Targeting NMDAR2A: Esketamine's Impact on BV2 Microglial Pro-inflammatory Polarization

doi:10.21203/rs.3.rs-5313533/v1

Download PDF

Research Article

Targeting NMDAR2A: Esketamine's Impact on BV2 Microglial Pro-inflammatory Polarization

https://doi.org/10.21203/rs.3.rs-5313533/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

The polarization of microglia to the pro-inflammatory phenotype plays a crucial role in the initiation and progression of neuroinflammatory diseases, primarily owing to the secretion of pro-inflammatory cytokines that exacerbate damage within the central nervous system (CNS). Investigating the mechanisms underlying the inhibition of microglial pro-inflammatory polarization could be the potential targets for the prevention of neuroinflammatory diseases. The activation of N-methyl-D-aspartic acid (NMDA) receptors can mediate the over-activation and toxicity of microglia. Due to the unique structure of NMDA receptors, it has different subtypes and perform distinct functions. Our study indicates that Esketamine, a non-competitive antagonist of NMDAR, can up-regulate the phosphorylated Mammalian target of rapamycin (mTOR) and Brain-derived neurotrophic factor (BDNF)-Tropomyosin-related kinase receptor B (TrkB) signaling pathway by inhibiting the 2A subtype of NMDA receptors, attenuates LPS-induced pro-inflammatory polarization of BV2 microglia and reduces the expression of pro-inflammatory cytokines. However, the NMDAR2B subtype does not appear to be involved in this process.

NMDAR subtypes

Microglia

Neuroinflammation

Esketamine

Microglia, as resident immune cells in the CNS, perform dual roles in immune response. Upon encountering external stimuli, they have the ability to differentiate into either a pro-inflammatory phenotype or an anti-inflammatory phenotype (Wright-Jin & Gutmann 2019). The pro-inflammatory phenotype, which is detrimental to neurons, releases pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), among others. anti-inflammatory microglia are typically induced by anti-inflammatory cytokines to differentiate and secrete interleukin-10 (IL-10), neurotrophic factors, and other molecules, thereby contributing to neuronal protection and damage repair (Lucin & Wyss-Coray 2009, Yu et al. 2023). The polarization of microglia is critically implicated in a range of neuroinflammatory diseases (Castro et al. 2022, Yan et al. 2022, Yu et al. 2023). Evidence indicates that microglia are capable of transitioning from the pro-inflammatory phenotype to the anti-inflammatory phenotype through the modulation of signaling pathways, transcription factors, surface receptors, and cytokines (Correale 2014, Yan et al. 2022). Consequently, the inhibition of polarization and the promotion of anti-inflammatory phenotype transformation in microglia represent potential therapeutic targets for the prevention and treatment of neuroinflammatory conditions.

Esketamine, the right-handed optical isomer of ketamine, functions as a non-competitive antagonist of NMDA receptors. It exhibits a higher affinity for NMDAR compared to ketamine and demonstrates analgesic and hypnotic effects that are twice as potent, additionally, Esketamine is associated with fewer psychiatric side effects. Our prior research has demonstrated that it can inhibit neuroinflammation and exert a neuroprotective effect in sepsis-associated encephalopathy(SAE) through the regulation of microglial function (Li et al. 2024). Numerous studies have demonstrated that the neuroprotective effects of Esketamine may be associated with the up-regulation of Mammalian target of rapamycin (mTOR) and Brain-derived neurotrophic factor (BDNF)-Tropomyosin-related kinase receptor B (TrkB) signaling pathway (Yang et al. 2018, Schatzberg 2021, Wang et al. 2022). On the other hand, Research findings suggest that activation of NMDAR can trigger microglial activation and the subsequent release of pro-inflammatory mediators, resulting in neurotoxic effects. Conversely, pharmacological inhibition of NMDAR may regulate the excessive activation of microglia and their pro-inflammatory characteristics through various mechanisms (Kaindl et al. 2012, Lu et al. 2020). Consequently, we hypothesize that Esketamine may also attenuates the pro-inflammatory polarization of BV2 microglia.

NMDARs are ion channels regulated by the excitatory neurotransmitter glutamate and are ubiquitously distributed throughout the CNS. Structurally, NMDARs are tetrameric complexes consisting of two GluN1 subunits in combination with either two GluN2 or two GluN3 subunits. The GluN2 subunits are further categorized into four subtypes: GluN2A, GluN2B, GluN2C, and GluN2D, whereas the GluN3 subunits are classified into two subtypes: GluN3A and GluN3B. The expression of the GluN3 subunit is developmentally regulated and is minimally present in the adult brain. Owing to its distinctive structural attributes, NMDARs encompasses various subtypes, each exhibiting distinct patterns of brain distribution, expression, and electrophysiological properties (Paoletti et al. 2013). These variations may consequently influence the pharmacological efficacy and side effects profile of Esketamine-based therapeutics. However, the absence of specific inhibitors and the limited selectivity of existing inhibitors have hindered previous studies to investigating the role of each subtype in microglia (Frizelle et al. 2006). Consequently, the specific NMDAR subtype responsible for regulation of polarization in microglia by Esketamine has arouses our concern.

Therefore, this study investigated the regulatory influence of Esketamine on pro-inflammatory polarization of BV2 microglia and elucidated the underlying mechanisms, with a particular focus on the role of the NMDAR2A subtype.

2.1 Cell culture drug treatments

BV2 microglia (Shanghai Zhong Qiao Xin Zhou Biotechnology Co.) were cultured in DMEM medium (#C11995500BT, Gibco) with 10% FBS (#11011 − 8611, Sijiqing) and 1% streptomycin (#15070063, Gibco). The cells were cultured at 37°C in 5% CO2 and used for experiments after reaching 80–90% confluence. The inflammatory model of BV2 microglia was developed by utilizing lipopolysaccharide (LPS), as evidenced by prior research (Sun et al. 2021). The drug concentration of LPS (#L8880, solarbio) was 1µg/ml. According to previous studies (Li et al. 2022), the drug concentration of Esketamine (#200228BL, Jiangsu Hengrui Medicine Co., Ltd) is 50µM. The concentration of MK801(#HY-15084A, MCE) and Rapastinel(#HY-16728B, MCE) were both 10µM (Wu et al. 2018, Wu et al. 2023). The concentration of Rapamycin (#HY-10219, MCE) is 1nM (Yang et al. 2017).

2.2 Small interfering RNA (siRNA) transfection

BV2 microglial cells were seeded on a 12-well plate 1 day prior to transfection. siRNA, against mouse Grin2a, Grin2b and negative control, were designed and synthesized by Haixing shengwu Co. (Jiangsu, China). The cells were transfected with appropriate siRNA accompanied by transfection reagents (Lipofectamine 3000) according to the manufacturer’s instructions. After 48 h, the cells were collected for subsequent experiments. The siRNA sequences are shown in Supplementary table 1. Western blot analysis is used to evaluate the efficiency of siRNA. Next, the BV2 microglia cells were exposed to LPS (1µg/mL) with or without Esketamine (50µM).

2.3 Enzyme-Linked Immunosorbent Assay (ELISA)

Cell culture medium concentrations of cytokines, including IL-4(#JM-02448M1, JingMei Bio-Technology Co.) and TNF-α (#JM-02415M1, JingMei Bio-Technology Co.), were measured using the quantitative ELISA kits according to the manufacturer's instructions. The optical density was measured at 450nm. The standard curve of each cytokine determined the cytokine concentrations.

2.4 Western blot

BV2 microglia were collected from each treatment group, homogenized and extracted on ice using RIPA lysis buffer supplemented with phosphatase and protease inhibitors. The mixture was centrifuged at 12000 r/min at 4℃ for 10 minutes. The protein concentration in the supernatant was determined using the BCA Protein Assay Kit (#PC0020, Solarbio). Equal amounts of protein were mixed with 5X loading buffer and boiled for 6 minutes. Protein mixtures underwent separation through SDS-PAGE and were subsequently transferred onto PVDF membranes. The membrane was subsequently blocked with a 5% non-fat milk solution for 60 minutes at room temperature, and then rinsed with TBST. The membrane was then probed with primary antibodies at 4 ℃ overnight, followed by secondary antibodies at room temperature for 1 hour. The visualization of protein bands was achieved using the Intelligent Image Workstation (#GelView 6000Plus, BLT Photon Technology). The experiments were conducted independently on four occasions, and the resulting data was analyzed using ImageJ software. A horizontal membrane incision obtained mTOR, p-mTOR, BDNF, TrkB, p-TrkB, NMDAR2A, NMDAR2B, NMDAR1 and corresponding GAPDH protein bands. ARG-1, CD32, and corresponding GAPDH protein bands were obtained by stripping and reprobing the uncut membrane. The antibody dilution ratio is as follows: The antibody against TrkB (#AF6461, Affinity, 1:1000), the antibody against phosphor-TrkB (#AF3461, Affinity, 1:1000), the antibody against ARG-1 (#DF6657, Affinity, 1:1000). The antibody against BDNF (#ab108319, Abcam, 1:5000). The antibody against mTOR (#ET1608-5, HUABIO, 1:2500), the antibody against phosphor-mTOR (#HA600094, HUABIO, 1:1000). The antibody against NMDAR2A (#ET1704-80, HUABIO, 1:500), The antibody against NMDAR2B (#AF6426, Affinity, 1:1000), The antibody against NMDAR1 (AF6406, Affinity, 1:1000). The antibody against CD32 (#15625-1-AP, Proteintech, 1:5000).

2.5 Immunofluorescence

Cells that were treated and cultured on coverslips were fixed in precooled 4% paraformaldehyde (PFA) for 20 minutes. After washed with phosphate-buffered saline (PBS), the cells were then blocked with 5% bovine serum albumin (BSA) at room temperature for 1 hours. Subsequently, the cells were incubated overnight at 4 ℃ with the primary antibodies. After rinsing with PBS, the cells were stained with the corresponding secondary antibodies in the dark for 1 hour at room temperature. Finally, the nuclei were counterstained with 4’,6-diamidino-2-phenyl-indole (DAPI, #C0065, Solarbio). The antibody dilution ratio is as follows: The antibody against CD32 (1:200), The antibody against ARG-1(1:200).

2.6 Statistical analysis

All results were expressed as mean ± standard deviation derived from at least 3 independent experiments, and statistical analyses were performed using GraphPad Prism9.0 (GraphPad Software, Inc.). The Shapiro-Wilk test was applied to all datasets to test for a normal distribution. Based on this test, multiple groups were compared using one-way ANOVA and Tukey’s post hoc tests. P < 0.05 was considered statistically significant.

3.1 Esketamine suppresses the proinflammatory activation of microglia through the inhibition of NMDAR

The non-specific NMDAR inhibitor MK801 and the NMDAR agonist Rapastinel were utilized in BV2 microglia. The levels of TNF-α and IL-4 in the BV2 cell culture medium of each experimental group were quantified by Elisa. Both Esketamine and MK801 demonstrated an increase in IL-4 expression in LPS-induced BV2 microglia, however, the NMDAR activator Rapastinel counteracted the effect of Esketamine (Fig. 1A). The levels of TNF-α in the BV2 cell culture medium of the LPS group were found to be significantly elevated. Both Esketamine and MK801 demonstrated inhibitory effects on TNF-α expression, however, the suppressive impact of Esketamine was counteracted by Rapastinel (Fig. 1B). The findings from the Western blot analysis indicate that both Esketamine and MK801 were found to decrease CD32 protein expression, with the effect of Esketamine being reversed by rapastinel (Fig. 1C-D). Esketamine and MK801 led to an increase in ARG-1 protein expression in LPS-induced inflammatory cell models, However, the impact of Esketamine on ARG-1 expression was counteracted by rapastinel (Fig. 1C, E). Furthermore, The fluorescence intensity of the Pro-inflammatory microglia marker CD32 and anti-inflammatory microglia marker ARG-1 in each group was assessed via immunofluorescence analysis. The fluorescence intensity of CD32 increased in BV2 cells in the LPS group. Esketamine and MK801 decreased CD32 fluorescence intensity, but Rapastinel reversed the effect of Esketamine (Fig. 1F-G). Both Esketamine and MK801 significantly increased the fluorescence intensity of ARG-1 in LPS-induced BV2 cells. However, Rapastinel reversed the effect of Esketamine (Fig. 1H-I).

3.2 Esketamine induces the up-regulation of p-mTOR and BDNF-TrkB through the inhibition of NMDAR.

The protein levels of mTOR, p-mTOR, BDNF, TrkB, and p-TrkB in BV2 cells from each group were assessed by western blot analysis. The p-mTOR/ mTOR ratio was decreased in LPS treated BV2 microglia, while Esketamine, similar to MK801, increased the p-mTOR/mTOR ratio. However, the NMDAR agonist Rapastinel reversed the effects of Esketamine (Fig. 2A-C). The BDNF protein expression in the LPS group exhibited a significant decrease. Both MK801 and Esketamine were able to increase the expression of BDNF, however, Rapastinel was able to counteract the effect of Esketamine (Fig. 2A, D). TrkB protein expression was found to be significantly decreased in the LPS group. Both MK801 and Esketamine were observed to up-regulate TrkB expression, however, the effect of Esketamine was reversed by Rapastinel (Fig. 2A, E). Additionally, p-TrkB protein expression was also decreased in the LPS group, with Esketamine and MK801 showing the ability to up-regulate the expression of p-TrkB. Notably, Rapastinel was able to reverse the effect of Esketamine (Fig. 2A, F).

3.3 Inhibiting mTOR may counteract Esketamine's impact on microglia pro-inflammatory polarization.

To verify the effect of mTOR, the mTOR inhibitor rapamycin was administered to BV2 microglia, and the expression of inflammatory cytokines in BV2 cell culture was detected by Elisa. In LPS-induced BV2 inflammatory cell models, Esketamine was found to significantly increase IL-4 secretion in the culture medium, however, Rapamycin was able to counteract this effect (Fig. 3A). Furthermore, in the LPS-induced inflammatory model of BV2 microglia cells, the expression of TNF-α was observed to increase in the culture medium. Interestingly, Esketamine was able to decrease the expression of TNF-α, but Rapamycin reversed the effect of Esketamine (Fig. 3B). Western blot was used to detect the expression levels of CD32 and ARG-1 protein in BV2 microglia of each group. In the LPS-induced inflammatory model of BV2 microglia, CD32 protein expression was increased. Esketamine down-regulated CD32 protein expression, but Rapamycin reversed the effect of Esketamine (Fig. 3C-D). In the LPS-induced inflammatory model of BV2 microglia cells, Esketamine increased ARG-1 protein expression, but Rapamycin reversed the effect of Esketamine (Fig. 3C, E).

3.4 Inhibition of mTOR reversed the upregulation of BDNF-TrkB induced by Esketamine

The effects of Rapamycin, an mTOR inhibitor, on the expression levels of BDNF, TrkB, and p-TrkB in BV2 microglia cells were assessed using western blot analysis. In the LPS-induced inflammatory model of BV2 microglia, the down-regulation of BDNF protein expression was observed, treatment with Esketamine resulted in a significant increase in BDNF protein expression, however, this effect was reversed by Rapamycin (Fig. 4A-B). Similarly, in the same model, the expression of TrkB and p-TrkB proteins was down-regulated. Esketamine treatment led to a significant increase in the expression of TrkB and p-TrkB proteins, but this effect was reversed by Rapamycin (Fig. 4A, C-D).

3.5 Esketamine attenuate BV2 microglial cell pro-inflammation polarization through NMDAR2A.

Neither LPS nor Esketamine had a significant impact on the expression levels of NMDAR2A, NMDAR2B, and NMDAR1 in BV2 microglia cells (Supplementary Fig. 1). To verify the function of each receptor subtype, the NMDAR2A and NMDAR2B genes were were interfered with siRNA specific to Grin2a and Grin2b, respectively. 48 hours post-transfection, cells were harvested, and transfection efficiency was assessed via western blot analysis (Supplementary Fig. 2, Supplementary Fig. 3). The levels of IL-4 and TNF-α in BV2 cell culture medium were quantified using Elisa. In the LPS-induced inflammatory model of BV2 microglia, Esketamine and the downregulation NMDAR2A were both resulted in an upregulation of IL-4 expression in the cell culture medium. however, the addition of Esketamine following siRNA interference with NMDAR2A did not lead to a further increase in IL-4 expression (Fig. 5A). In the LPS-treated BV2 microglia, the up-regulation of TNF-α in the cell culture medium was statistically significant, similar to Esketamine, siRNA-Grin2a demonstrated a down-regulation of TNF-α expression, nevertheless, the subsequent administration of Esketamine following siRNA interference with NMDAR2A did not result in a further decrease in TNF-α expression (Fig. 5B). Western blot analysis was utilized to assess the expression levels of CD32 and ARG-1 proteins in BV2 cells across different experimental groups. The results showed a significant up-regulation of CD32 protein expression in BV2 cells subjected to LPS-induced inflammation. Interestingly, siRNA-Grin2a led to a down-regulation of CD32 protein expression in the inflammatory model, similar to the effects observed with Esketamine treatment. However, the administration of Esketamine did not result in further down-regulation of CD32 protein expression following siRNA interference with NMDAR2A (Fig. 5C-D). In the LPS-induced BV2 cell inflammatory model, both Esketamine and siRNA-Grin2a were found to up-regulate ARG-1 protein expression. but Esketamine administration did not further up-regulate ARG-1 protein expression after siRNA interference with NMDAR2A (Fig. 5C, E). However, modulation of the NMDAR2B receptor subtype did not influence the expression levels of the inflammatory cytokines IL-4 and TNF-α in BV2 cell cultures, nor did it alter the protein expression of CD32 and ARG-1 in BV2 cells (Supplementary Fig. 4)

3.6 Esketamine regulates the expression of mTOR and BDNF-TrkB proteins through NMDAR2A

The protein expression levels of mTOR, p-mTOR, BDNF, TrkB, and p-TrkB in BV2 microglia cells of each experimental group were assessed via western blot analysis. The p-mTOR/ mTOR ratio was found to be significantly decreased in the LPS-induced inflammatory cell model of BV2 microglia cells. Similar to Esketamine, NMDAR2A knockdown resulted in an upregulation of the p-mTOR/ mTOR ratio in the LPS-induced inflammatory cell model. However, the administration of Esketamine following siRNA interference with NMDAR2A did not lead to a further increase in the ratio (Fig. 6A-C). In the LPS-induced inflammatory model of BV2 microglia, the down-regulation of BDNF protein expression was observed. Similar to Esketamine, the knockdown of NMDAR2A resulted in an up-regulation of BDNF protein expression, nevertheless, the administration of Esketamine did not lead to a further up-regulation of BDNF protein expression following siRNA interference with NMDAR2A (Fig. 6A,D). In the LPS-induced inflammatory model of BV2 microglia, a significant decrease in the expression of TrkB protein was observed. Similar to Esketamine, interference with the NMDAR2A gene led to an up-regulation of TrkB protein expression, nevertheless, the administration of Esketamine following siRNA interference with NMDAR2A did not result in a further increase in TrkB protein expression (Fig. 6A, E). In the LPS-induced inflammatory model of BV2 microglia, a significant decrease in the expression of p-TrkB protein was observed. Similar to Esketamine, interference with the NMDAR2A gene resulted in an up-regulation of p-TrkB protein expression. However, administration of Esketamine did not lead to further upregulation of p-TrkB protein expression following siRNA interference with NMDAR2A (Fig. 6A, F). However, modulation of the NMDAR2B receptor subtype did not influence the protein expression of mTOR, p-mTOR, BDNF, TrkB, and p-TrkB in BV2 microglia cells (Supplementary Fig. 5).

Esketamine has gained widespread clinical use due to its rapid and potent antidepressant properties, with its potential application in neuroinflammatory conditions being increasingly acknowledged in recent years. Studies have demonstrated its ability to mitigate depression-like symptoms in mice following surgery and anesthesia through the attenuation of neuroinflammation (Wang et al. 2022), alleviate post-stroke anxiety by suppressing microglial activation in the mouse cortex (Huang et al. 2023), and mitigate postoperative cognitive impairment by modulating astrocyte function (Li et al. 2022). Numerous studies have substantiated the significant involvement of microglia in the pathogenesis of neuroinflammatory diseases (Wright-Jin & Gutmann 2019). Activation of the NMDAR receptor has been shown to stimulate microglial activation and subsequent release of pro-inflammatory mediators, culminating in neurotoxic effects. Conversely, inhibition of NMDAR activity may effectively mitigate the pro-inflammatory phenotype exhibited by microglia (Wu et al. 2018, Wu et al. 2023). Although numerous studies have indicated that ketamine and its enantiomers may exhibit pharmacological mechanisms that are both reliant on and independent of NMDAR (Zorumski et al. 2016, Wei et al. 2022). However, our results confirm that Esketamine inhibits the pro-inflammatory polarization of BV2 microglia through an NMDAR-dependent pathway, reducing the release of proinflammatory cytokines while increasing the release of anti-inflammatory cytokines.

Prior research has suggested a potential relationship between the therapeutic efficacy of Esketamine in treating neuroinflammatory conditions and the increased expression of BDNF (Wen et al. 2024), yet the precise regulatory mechanisms are not fully understood. Furthermore, research has demonstrated that BDNF plays a significant role in the functional regulation of LPS-mediated microglial inflammation models (Charlton et al. 2023). Our research found that that Esketamine has the potential to increase the expression of BDNF via the mTOR pathway, leading to a decrease in pro-inflammatory polarization of BV2 microglia. Prior research has primarily examined the impact of ketamine on neuronal function, revealing that ketamine activates the mTOR pathway by inhibiting NMDAR, leading to heightened levels of synaptic signaling proteins and enhanced synaptic activity (Li et al. 2010). But Wanda et al (Nowak et al. 2019) showed that ketamine acts on monocytes by inducing a M2-like anti-inflammatory phenotype via the NMDAR and mTOR pathway but also by promoting a particular gene expression profile that supports an independant way to induce the M2 program. Further investigation revealed that Esketamine, one of the two enantiomers of ketamine, exhibited a regulatory effect on mTOR, whereas arketamine did not (Yang et al. 2018). Consistent with prior research (Zhang et al. 2024), our findings indicate that increased mTOR signaling can influence the production and secretion of BDNF, however, additional investigation is required to elucidate the specific underlying mechanism.

In the examination of ketamine's mechanism of action, initial beliefs suggested that it targeted the neuronal NMDAR2B receptor subtype to produce its antidepressant properties (Miller et al. 2014). However, a recent investigation conducted by Su et al (Su et al. 2023). revealed that the NMDAR2A receptor subtype is essential for the rapid ketamine-induced antidepressant effects. Consequently, the significance of NMDAR subtype in the modulation of microglia polarization arouses our attention. Previous studies have confirmed the presence and functionality of NMDAR subtypes on microglia in both developing and mature CNS of mice and humans (Kaindl et al. 2012). Our study found that the impact of LPS and Esketamine on the expression of NMDAR1, NMDAR2A, and NMDAR2B proteins was not significant. Subsequently, siRNA was used to target interference NMDAR2A and NMDAR2B in BV2 microglia, indicating that Esketamine's modulation of microglia polarization depends on NMDAR2A rather than NMDAR2B. Given the various functions of microglia in the CNS, further investigation is needed to understand the specific role of NMDAR2B in regulating microglia phenotype or function.

In summary, our research demonstrated that Esketamine has the ability to attenuate pro-inflammatory polarization of BV2 microglia and decrease the expression of proinflammatory cytokines through the inhibition of NMDAR. This mechanism may be associated with the upregulation of mTOR and BDNF-TrkB pathways. Further investigation into the specific NMDAR subtypes involved revealed that NMDAR2A plays a predominant role in regulating the polarization of BV2 microglia, but not NMDAR2B.

CNS:central nervous system; NMDA:N-methyl-D-aspartic acid; TNF-α:tumor necrosis factor-α; IL-1β: interleukin-1β; IL-10:interleukin-10; SAE: sepsis-associated encephalopathy; mTOR: Mammalian target of rapamycin; BDNF: Brain-derived neurotrophic factor; TrkB: Tropomyosin-related kinase receptor B；siRNA:Small interfering RNA; ELISA:Enzyme-Linked Immunosorbent Assay

Funding information: This study was supported by the National Natural Science Foundation of China (No. 82160224), Guizhou Provincial Department of Education Youth Science and Technology Talent Growth Project (No. QIANJIAOHE KY[2022]238), Science and Technology Fund of Guizhou Provincial Health Commission(grant number gzwkj2023-399), Basic Research Program of Guizhou Province (Natural Sciences)(No. QIANKEHE ZK[2023]391), Start-up Fund of Affiliated Hospital of Guizhou Medical University(grant number gyfybsky-2022-05), Key Laboratory of Anesthesia and Pain Research, Guizhou Medical University (No.[2024]fy003).

Author contributions: H.L., XH. Z., YM.R. was the main designer of this study and drafted the manuscript. XY.Z., YS.L., W.H., B.T., and YM.R.performed the experiments and analyzed the data. All authors read and approved the final manuscript.

Conflict of interest: The authors have no conflicts of interest to declare

Data availability statement: The data that support the findings of this study are contained within the article and the Supporting Information. All source data generated for this study are available from the corresponding author (Zou Xiaohua; [email protected]) upon reasonable request.

Acknowledgment: Not applicable.

Castro L, Goncalves-De-Albuquerque CF, Silva AR (2022) Polarization of microglia and its therapeutic potential in sepsis. Int J Mol Sci 23
Charlton T, Prowse N, Mcfee A, Heiratifar N, Fortin T, Paquette C, Hayley S (2023) Brain-derived neurotrophic factor (bdnf) has direct anti-inflammatory effects on microglia. Front Cell Neurosci 17:1188672
Correale J (2014) The role of microglial activation in disease progression. Mult Scler 20:1288-1295
Frizelle PA, Chen PE, Wyllie DJ (2006) Equilibrium constants for (r)-[(s)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (nvp-aam077) acting at recombinant nr1/nr2a and nr1/nr2b n-methyl-d-aspartate receptors: implications for studies of synaptic transmission. Mol Pharmacol 70:1022-1032
Huang A, Chen Y, Wang S, Du H, Guan A, Wu H, Zhai Q, Duan N, Li X, Zhao P, Zhu Y, Bai J, Xiao Y, Yang T, Wang Q, Deng B (2023) Esketamine ameliorates post-stroke anxiety by modulating microglial hdac3/nf-κb/cox1 inflammatory signaling in ischemic cortex. Eur J Pharmacol 947:175667
Kaindl AM, Degos V, Peineau S, Gouadon E, Chhor V, Loron G, Le Charpentier T, Josserand J, Ali C, Vivien D, Collingridge GL, Lombet A, Issa L, Rene F, Loeffler JP, Kavelaars A, Verney C, Mantz J, Gressens P (2012) Activation of microglial n-methyl-d-aspartate receptors triggers inflammation and neuronal cell death in the developing and mature brain. Ann Neurol 72:536-549
Li H, Hu W, Wu Z, Tian B, Ren Y, Zou X (2024) Esketamine improves cognitive function in sepsis-associated encephalopathy by inhibiting microglia-mediated neuroinflammation. Eur J Pharmacol 983:177014
Li N, Lee B, Liu RJ, Banasr M, Dwyer JM, Iwata M, Li XY, Aghajanian G, Duman RS (2010) Mtor-dependent synapse formation underlies the rapid antidepressant effects of nmda antagonists. Science 329:959-964
Li Y, Wu Z, Zheng W, Wang J, Yue-Xin, Song R, Gao J (2022) Esketamine alleviates postoperative cognitive decline via stimulator of interferon genes/ tank-binding kinase 1 signaling pathway in aged rats. Brain Res Bull 187:169-180
Li Y, Wu ZY, Zheng WC, Wang JX, Yue-Xin, Song RX, Gao JG (2022) Esketamine alleviates postoperative cognitive decline via stimulator of interferon genes/ tank-binding kinase 1 signaling pathway in aged rats. Brain Res Bull 187:169-180
Lu Y, Ding X, Wu X, Huang S (2020) Ketamine inhibits lps-mediated bv2 microglial inflammation via nmda receptor blockage. Fundam Clin Pharmacol 34:229-237
Lucin KM, Wyss-Coray T (2009) Immune activation in brain aging and neurodegeneration: too much or too little? Neuron 64:110-122
Miller OH, Yang L, Wang CC, Hargroder EA, Zhang Y, Delpire E, Hall BJ (2014) Glun2b-containing nmda receptors regulate depression-like behavior and are critical for the rapid antidepressant actions of ketamine. Elife 3:e3581
Nowak W, Grendas LN, Sanmarco LM, Estecho IG, Arena AR, Eberhardt N, Rodante DE, Aoki MP, Daray FM, Carrera SE, Errasti AE (2019) Pro-inflammatory monocyte profile in patients with major depressive disorder and suicide behaviour and how ketamine induces anti-inflammatory m2 macrophages by nmdar and mtor. Ebiomedicine 50:290-305
Paoletti P, Bellone C, Zhou Q (2013) Nmda receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nat Rev Neurosci 14:383-400
Schatzberg AF (2021) Mechanisms of action of ketamine and esketamine. Am J Psychiatry 178:1130
Su T, Lu Y, Fu C, Geng Y, Chen Y (2023) Glun2a mediates ketamine-induced rapid antidepressant-like responses. Nat Neurosci 26:1751-1761
Sun S, Zou Y, Hao S, Niu Z, Wu L (2021) Polydatin inhibits lps-induced inflammatory response in bv2 microglia by disrupting the formation of lipid rafts. Immunopharmacol Immunotoxicol 43:138-144
Wang T, Weng H, Zhou H, Yang Z, Tian Z, Xi B, Li Y (2022) Esketamine alleviates postoperative depression-like behavior through anti-inflammatory actions in mouse prefrontal cortex. J Affect Disord 307:97-107
Wei Y, Chang L, Hashimoto K (2022) Molecular mechanisms underlying the antidepressant actions of arketamine: beyond the nmda receptor. Mol Psychiatry 27:559-573
Wen Y, Xu J, Shen J, Tang Z, Li S, Zhang Q, Li J, Sun J (2024) Esketamine prevents postoperative emotional and cognitive dysfunction by suppressing microglial m1 polarization and regulating the bdnf-trkb pathway in ageing rats with preoperative sleep disturbance. Mol Neurobiol
Wright-Jin EC, Gutmann DH (2019) Microglia as dynamic cellular mediators of brain function. Trends Mol Med 25:967-979
Wu CC, Tzeng CY, Chang CY, Wang JD, Chen YF, Chen WY, Kuan YH, Liao SL, Wang WY, Chen CJ (2023) Nmda receptor inhibitor mk801 alleviated pro-inflammatory polarization of bv-2 microglia cells. Eur J Pharmacol 955:175927
Wu Q, Zhao Y, Chen X, Zhu M, Miao C (2018) Propofol attenuates bv2 microglia inflammation via nmda receptor inhibition. Can J Physiol Pharmacol 96:241-248
Yan X, Yang K, Xiao Q, Hou R, Pan X, Zhu X (2022) Central role of microglia in sepsis-associated encephalopathy: from mechanism to therapy. Front Immunol 13:929316
Yang C, Ren Q, Qu Y, Zhang JC, Ma M, Dong C, Hashimoto K (2018) Mechanistic target of rapamycin-independent antidepressant effects of (r)-ketamine in a social defeat stress model. Biol Psychiatry 83:18-28
Yang MT, Lin YC, Ho WH, Liu CL, Lee WT (2017) Everolimus is better than rapamycin in attenuating neuroinflammation in kainic acid-induced seizures. J Neuroinflammation 14:15
Yu H, Chang Q, Sun T, He X, Wen L, An J, Feng J, Zhao Y (2023) Metabolic reprogramming and polarization of microglia in parkin

No competing interests reported.

supplement.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Targeting NMDAR2A: Esketamine's Impact on BV2 Microglial Pro-inflammatory Polarization

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Cell culture drug treatments

2.2 Small interfering RNA (siRNA) transfection

2.3 Enzyme-Linked Immunosorbent Assay (ELISA)

2.4 Western blot

2.5 Immunofluorescence

2.6 Statistical analysis

3.Results

3.1 Esketamine suppresses the proinflammatory activation of microglia through the inhibition of NMDAR

3.2 Esketamine induces the up-regulation of p-mTOR and BDNF-TrkB through the inhibition of NMDAR.

3.3 Inhibiting mTOR may counteract Esketamine's impact on microglia pro-inflammatory polarization.

3.4 Inhibition of mTOR reversed the upregulation of BDNF-TrkB induced by Esketamine

3.5 Esketamine attenuate BV2 microglial cell pro-inflammation polarization through NMDAR2A.

3.6 Esketamine regulates the expression of mTOR and BDNF-TrkB proteins through NMDAR2A

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1