the mechanisms underlying PND have been the subject of several theories, with neuroinflammation being one of the most extensively investigated^[3]. There exists compelling evidence linking neuroinflammatory processes to the development of various neurodegenerative conditions, including PND^[4]. The inflammatory hypothesis was initially proposed by Hensley et al., who demonstrated the overexpression or activation of pro-inflammatory signal transduction pathways in the brains of individuals with Alzheimer's disease (AD), notably emphasizing the involvement of P38 mitogen-activated protein kinase (P38 MAPK)^[5].

According to a recent study, individuals with AD and Parkinson's disease (PD) exhibit elevated levels of phosphorylated P38 MAPK in both their peripheral blood leukocytes and neural cells. Notably, the severity of AD appears to be correlated with the expression of phosphorylated P38 MAPK in peripheral blood^[6]. Our earlier research findings suggested a potential connection between phosphorylated P38 MAPK and reduced neuronal degeneration in mood swings and cognitive issues observed in aged mice treated 30 days following abdominal exploratory laparotomy (AEL)^[7]. Another study indicated that blocking the P38 MAPK signaling pathway may enhance cognitive function in rats^[8]. Concurrently, it is worth noting that the P38 MAPK signaling pathway can activate GSK3β^[9]. The intricate role of GSK3β^[10] is well-documented in its association with the pathogenesis of AD, stroke, and certain neuropsychiatric disorders^[11]. GSK3β serves as a pivotal kinase involved in the development of Tau pathology within the CNS. Moreover, it exerts regulatory control over microtubules, neurofilament, myelin basic protein, and nerve growth factor and undergoes phosphorylation events within the CNS^{[12, 13]}. Importantly, the expression level of GSK3β tends to increase with advancing age.

Astrocytes are important regulators of neuroinflammation^[14]. Surgical trauma and pain trigger a systemic inflammatory response, leading to the release of systemic inflammatory mediators that can breach the relatively permeable blood-brain barrier. This, in turn, activates microglia and astrocytes, prompting the secretion of additional cytokines and culminating in a state of central inflammation^[15]. Recent findings from our research have indicated that NLRP3-GABA signaling within astrocytes contributes to the development of impulse-like behavior and cognitive impairment in aged mice^[16]. Additionally, it has been demonstrated that pharmacological inhibition or genetic deficiencies in P38 MAPK can result in the hyperactivation of the NLRP3 inflammasome^[17], and P38 MAPK signaling holds promise as a potential therapeutic target for mitigating arsenic-induced depression and anxiety disorders by modulating the NLRP3 inflammasome^[18]. However, it remains unclear whether the individual inhibition of P38 MAPK, GSK3β, and NLRP3 expression can ameliorate postoperative cognitive dysfunction in aged mice and elucidate the underlying mechanisms.

In this study, we constructed an AEL model to investigate whether inhibitors of P38 MAPK and GSK3β, as well as astrocyte-specific knockout of NLRP3, could alleviate perioperative neurocognitive dysfunction (PND) to provide insights into potential therapeutic approaches for the treatment of PND.

All animal experiments were conducted in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. The protocols involving animals were approved by the Animal Review Board of Hebei Province at the Cangzhou Hospital of Integrated Traditional and Western Medicine.

Group assignment

Conditional NLRP3 knockout (NLRP3-cKO) C57BL/6J mice (Saiye Biotechnology Co., Ltd.) were generated by mating heterozygote NLRP3 knockout mice carrying loxp-flanked NLRP3 genes. Then, 18-month-old male C57BL/6J mice (Liaoning Changsheng Biotechnology Co., Ltd.) were used to establish the AEL model. Prior to surgical procedures, the mice were individually housed under controlled conditions, including a temperature range of 23–25°C, a 12-hour light-dark cycle, humidity levels between 40% and 60%, and ad libitum access to food and water. The experimental design comprised three stages. In the first stage, aged wild-type mice were randomly assigned to four groups: Control + Vehicle (n = 30), AEL + Vehicle (n = 30), Control + SB202190 (n = 30), and AEL + SB202190 (n = 30). In the second stage, another cohort of aged wild-type mice were randomly divided into four groups: Control + Vehicle (n = 30), AEL + Vehicle (n = 30), Control + TWS119 (n = 30), and AEL + TWS119 (n = 30). In the third stage, both wild-type (WT) mice and NLRP3-cKO mice were selected and segregated into two groups: WT AEL (n = 30) and NLRP3-cKO AEL (n = 30).

AEL model

Briefly, the mice were induced with 7–8% sevoflurane in an anesthetic box and maintained under 3–4% sevoflurane anesthesia on a 36°C to 38°C warming pad. A 2-cm midline incision was made after shaving and chlorhexidine disinfection to establish the AEL model. During the procedure, sterile instruments were used to gently manipulate the liver, spleen, colon, and stomach, and the small intestine was exposed approximately 10 cm from the abdominal cavity, covered with damp gauze, and gently manipulated for 3 minutes. Subsequently, the muscle and skin were sutured separately, and tissue adhesive glue was employed to close the skin incision. Postoperative pain management involved the administration of 0.2% ropivacaine (300 µl). The surgical duration was standardized to 10 minutes for each procedure. In the control group, the mice underwent anesthesia, shaving, a 2-cm skin incision, and received ropivacaine in the same manner. Following surgery, the mice were allowed to recover in a 35°C incubator for 30 minutes before being returned to their respective cages.

Drug administration

SB202190 was purchased from Beyotime Co., Ltd. (Catalog # SC0380-25mg, Beyotime, Shanghai, China), while TWS119 was obtained from Slleckchem Co., Ltd. (Catalog # S1590, Slleckchem, Shanghai, China). Both SB202190 and TWS119 were initially dissolved in dimethyl sulfoxide (DMSO) and subsequently diluted in saline to attain a final concentration of 1% (v/v). Following the surgical procedures, the animal groups were administered either vehicle (1 mL/100 g), SB202190 (1 mg/kg), or TWS119 (10 mg/kg) for 7 days. Intraperitoneal injection (i.p.) was employed as the route of administration for SB202190, TWS119, and the vehicle solution.

Behavioral detection

The mice were divided into two groups, each consisting of 24 mice. One group underwent the open field test (OFT) on the 14th day after surgery and the force swimming test (FST) on the 16th and 17th days following surgery. The second group underwent the novel object recognition (NOR) test on the 14th and 15th days post-surgery and the fear conditioning test (FC) test on the 16th and 17th days post-surgery. These assessments were utilized to evaluate anxiety-like and depression-like behaviors. A graphical representation of these tests is shown in Fig. 1 (generated using BioRender.com)^[19]. The NOR and FC tests were performed to evaluate cognitive impairment and memory differences in the mice. Prior to starting the behavioral tests, The mice were allowed to acclimatize to the testing environment for 30 minutes.

For OFT, each mouse was placed in the bottom right corner of a chamber measuring 60 × 60 × 40 cm, and their movements over five minutes were recorded using video tracking. Parameters such as total distance traveled and time spent in the center of the arena were recorded to assess locomotor activity and anxiety-related behaviors.

For the FST, on the first day, the mice were gently placed into a vertical cylindrical container (60 cm in height and 20 cm in diameter) filled with 12 cm of water maintained at a temperature of 25℃ for a 10-minute adaptive phase. On the second day, individual mice were subjected to a 5-minute swimming session, during which a video camera recorded their movements positioned 50 cm in front of the cylinder. The data were subsequently analyzed using a SuperMaze tracking system.

In the NOR test, the mice were allowed to explore two identical cubic objects situated in the left and right corners of a black box (60 cm × 60 cm × 40 cm) for 5 minutes. Subsequently, on the following day, one of the cubic objects (specifically, the one on the right) was replaced with a novel spherical object, and continuous monitoring was conducted for 5 minutes (ZHONGSHI SCIENCE & TECHNOLOGY, Beijing, China). The mice's cognitive ability was assessed using the Recognition Index (RI), calculated as follows: RI = novel object exploration time / (novel object exploration time + familiar object exploration time).

In the FC test, the mice were initially subjected to three-tone-foot shock pairings. The tone had a frequency of 3 kHz, an intensity of 70 dB, and a duration of 30 seconds, while the foot shock was set at 0.4 mA and lasted for 2 seconds. On the subsequent day, the mice were placed in the same chamber without the tone-foot shock pairings for 3 minutes. Two hours later, the mice were introduced to a new chamber characterized by distinct contextual features, including transparent walls, lighting conditions, and a unique odor achieved by wiping the chamber with a 1% acetic acid solution. The mice's "freezing" behaviors, reflective of memory dysfunction, were recorded during two 3-minute intervals: one in the dark chamber (context-related) and the other in the transparent chamber (tone-related).

Local field potential (LFP) recording

Theta oscillations observed in the LFP within the hippocampus are known to emerge during translational movement and periods of heightened arousal. These oscillations play a pivotal role in modulating the activity of principal cells and are closely associated with functions related to spatial cognition and episodic memory. Any reduction in the frequency of theta oscillations may significantly impact synaptic plasticity and mnemonic function^[20].

To assess these theta oscillations, six mice were selected using a computer-generated random number table. They were anesthetized using isoflurane, and a stainless-steel electrode (Catalog No. 2307051101MWA08-2, Kedou Brain-Computer Technology Co., Ltd., Suzhou, China) was precisely positioned within the principal cell layer of CA1 in the dorsal hippocampus (coordinates: AP: -2.0 mm; ML: +1.2 mm; DV: -1.3 mm). Then, the mice were allowed to recover for 7 days. The recorded LFP signals were digitized using the NeuroLego System (Beijing Biogene Biological Technology Co., Ltd.) for subsequent LFP analysis. The LFP data were analyzed using the NeuroExplorer 5 software (Nex Technologies, Alabama, USA). The time window of the NOR event was defined as the period of 5 seconds before and after the nose-poke of the familiar object in the novel location. The normalized power change of the theta (3–8 Hz) rhythm was defined as the mean power at every point divided by the mean of the baseline.

Immunofluorescence

During sevoflurane anesthesia, the mice underwent perfusion with ice-cold saline and 10% neutral-buffered formalin, administered through the ventriculus sinister-aorta. Then, the tissues were embedded in paraffin and subsequently sectioned into 4 µm-thick slices, underwent sodium citrate treatment to facilitate antigen retrieval and were subsequently blocked with Quickblock (Catalog # P0260, Beyotime, Shanghai, China), followed by incubation with the following antibodies: monoclonal mouse antibodies against GFAP (dilution 1:100, Catalog # AF0156, Beyotime), monoclonal rabbit antibodies against GSK3β (dilution 1:100, Catalog # AF1543, Beyotime), rabbit antibodies against phosphorylated GSK3β (dilution 1:100, Catalog # GB13160-1-50, Servicebio, China), polyclonal rabbit antibodies against NLRP3 (dilution 1:100, Catalog # K004108P, Solarbio, China), monoclonal rabbit antibodies against P38 MAPK (dilution 1:100, Catalog # AF5887, Beyotime, China) and polyclonal rabbit antibodies against phosphorylated P38 MAPK (dilution 1:100, Catalog # AF5887, Beyotime), overnight at 4°C. On the following day, the sections were treated with corresponding secondary antibodies (CyTM3-conjugated goat anti-rabbit IgG, 1.5 mg/mL, Catalog # A0516, Beyotime; Alexa Fluor 488-conjugated goat anti-mouse IgG, 1.5 mg/mL, Catalog # A0428, Beyotime) in the dark for 1 hour. After rinsing with PBS, the cell nuclei were stained with DAPI at a concentration of 5 µg/mL (Catalog # P0131, Beyotime). A pathologist blinded to the experimental groups assessed these sections using a fluorescence microscope (Model: RVL-100-M; Echo, USA). For each group, six fields at a magnification of ×200 were randomly selected from three slices. The intensity and percentage of cells positive for phospho-P38 MAPK, GSK3β, NLRP3 and GFAP were quantified using the Image-Pro Plus 6.0 software (NIH, Bethesda, MD, USA).

Western blot assay

The mice were anesthetized using 7–8% sevoflurane and perfused with 0.9% cold saline through the left ventricular aorta. Total protein was extracted from the hippocampus using a stereoscopic microscope. A sample containing 30 µg of protein was loaded onto a 12% SDS-PAGE gel for electrophoretic separation, then the separated proteins were transferred onto a PVDF membrane. Following this, the membranes were blocked using a QuickBlock™ Blocking Buffer (Catalog # P0252, Beyotime) for 10 minutes at room temperature and incubated overnight at 4°C with primary antibodies, which included monoclonal rabbit antibodies against GSK3β (dilution 1:800, Catalog # AF1543, Beyotime), rabbit antibodies against phosphorylated GSK3β (dilution 1:800, Catalog # GB13160-1-50, Servicebio, China), polyclonal rabbit antibodies against NLRP3 (dilution 1:800, Catalog # K004108P, Solarbio, China), monoclonal rabbit antibodies against P38 MAPK (dilution 1:800, Catalog # AF5887, Beyotime, China), and polyclonal rabbit antibodies against phosphorylated P38 MAPK (dilution 1:800, Catalog # AF5887, Beyotime). The next day, the membranes were incubated with goat anti-rabbit secondary antibodies (dilution 1:1000, Catalog # A0208, Beyotime) for 1 hour at 25°C, followed by exposure to ECL (Catalog # P0018AM, Beyotime) for 5 minutes. As an internal reference, polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (dilution 1:1000, Catalog # K106389P, Solarbio) were employed. The optical density (OD) values of the protein bands were determined using ImageJ software version 1.8.0 (National Institutes of Health, Bethesda, MD, USA). All experiments were independently repeated three times.

Statistical analysis

The data are presented as mean ± standard deviation (mean ± SD). All statistical analyses were conducted using GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA). To assess data distribution, normality and lognormality tests were employed. For data that followed a normal distribution, differences were evaluated using either a one-way analysis of variance (ANOVA) or t-test, followed by Tukey's multiple comparison test when applicable. In cases where data did not adhere to a normal distribution, the Kruskal-Wallis test or nonparametric test was utilized to assess differences, followed by the Dunn-Bonferroni test. Statistical significance was determined using P < 0.05. The detailed statistical results are shown in Supplemental File 1.

P38 MAPK inhibitors can improve postoperative cognitive impairment in aged mice but may increase anxiety and depression-like behaviors in mice

This study involved 312 mice, with twelve fatalities recorded in the AEL group postoperatively (Supplemental File 2). In the OFT, there were no notable differences in the total distance traveled by aged mice exposed to either the control condition or AEL surgery, indicating that AEL surgery may not affect their locomotor abilities (Fig. 2A, B). Intriguingly, the NOR test revealed a significant reduction in the ratio of time spent exploring the novel object in AEL-exposed mice compared to the control group. However, this ratio increased following SB202190 treatment (Fig. 2D, E). Furthermore, the results of the FC test indicated that mice subjected to AEL surgery exhibited a substantial reduction in cue-related freezing time compared to the control group, while mice treated with SB202190 displayed an extended duration of cue-related freezing time (Fig. 2F). These findings suggest that both anesthesia and surgery could impair the cognitive function of aged mice.

Additionally, the OFT results demonstrated that mice exposed to AEL spent significantly less time in the central area of the testing arena, which was further exacerbated in mice receiving SB202190 treatment (Fig. 2A, C). Moreover, in FST, the mice exposed to AEL surgery exhibited a reduced duration of struggling behavior. Notably, the control group receiving SB202190 also displayed decreased struggling duration compared to the control group who received the vehicle (Fig. 2G, H). To assess the effectiveness of P38 inhibitors in suppressing P38 expression, we examined the protein levels of phosphorylated P38, and the results revealed no significant alterations in total P38 expression levels, while phosphorylated P38 exhibited a significant reduction following SB202190 treatment (Fig. 2I, J).

In our prior study, we observed a notable increase in astrocyte expression following AEL surgery. Considering the critical role of the hippocampus in PND development^{[7, 21]}, we focused on the pathological changes in phosphorylated P38 and GFAP in this region 14 days post-surgery. The results revealed an upregulation of phosphorylated P38 expression in astrocytes, evident through increased phosphorylated P38-occupied regions within GFAP-positive cells in the CA1 region of mice undergoing AEL surgery compared to control groups. Notably, SB202190 treatment led to a reduction in phospho-P38 fluorescence expression within GFAP-positive cells (Fig. 3A, B). Local Field Potential (LFP) data provided insights into the mean power within the theta band, showing a decrease after AEL surgery. Interestingly, SB202190-treated mice exhibited an increase in mean power within the theta band (Fig. 3C, D). These findings suggest that P38 MAPK inhibitors have the potential to mitigate postoperative cognitive dysfunction in aged mice. However, it's important to note that AEL surgery may induce anxiety and depression-like behavior in aged mice, and the use of P38 MAPK inhibitors could exacerbate these effects.

GSK3β inhibitors ameliorate postoperative cognitive impairment in aged mice but may cause impulsive-like behavior in mice

Similar to the previous findings, the OFT results indicated no difference in the total distance traveled between the control and AEL groups (Fig. 4A, B). In contrast, the NOR and FC results showed a decrease in the ratio of time spent exploring the novel object and cue-related freezing time in the AEL-exposed mice. However, both of these parameters improved following TWS119 treatment (Fig. 4D–F). Interestingly, the OFT results revealed an increase in the time spent in the central area in the control + TWS119 group compared to the control + vehicle group (Fig. 4A, C). In FST, the control + TWS119 group exhibited a longer struggling duration compared to the control + vehicle group and even surpassed that of the AEL + TWS119 group (Fig. 4G, H). These results suggest that GSK3β inhibitors may be associated with impulsive-like behavior in aged mice. To assess the effectiveness of GSK3β inhibitors in suppressing GSK3β expression, we examined the protein levels of GSK3β, and the results revealed no significant change in the total GSK3β expression level, while phosphorylated GSK3β was significantly reduced after TWS119 treatment (Fig. 4I, J).

Furthermore, we examined the pathological changes in phosphorylated GSK3β and GFAP within the CA1 region at 14 days post-surgical exposure (Fig. 5A). The findings revealed an upregulation of phosphorylated GSK3β within GFAP-positive cells following AEL surgery compared to the control groups. Notably, there was no significant difference in phosphorylated GSK3β expression between the control + TWS119 and AEL + TWS119 groups. However, phosphorylated GSK3β was downregulated in the GFAP-positive cells of the control + TWS119 group compared to the control + vehicle group (Fig. 5B). LFP data showed that the mean power within the theta band was decreased after AEL surgery. Intriguingly, TWS119-treated mice showed upregulated mean power within the theta band (Fig. 5C, D). These findings indicate that GSK3β inhibitors could alleviate postoperative cognitive dysfunction in aged mice, with the possibility of inducing postoperative impulsive-like behavior in this age group.

Astrocyte-specific NLRP3 knockout improves postoperative cognitive impairment in aged mice

The preceding results demonstrate that while inhibiting the phosphorylation of P38 and GSK3β can improve postoperative cognitive dysfunction in aged mice, it may concurrently induce or exacerbate other mood-related changes in these animals. Previous research has established P38 MAPK's role in mediating NLRP3 inflammasome activation^{[17, 18]}, prompting us to investigate whether targeting NLRP3 could mitigate surgical cognitive impairment in aged mice. Astrocyte-specific NLRP3 knockout mice were generated by using a crossbreeding strategy involving NLRP3^f/f mice and GFAP-Cre mice for subsequent experiments (Fig. 6A, B). The results showed that astrocyte-specific NLRP3 knockout mice exhibited no significant difference in total distance covered during OFT. However, there was a significant increase in the time spent in the central area (Fig. 6C-E). Furthermore, these knockout mice displayed significant improvements in both the RI in the NOR test and the freezing time during cue-related FC compared to wild-type mice (Fig. 6F-H). Struggling duration in FST showed no significant difference between the two groups (Fig. 6I, J). Western blot results illustrated a downregulation in NLRP3 protein expression in astrocyte-specific NLRP3 knockout mice compared to their wild-type counterparts (Fig. 6K, L).

Immunofluorescence analysis revealed the absence of NLRP3 expression in astrocyte-specific NLRP3 knockout mice, whereas NLRP3 expression was observed in GFAP-expressing cells in WT mice (Fig. 6M, N). Likewise, NLRP3-specific knockout mice subjected to AEL surgery exhibited higher mean theta-band power in the LFP compared to WT mice (Fig. 6O, P). In summary, these findings indicate that astrocyte-specific NLRP3 knockdown has the potential to alleviate postoperative cognitive impairment in aged mice.

In this present study, our results highlight the significant role of NLRP3 in PND induced by anesthesia and surgery. While P38 MAPK inhibitors may enhance cognition, they may exacerbate depressive-like behavior in mice. Similarly, GSK3β inhibitors can improve cognition but might induce impulsive-like behavior in mice. Moreover, astrocyte-specific knockout of NLRP3 effectively reversed cognitive impairment in mice and downregulated the protein expression of phosphorylated P38 MAPK and phosphorylated GSK3β. Collectively, our findings suggest the importance of inhibiting NLRP3 in astrocytes as a promising avenue for clinical PND management.

Inflammation plays a pivotal role in the pathogenesis of various diseases, including autoimmune disorders, AD, neurological conditions, and cancer, among others. Following surgery, inflammatory agents from the periphery can access the brain through multiple pathways, ultimately triggering inflammatory responses within the CNS^{[22, 23]}. Similarly, in the PND model, pro-inflammatory molecules are expressed both centrally and peripherally, contributing to cognitive impairment^[24]. MAPK is a major factor acting upstream of many pro-inflammatory pathways, and in previous studies, P38 MAPK was found to have an important role in the development of PND in aged mice^[7]. Notably, the increased expression of phosphorylated P38 MAPK in peripheral blood has been linked to disease severity in AD^[6]. Therefore, the inhibition of P38 MAPK has emerged as a potential therapeutic target for managing autoimmune and inflammatory diseases. It is worth noting that earlier studies have indicated increased anxiety levels in behavioral tests in P38 MAPK knockout mice, potentially linked to impaired synapse formation^[25]. Therefore, this study sought to evaluate the impact of P38 MAPK inhibitors on aged mice with PND, given the current clinical trials exploring various inhibitors^{[26, 27]}. Recent research has investigated the impact of P38 MAPK inhibitors in AD, demonstrating that pyridyl imidazole compounds such as SB202190 and SB203580 exhibit excellent anti-apoptotic effects in vivo and can delay cognitive decline in animal models^[28], consistent with our results. Specifically, our data indicates that SB202190 downregulates the expression of phosphorylated P38 MAPK. In aged mice subjected to AEL, there was a decrease in both the recognition index in the NOR test and the freezing time in the FC test. However, SB202190 treatment increased the RI in NOR and the freezing time in FC. Immunofluorescence analysis revealed reduced phosphorylated P38 expression in GFAP-positive cells in mice treated with SB202190, along with increased mean theta power in LFP during the exploration of novel locations. These results suggest that SB202190 may ameliorate postoperative cognitive impairment in elderly mice by inhibiting P38 MAPK expression. Additionally, AEL-exposed mice displayed reduced central time in OFT and reduced struggling time in FST, indicating the presence of anxiety and depression-like behavior. Mice exposed to SB202190 exhibited further reductions in central time in OFT and struggling time in FST, implying that AEL mice experience anxiety and depression, and the inhibition of phosphorylated P38 expression by SB202190 may exacerbate these anxiety and depression-like behaviors, similar to the outcomes observed in P38 MAPK knockout mice. Thus, inhibiting phosphorylated P38 expression could improve postoperative cognitive dysfunction but at the expense of potentially inducing or worsening postoperative depression and anxiety-like behavior in mice.

Glycogen synthase kinase 3 (GSK3) represents a class of serine/threonine protein kinases pivotal in regulating numerous fundamental cellular pathways, some of which are related to neurodegenerative processes^[11]. There are two isoforms of GSK3, namely GSK3α and GSK3β. GSK3β, in particular, plays a multifaceted role, participating in various signaling pathways within tissue cells, and serves as a critical node in both mitogen-activated protein kinase (MAPK) and Wnt signaling pathways^[10]. GSK3β has been demonstrated to hold significant importance in neuronal survival, synaptic plasticity, and memory processes. Moreover, it has been implicated in the pathophysiology of conditions such as depression and AD^[29]. P38 MAPK may directly influence the activity of GSK3β, leading to an increase in GSK3β kinase activity^{[30, 31]}. This interaction between P38 MAPK and GSK3β and its impact on memory deficits and depression-like behavior offers a plausible mechanism underlying the development of AD and depression mediated by both regulators^[32]. Therefore, we explored the inhibition of GSK3β phosphorylation using TWS119 to evaluate its potential in improving postoperative cognitive function in aged mice. The outcomes of our study demonstrated that mice exposed to TWS119 had reduced levels of phosphorylated GSK3β protein expression, suggesting effective inhibition of phosphorylated GSK3β expression by TWS119. Furthermore, the TWS119 treatment improved the recognition index of NOR and increased the freezing time during FC testing. Immunofluorescence analysis revealed a decrease in phosphorylated GSK3β expression in GFAP-positive cells in response to TWS119 treatment, while LFP data indicated an increase in mean theta power during exploration of the novel location. These findings collectively suggest that inhibiting GSK3β expression could effectively enhance postoperative cognitive function in mice. However, in contrast to SB202190, both the central time in the OFT and the struggling duration in the FST were elevated in mice exposed to TWS119, implying that inhibiting GSK3β phosphorylation expression with TWS119 may induce impulsive-like behavior in mice. Thus, our results indicate that the inhibition of phosphorylated GSK3β expression can also ameliorate postoperative cognitive dysfunction but may precipitate impulsive-like behavior in mice.

Aberrant activation of NLRP3 inflammasome is linked to chronic inflammation in the body and is implicated in inflammation-related conditions, including neurobehavioral disorders. Additionally, the P38 MAPK signaling pathway can trigger the activation of the NLRP3 inflammasome^[18]. Excessive NLRP3 inflammasome activation is implicated in numerous diseases, including various acute and chronic neurological disorders^[33]. Astrocytes, the most abundant type of glial cells in the CNS, play diverse and vital roles in maintaining homeostasis, enhancing synaptic plasticity, providing neuroprotection, and preserving normal brain function^[34]. Simultaneously, astrocytes can participate in inflammatory responses, which are pivotal in the progression of mental and neurodegenerative disorders^{[35, 36]}. They can also assemble various inflammasomes and contribute to a range of nervous system diseases^{[37, 38]}. Numerous studies have demonstrated that the extent of neuroinflammation and synaptic damage in the hippocampus of depressed rats is associated with the activation of the NLRP3 inflammasome and that inhibiting or knocking down NLRP3 can exert central neuroprotection^{[39, 40]}. Therefore, in this study, we used astrocyte-specific knockout of the NLRP3 gene to investigate its potential in ameliorating postoperative cognitive dysfunction. Immunofluorescence analysis revealed the absence of NLRP3 expression in GFAP-positive cells within the CA1 region of astrocyte-specific NLRP3 knockout mice, confirming the successful NLRP3 knockout in astrocytes. Behavioral tests demonstrated that astrocyte-specific NLRP3 knockout mice exhibited increased recognition index in NOR, freezing time in FC, and center time in OFT compared to WT mice following AEL surgery, collectively indicating an effective improvement in postoperative cognitive function. Furthermore, Western blot results showed that astrocyte-specific NLRP3 gene knockout not only reduced NLRP3 protein expression but also decreased the levels of phospho-P38 MAPK and phospho-GSK3β after AEL surgery. Collectively, these findings suggest that NLRP3 may contribute to the development of PND by promoting the phosphorylation of P38 MAPK and GSK3β.

The above results indicate that while individual inhibition of P38 MAPK and GSK3β phosphorylation improved postoperative cognitive impairment, it concurrently induced mood changes in mice, likely implicating alterations in other signaling pathways. Conversely, the simultaneous inhibition of P38 MAPK and GSK3β phosphorylation by NLRP3 gene knockout in astrocytes effectively improved postoperative cognitive dysfunction and mood alterations in mice, suggesting the involvement of the P38 MAPK/NLRP3 pathway in these outcomes. Our study establishes the significance of the P38 MAPK/NLRP3 pathway in the progression of postoperative PND, and future investigations could further investigate its precise role in the development of this condition.

In conclusion, this present study highlights that the targeted knockdown of NLRP3 in astrocytes may effectively mitigate postoperative cognitive dysfunction in aged mice, likely through the reduction of phosphorylated P38 MAPK and phosphorylated GSK3β. These findings contribute to a better understanding of the therapeutic strategies for addressing PND and introduce innovative insights for targeting signaling pathways against PND.

Authors' contributions

XML and JML were responsible for the design of the study. LYW was responsible for statistical analysis. BDL, YLS, ZQW, QML, HYP, and TTZ were responsible for the experiments and data collection. LMZ, XML and JML confirmed the authenticity of all raw data. All authors have read and approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (No. 81701296, 82171455) and the Natural Science Foundation of Hebei Province (No. H2021110004).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

All experiments were approved by the Ethics Committee of Cangzhou Integrated Traditional Chinese and Western Medicine Hospital under the registration number CZX2023-KY-024.

Conflict of interest

The authors have no conflicts of interest to declare.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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Inhibition of the P38 MAPK/NLRP3 Pathway Mitigates Cognitive Dysfunction and Mood Alterations in Aged Mice after Abdominal Surgery Plus Sevoflurane

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Abstract

Background

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Introduction

Methods

Group assignment

AEL model

Drug administration

Behavioral detection

Local field potential (LFP) recording

Immunofluorescence

Western blot assay

Statistical analysis

Results

Astrocyte-specific NLRP3 knockout improves postoperative cognitive impairment in aged mice

Discussion

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References

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Supplementary Files

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