Esketamine suppresses astrocyte-driven neuroinflammation in traumatic brain injury via the METTL5/c-Myc/PD-L1

Esketamine suppresses astrocyte-driven neuroinflammation in traumatic brain injury via the METTL5/c-Myc/PD-L1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Esketamine suppresses astrocyte-driven neuroinflammation in traumatic brain injury via the METTL5/c-Myc/PD-L1 Lan Luo, Miao Yu, Xiaoyan Li, Yonghong Bi, Pengyu Duan, Yao Meng, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7330976/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2026 Read the published version in Neurochemical Research → Version 1 posted 10 You are reading this latest preprint version Abstract Background Traumatic brain injury (TBI) is a major global health issue leading to high mortality and disability. Activated astrocytes are one of the pivotal driving factors in the neuroinflammatory cascade following TBI. Our study investigates the role of esketamine in modulating astrocyte-induced neuroinflammation, a key factor in enhancing TBI patient outcomes. Method Mice received a mouse weight-drop cortical impact or sham surgery and TBI mice were treated with either vehicle or esketamine at 2 h post-injury for 7 consecutive days. The modified Neurological Severity Scoring system, Rotarod test, Open Field test and Novel Object Recognition test were used to assess the neurological function after TBI. And cortical tissues surrounding focal trauma were obtained for Nissl staining, immunofluorescence, ELISA assay and western blotting. In vitro, astrocytes were induced with LPS, followed by the addition of esketamine to the culture medium. After a 24-hour exposure, the astrocytes were collected for CCK-8 assay, qRT-PCR, western blotting, immunofluorescence and Co-IP analysis. Results Esketamine dramatically improved the neurological outcome of mice and reduced neuronal cell death ( P < 0.05) and neuroinflammation after TBI. Its anti-inflammatory benefits stem from its ability to suppress astrocyte activation ( P < 0.05), inhibit pro-inflammatory A1 astrocyte differentiation ( P < 0.01), and promote the formation of protective A2 astrocytes ( P < 0.01). Esketamine exerts its effects by inhibiting the METTL5/c-Myc/PD-L1 signaling pathway. Conclusions Esketamine can effectively alleviate activated astrocytes and promote the polarization of activated astrocytes toward A2 following TBI by inhibiting the METTL5/c-Myc/PD-L1, demonstrating significant anti-inflammatory and neuroprotective effects. Esketamine traumatic brain injury neuroinflammation activated astrocyte A1/A2 astrocytes METTL5/c-Myc/PD-L1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Traumatic brain injury (TBI) is a serious global public health problem that contributes considerably to mortality and disability. 1 It is estimated that 55 million new cases occur each year and this number continues to rise, particularly among young and middle-aged men. 1, 2 TBI is categorized into primary injury caused by direct trauma and secondary injury caused by pathological processes such as excitotoxicity and inflammation. Secondary injury is one of the main causes of poor prognosis in patients, and inhibiting secondary brain injury is the key to treating TBI. 3 Recent studies have demonstrated that N6-methyladenosine (m6A) modification plays an important role in the pathophysiology of TBI. 4 Previous studies on the function of m6A modification in TBI have mainly focused on m6A modification of messenger RNA (mRNA), but recent human genetic studies have indicated that methylation of ribosomal RNA (rRNA m6A) has also been associated with brain development and cognition. 4, 5 However, the specific role of rRNA m6A in TBI remains unclear and further in-depth studies are required. Numerous studies have been conducted on microglia-mediated neuroinflammation after TBI, but the relationship between astrocytes and neuroinflammation after TBI remains controversial, and the mechanisms need to be further revealed. In a recent study, the newly discovered methyltransferase METTL5 together with TRMT112 promoted the m6A methylation of 18S rRNA. 6 In TBI models, the expression of c-Myc gene is significantly upregulated. 7 Knockdown of METTL5 modulates the c-Myc signaling pathway and inhibits PD-L1 production. 8 PD‐L1 is expressed on both microglia and astrocytes and plays an important role in central nervous system inflammation. 9 Ketamine is a noncompetitive NMDA receptor antagonist commonly used in clinical practice for anesthetic, analgesia, antidepressant, and anti-anxiety treatment. 10 Research indicates that ketamine may reduce the viability and migration of colon cancer cells by blocking c-Myc. 11 Esketamine, the S-enantiomer of racemic ketamine, has been shown to have higher efficacy and plays a neuroprotective role in neurological deficits and cerebral edema caused by TBI. 12 However, the effect of esketamine on astrocyte-driven neuroinflammation after TBI is still unknown, and more research is needed to reveal the impact of esketamine on TBI induced astrocyte activation and whether the mechanism is related to regulating rRNA methylation through the METTL5/c-Myc/PD-L1 signaling pathway to exert neuroprotective effects. Materials and Methods 2.1. Animals Male C57BL/6J mice (22-25 g in weight, 8-12 weeks old) were ordered from the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University. Mice were maintained at 22 ± 1 °C, 50 ± 1% relative humidity, 12/12 light/dark cycle, and free access to water and food. The experimental procedures were conducted in accordance with the ethical guidelines set by the Institutional Review Board of the Second Affiliated Hospital of Harbin Medical University (Reference No.:YJSDW2024-115), which followed the ARRIVE guidelines and AVMA guidelines for the euthanasia of animals in the experiment. Mice were randomly divided into four groups (n = 10 per group): (1) Sham + 0.9% Nacl, (2) TBI + 0.9% Nacl, (3) TBI + 4 mg kg -1 esketamine, and (4) TBI + 8 mg kg -1 esketamine. A researcher who was blinded to the experimental conditions and treatment collected the data. All reagents used in the experiment are listed in Table S1. 2.2. TBI Model In our study, we used the Flierl et al. weight-drop model for TBI. 13 Mice were anesthetized with 50 mg kg -1 pentobarbital sodium, and their skulls were shaved and disinfected. Anesthesia depth was assessed by monitoring respiratory rate, pedal withdrawal reflexes, and maintaining body temperature at 37°C to ensure optimal conditions prior to surgery. Local anesthesia with 0.2% ropivacaine (1 mg/kg) was administered along the incision line at least 5 minutes before skin incision and reapplied postoperatively. A 4 mm bone window was made on the right side of the skull, 1.5 mm behind the bregma and beside the central sulcus (Figure 1b). A 20 g weight was dropped from a distance of 20 cm to cause a 3 mm diameter, 2 mm deep cortical injury. The mice convulsed and stopped breathing, but they recovered spontaneously after a few seconds. The sham group received identical procedures to the TBI group, excluding the 20-gram weight-drop impact. They were sutured and recovered from anesthesia on a warming pad. All mice were given an intraperitoneal injection of either vehicle or esketamine for 7 consecutive days. The first treatment begins at 2 hours after TBI. Postoperative analgesic criteria, aligned with national and international guidelines, were predefined to include signs of severe pain or discomfort (e.g., aggression, piloerection, hunched posture, or rapid shallow breathing). All animals in both the TBI and sham groups were monitored for pain multiple times daily for 7 consecutive days post-surgery. No animals required postoperative analgesic treatment during this period. By the 7th day, mice were deeply anesthetized with pentobarbital (70 mg/kg, i.p.) and then euthanized via cervical dislocation upon loss of consciousness and absence of pain reflexes. 2.3. Neurobehavioral evaluation The modified Neurological Severity Scoring System (mNSS) is employed to assess and grade various aspects of neurological function, including motor abilities, sensory responses, and reflex actions. 14 A score of 10 to 14 indicates severe injury; 5 to 9 indicates moderate injury; and 1 to 4 indicates mild injury. The maximum possible score is 14. The Table S2 provides a detailed scoring guideline. 2.4. Rotarod test Motor coordination and balance were determined by the rotarod test. 15 Before TBI, the mice were introduced to the rotarod apparatus, starting at a slow speed of 4 r min -1 , and gradually increasing to 40 r min -1 . The mice were required to remain their balance on the rotarod for at least 150 seconds. Data were collected at 1, 3, 5 and 7 days after TBI. Each trial ended when the mouse fell off the rod or after a maximum of 5 minutes. All animals were tested 3 times with a break of at least 10 minutes between tests. Data were presented as the mean of 3 tests. 2.5. Open field test Open field test (OFT) was used to evaluate the anxiety levels of the mice on the 7th day after TBI. 16 Mice were acclimated for 10 minutes in an opaque cage of 40 × 40 × 40 cm the day before the experiment. Before each experiment, mouse excrement was removed, and the field was wiped with 75% alcohol and left to air dry naturally. During the experiment, there should be no other sounds and no unintended interference. The mouse was placed in the center of the field, and its free movement was captured with a camera during the next five minutes. The center time and total moving distance of each mouse were analyzed by the automated behavioral tracking system. 2.6. Novel Object Recognition test On the 7th day post-TBI, the Novel Object Recognition (NOR) test was used to assess mice's learning and memory recovery. 17 Two 4 cm diameter spherical wooden objects were placed at the box's bottom corners. The mouse was positioned at the bottom facing the wall, and its movements were recorded for 5 minutes. After 1 hour, one sphere was replaced with a cubic object of equal size, and the mouse's interactions were recorded for another 5 minutes. The results were expressed using the novel object recognition index, which was calculated as follows: Novel Object Recognition Index = Time spent exploring the novel object / (Time spent exploring the novel object + Time spent exploring the familiar object). 2.7. Western blot Cortical tissue surrounding the lesion or cell lysate (n = 5 per group) were lysed, and total protein was assessed by the BCA method. Proteins were separated using SDS‐PAGE and then transferred to 0.45 μm PVDF membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 2 hours before incubating with primary antibodies overnight. The membranes were incubated with secondary antibodies for 1 h at room temperature the next day. Enhanced chemiluminescence (ECL) kits were used to observe immunoblots and for imaging on a GenoSens 2250 instrument (Shanghai, China), and the results were quantified using ImageJ software (National Institutes of Health, MD, USA). 2.8. Enzyme‐linked immunosorbent assay The levels of IL‐1β, TNF‐α, and IL‐10 in cortical tissue were quantified using Enzyme‐linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. 2.9. Nissl staining Nissl staining was performed according to the manufacturer’s instructions. After deparaffinizing and rehydrating 20 μm coronal sections, the slides were stained with Nissl Staining Solution at 37 °C for 5 minutes. A typical neuron has a large cell body, plenty of cytoplasm, and significant Nissl body levels. Other cell types, such as a shrunken cell body, condensed nuclei, and decreased or absent Nissl bodies, indicate damaged cells. Three randomly selected fields in the surrounding ipsilateral injured cortex were examined. Cell counting was performed by using the ImageJ software, and 4 mice from each group were quantified. 2.10. Immunofluorescence and confocal microscopy analysis Fix cells or tissue samples with 4% paraformaldehyde for 10 minutes. Then, wash the samples three times with PBS for 5 minutes each. To enhance antibody penetration, treat the samples with 0.3% Triton X-100 for 10 minutes. After that, block the samples with 2% BSA at room temperature for 2 hours. Add the diluted primary antibody solution and incubate overnight at 4 °C. The next day, wash the samples three times with PBS. Incubate with the fluorescently labeled secondary antibody at room temperature for 1 hour. Wash the samples three times with PBS again. Stain the second target antibody with the same way, and then mount the samples with an anti-fade reagent. Finally, use a confocal microscope to observe and record the fluorescence signals. 2.11. Cell culture The murine astrocyte cell lines, C8-D1A, were obtained from Procell Life Technology (Wuhan, China). The cells were routinely maintained in C8-D1A-specific culture medium and incubated in a humidified incubator at 37 °C with 5% CO 2 . The astrocyte cell was pretreated with 1μg ml -1 of lipopolysaccharide (LPS) for 24 h and then treated with esketamine for 24 h. 2.12. Plasmid transfection All of the small interfering RNAs (siRNAs), including METTL5-1, METTL5-2, METTL5-3 as well as the negative controls (NC siRNA) and positive controls (GAPDH siRNA) were purchased from Genecreate biological engineering (Wuhan, China). The astrocytes were transfected using Lipo2000 reagent (Invitrogen, USA), according to the manufacturer’s instructions. Briefly, 9 μl Lipo2000 reagent and 5 μl plasmid were diluted into 50 μl Opti-MEM and incubated at room temperature for 5 minutes, then mix and incubate for an additional 20 minutes. After 24 hours of transfection, cells were used for follow-up experiments. 2.13. The viability of astrocytes The Cell Counting Kit-8 (CCK-8) assay was used to evaluate the viability of astrocytes. Astrocytes were cultured in 96-well plates at a density of 5 × 10 3 cells per well. After adding 10 μl of the CCK-8 solution to each well, and the cells were incubated at 37 °C for 1 hour. The absorbance was measured at 450 nm using a Thermo Scientific microplate reader. 2.14. Quantitative reverse transcription‐polymerase chain reaction (qRT-PCR) Total RNA was extracted from cells using Trizol according to the manufacturer’s instructions (Invitrogen, California, USA). cDNA was synthesized using the Reverse Transcription kit (Servicebio, Wuhan, China), and quantitative polymerase chain reaction was performed using SYBR Green (Servicebio, Wuhan, China). All results were normalized to the mRNA expression level of GAPDH. Gene-specific primers were shown in Table S3. 2.15. Co-immunoprecipitation Incubate 20 μl of magnetic beads with 4.0 μg METTL5-specific primary antibody or 2 μg mouse IgG control antibody for 2 hours at 4 °C. Subsequently, add the protease inhibitor-treated cell lysate to the antibody-magnetic bead complex and incubate overnight at 4 °C. After incubation, place the antigen-antibody-magnetic bead complex on a magnetic stand to facilitate separation, and carefully discard the supernatant. To eliminate any unattached proteins, wash the magnetic beads with PBS three times. Finally, add 20 μl of 1 × SDS loading buffer to the beads, boil for 5 minutes to denature the proteins, and proceed with the Western blot analysis. 2.16. Statistical Analysis Data are presented as n (%), mean ± standard deviation (SD), median (interquartile range), or mean ± standard error of the mean (SEM), as appropriate. All statistical analyses were performed using GraphPad Prism 10.0. The normality of continuous data was assessed using the Shapiro-Wilk or Kolmogorov-Smirnov test. For data satisfying the assumptions of normal distribution and homogeneity of variance, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used for multiple comparisons. Non-normally distributed data were analyzed using the Kruskal-Wallis test, with Mann-Whitney U test for pairwise comparisons. For longitudinal data involving repeated measures, a two-way repeated measures ANOVA was applied to account for within-subject correlations and the interaction between group and time, with Bonferroni correction used for post hoc analyses. A P-value < 0.05 was considered statistically significant. Results 3.1. Esketamine improves neurological deficits and behavioral outcomes after TBI To assess the effects of different doses of esketamine on neurological function after TBI, we performed the mNSS, rotarod test, OFT, and NOR. These tests were administered in accordance with the research procedure (Figure 1a) to evaluate sensory-motor, cognitive, and anxiety-related behaviors. There were no significant differences among the groups before the TBI ( P > 0.05). On the first day following TBI, both the TBI group and esketamine-treated groups (4 mg kg -1 and 8 mg kg -1 ) exhibited severe neurological deficits, as evidenced by significantly elevated modified Neurological Severity Score (mNSS) (P < 0.0001), and demonstrated marked motor coordination impairments, indicated by a significantly reduced latency to fall in the rotarod test (P < 0.0001). However, on subsequent days, neurological deficits and motor coordination deficits started to recover in all the groups. By the 7th day, the esketamine-treated group demonstrated a significantly greater reduction in neurological deficit scores (P < 0.0001) (Figure 1c) and a more pronounced improvement in motor coordination (P < 0.0001) (Figure 1d) compared to the TBI group. Besides, mice treated with esketamine displayed significantly increased total distance moved ( P < 0.0001) and time in the center of the open field ( P < 0.01), suggesting improved activity and exploration (Figure 1e, 1g-h). In comparison to the TBI group, there was a significant difference in exploration time between novel and familiar objects ( P 0.05). In summary, esketamine has significantly promoted the recovery of neurological function after TBI, manifested by increased sensory and motor coordination, spatial learning and short-term memory, and a decrease in anxiety-related behaviors. The experimental data indicate that the therapeutic efficacy is optimum at a dose of 4 mg kg -1 for esketamine. Consequently, we decided to utilize the 4 mg kg -1 of esketamine in the subsequent experiments. 3.2. Esketamine significantly reduces neuronal cell death and neuroinflammation following TBI In order to evaluate the neuroprotective potential of esketamine, Nissl staining was employed on the mouse brain tissue seven days following TBI. The findings revealed that the brain tissue morphology of the Sham group mice remained unaffected, characterized by a contiguous and intact cortex. However, both the TBI group and the esketamine group exhibited severe parietal cortex damage and cortical morphology disruption. Microscopic examination revealed that neurons in the Sham group were neatly arranged with uniform, round shapes and centrally located, large, circular nuclei, surrounded by many Nissl bodies. In stark contrast, the TBI group displayed injured neurons with disorganized arrangements, irregular shapes, smaller sizes, and condensed chromatin. Nissl bodies were notably decreased, fragmented, and sometimes absent, indicating severe cellular damage. Notably, the intervention with esketamine significantly decreased the number of damaged neurons in the TBI group ( P < 0.05), indicating its potential to mitigate injury and promote recovery in TBI-damaged brain tissue (Figure 2a-b). The in vitro CCK8 experiment revealed treatment with esketamine significantly enhanced the viability of astrocytes and effectively suppressed cell death induced by LPS ( P < 0.0001) (Figure 2f-h). This finding is consistent with the results observed through Nissl staining in vivo. To investigate the effect of esketamine on inflammation in mice, levels of pro-inflammatory cytokines IL-1β and TNF-α, as well as the anti-inflammatory cytokine IL-10, were evaluated. Mice with TBI showed significantly higher levels of pro-inflammatory cytokines including IL-1β ( P < 0.001) and TNF-α, ( P < 0.01). Concurrently, there was a decrease in the levels of the anti-inflammatory cytokine IL-10 ( P < 0.05). Esketamine delivery decreased IL-1β and TNF-α, levels while increasing IL-10 levels (Figure 2c-e). 3.3. Esketamine inhibits METTL5 expression in activated astrocytes and modulates the polarization of activated astrocytes In physiological conditions, astrocytes in the adult human brain are typically in a non-migratory and quiescent state. However, under pathological conditions such as trauma and inflammation, astrocytes can be activated, characterized by hypertrophy, adoption of a fibroblast-like amoeboid morphology, increased migratory potential, and a concomitant increase in the expression of GFAP. 18 In this study, we employed specific antibodies to label METTL5 and GFAP for the identification of activated astrocytes. We observed an increase in METTL5 and GFAP double positive (METTL5 + GFAP + ) astrocytes surrounding the damage region in the TBI group. After treatment with esketamine, the number of METTL5 + GFAP + astrocytes decreased (Figure 3a). The Western blot analysis demonstrated that, compared to the sham group, GFAP and METTL5 expression increased in the injury area in the TBI group, but decreased after esketamine intervention (Figure 4e, 4l). Therefore, esketamine may suppress neuroinflammation by inhibiting the activated astrocytes. Astrocytes can be activated into two polarization states: the neurotoxic or pro-inflammatory phenotype (A1) marked by C3 and the neuroprotective or anti-inflammatory phenotype (A2) marked by S100A10 in response to pathological conditions such as trauma and inflammation. 19 In vitro, we used specific antibodies against C3, S100A10, and GFAP to mark A1 astrocytes, A2 astrocytes, and activated astrocytes. In the LPS group, the average fluorescence intensity of C3 increased while S100A10 decreased. esketamine treatment inhibited both the increase in C3 expression and the decrease in S100A10 expression (Figure 3b-e). These results suggest that esketamine reduces the activation of A1 astrocytes while increasing the formation of A2 astrocytes, which might be the mechanism behind its protective effect. 3.4. Esketamine suppresses the upregulation of METTL5/c-Myc/PD-L1 after TBI. To investigate the role of the METTL5/c-Myc/PD-L1 pathway in the neuroprotection of esketamine, western blot analysis was performed on cortical protein samples from the wounded region seven days after TBI. Our findings showed a considerable increase in METTL5, c-Myc, and PD-L1 protein levels in the TBI group versus sham surgery. This suggests that TBI may activate the METTL5/c-Myc/PD-L1 signaling pathway. The expression levels of these three proteins were dramatically reduced after esketamine treatment (Figure 4a-d), indicating that esketamine suppresses the METTL5/c-Myc/PD-L1 signaling pathway following TBI. To further understand the mechanism of action of esketamine, we conducted in vitro experiments using LPS. The in vitro results were comparable to those of the in vivo experiments, showing that esketamine administration significantly suppressed the expression of METTL5, c-Myc, and PD-L1 (Figure 4h-k). This result is consistent with the results from in vivo experiments. In subsequent in vitro experiments, we employed plasmid transfection technology to knock down the expression of the METTL5 gene. Firstly, a plasmid, METTL5-1 siRNA, which effectively knocked down the expression of METTL5 protein, was selected for use in following investigations through Western blot analysis (Figure 4f-g). We then assessed the transcriptional and protein levels of METTL5, c-Myc, and PD-L1. The results showed that after knocking down METTL5, the expression levels of c-Myc and PD-L1 were significantly reduced, which was consistent with the effects of esketamine administration (Figure 4h-k, 4m-o). This provides strong evidence that esketamine can inhibit the METTL5/c-Myc/PD-L1 signaling pathway after TBI. 3.5. Interaction of METTL 5, c-Myc, and PD-L1 in TBI Model In the TBI model, we utilized specific antibodies to label METTL5, c-Myc, and PD-L1 individually, while concurrently employing the GFAP antibody to identify activated astrocytes. We observed colocalization of METTL5, c-Myc, and PD-L1 within astrocytes, suggesting a potential interaction between METTL5 and c-Myc, PD-L1 (Figure 5a). To further validate the interaction between METTL5 and c-Myc, PD-L1, we conducted Co-IP analysis in vitro. Using a METTL5-specific antibody for the immunoprecipitation, the experimental results confirmed the existence of an actual interaction between METTL5 and c-Myc, as well as PD-L1 (Figure 5b). Discussion The activated astrocytes are strongly linked to the development of the neuroinflammatory cascade. Hence, efficient management of the neuroinflammatory cascade triggered by activated astrocytes is crucial for improving the prognosis of TBI patients. 20 In the current work, we found that esketamine exerted a protective effect on neurological function after TBI by blocking the METTL5/c-Myc/PD-L1 signaling axis, promoting the polarization of activated astrocytes toward A2, and significantly reducing astrocyte-mediated neuroinflammation. For a long time, it has been believed that ketamine may increase in intracranial pressure in TBI patients, leading to severe complications and limiting its use in TBI treatment. 21 However, a recent systematic review of 11 studies found that ketamine use does not cause sustained increases in intracranial pressure. Only one study in this analysis reported an intermittent rise of ICP while cerebral perfusion pressure remaining constant. More importantly, in these studies, there was no evidence of to suggest that ketamine causes any harm. 22 Besides, the multiple advantages of ketamine, including its excellent anesthetic and analgesic effects, antidepressant and antiepileptic properties, prevention of glutamate-induced neuronal necrosis, anti-inflammatory effects, as well as positive impact on airway resistance and hemodynamics, have rekindled interest in its use in patients with TBI. 10 The latest research shows that in the treatment of subarachnoid hemorrhage patients, esketamine has been confirmed to effectively and long-term inhibit the key process that leads to the worsening of brain injury, known as spreading depolarizations. 23 In addition, esketamine has neuroprotective effects against TBI by modulating autophagy and oxidative stress via AMPK/mTOR-dependent TFEB nuclear translocation. 12 In both in vivo and in vitro experiments, we observed significant improvements in neurological function and an increase in the number of surviving neurons in TBI mice treated with esketamine. In addition, the levels of inflammatory factors IL-1β and TNF-α in the mice's cortical tissue were significantly decreased, while the levels of the anti-inflammatory factor IL-10 increased. Our in vitro findings further revealed that treatment with esketamine significantly increased the viability of astrocytes. These findings confirm esketamine’s capacity to improve neuronal cell survival and neurological outcome by inhibiting the neuroinflammation after TBI, which enhances its potential for treatment in TBI. As the most dynamic and reversible chemical modification on ribonucleic acids, the m6A modification is found not only in mRNA but also in a wide range of non-coding RNAs (ncRNA). 4 m6A methylation plays an important role in disease mechanisms and therapy. The dynamic modulation of m6A methylation is achieved through the coordinated action of methyltransferases, demethylases, and RNA-binding proteins. 24 This intricate interplay ensures accurate regulation of RNA processing, such as splicing, translation, and degradation, which ultimately affects the stability and biological function of RNA molecules. 25 The m6A modification is well-documented for its roles in processes such as tumor immune responses 26 and metabolic reprogramming of tumor cells. 27 Emerging evidence suggests that the modulation of m6A modification can provide neuroprotective effects in central nervous system injuries. The neuroprotective effects of m6A modification have been attributed to its effects on improving neurological injury, suppressing inflammation, inhibiting apoptosis, reducing pyroptosis, and attenuating ferroptosis. 4 In Wu et al.'s study, acute brain injury resulted in upregulation of METTL3 protein expression in microglia. The increased METTL3 enhances the stability of BATF mRNA through the m6A-IGF2BP2-dependent pathway, which promotes BATF-dependent pathogenic genes expression in microglia and triggers both local and peripheral immune responses. 28 In a recent study, the newly discovered methyltransferase METTL5, in conjunction with TRMT112, facilitates the m6A methylation of 18S rRNA. 6 This modification not only improves rRNA metabolic stability, but also has a significant impact on ribosomal activity and cellular development processes. The dysregulation of METTL5 expression is notably linked to intellectual disability, cancer, and congenital dysplasia. 5 However, the exact role of METTL5 in TBI has yet to be fully elucidated. In both in vivo and in vitro experiments, we demonstrated for the first time that esketamine could inhibit the expression of METTL5 and improve the neurological function after TBI, which further reveals the mechanism of esketamine's protective effects against TBI. c-Myc is a common transcription factor that regulates the expression of multiple gene products involved in cell proliferation, growth, differentiation, and apoptosis. 29 Previous reports indicate that c-Myc can directly target the PD-L1 promoter, thereby regulating PD-L1 levels. 30 In a TBI model, reducing PD-L1 levels or deleting PD-1 signaling can ameliorate TBI-induced learning and memory deficits. 31 The expression of PD-L1 in senescent cells facilitates immune evasion, leading to the accumulation of senescent cells and enhanced inflammatory signaling. 32 In this study, we found that TBI leads to an increase in METTL5 protein expression in astrocytes, which enhances the stability of c-Myc rRNA and promotes the expression of c-Myc. Through plasmid-mediated knockdown of METTL5 or treatment with esketamine, we observed a reduction in METTL5 expression levels, along with a decrease in the expression of c-Myc and PD-L1. In addition, we verified the interaction relationship between METTL5, c-Myc, and PD-L1 through methods such as Co-IP and immunofluorescence colocalization. These findings suggest that the protective effect of esketamine after TBI may be achieved by suppressing METTL5/c-Myc/PD-L1. Neuroinflammation plays an important role in secondary brain injury after TBI. After TBI, acute and intense inflammatory cascade reactions occur, and the massive release of inflammatory mediators and cytokines can lead to secondary neuronal cell death and neurological dysfunction. Microglia have typically been the primary focus when exploring neuroinflammation after TBI. 33 However, with the increasing research on TBI in recent years, researchers have discovered that astrocytes also undergo significant structural changes in response to central nervous system injuries and diseases, a phenomenon known as astrocytes activation. 20 Although the role of activated astrocytes in TBI remains controversial, many clinical and experimental studies provide convincing evidence that activated astrocytes can exert a detrimental effect on central nervous system cells and tissues by producing inflammatory mediators and chemokines to exacerbate inflammation, hampering synapse sprouting or axon growth, the release of excitatory glutamate, and the disruption of the blood-brain barrier. 34-36 It has been now found that astrocytes can be activated into two polarization states: the neurotoxic or pro-inflammatory phenotype (A1) and the neuroprotective or anti-inflammatory phenotype (A2). A1 astrocytes lose their normal astrocyte functions of maintaining synapses due to the overexpression of complement cascade genes, and can exacerbate the progression of the disease by secretion of soluble neurotoxins to kill a portion of neurons and mature oligodendrocytes. In contrast, A2 astrocytes demonstrate a potential neuroprotective role by upregulating neurotrophic and anti-inflammatory genes, promoting neuronal survival and growth, and improving the reparative process. 19 In this study, we found that the expression of GFAP was significantly increased in the brain tissue surrounding the injury in mice after TBI, whereas esketamine treatment inhibited TBI-induced astrocyte activation. In vitro cellular experiments also confirmed that knockdown of METTL5 or treatment with esketamine produced similar results with reduced GFAP expression. Additionally, by using immunofluorescence labeling to detect subtype changes in activated astrocytes, we found that knockdown of METTL5 or treatment with esketamine suppressed the expression of A1 pro-inflammatory astrocytes and promoted the expression of A2 anti-inflammatory astrocytes. Taken together, we conclude that esketamine may exert a protective effect through the inhibition of activated astrocytes and the modulation of their polarization state. Our study has several limitations. First, in our experiments, we only used a single astrocyte without establishing a co-culture system, which prevented us from observing the interactions between astrocytes and other neuroimmune cells such as microglia. Second, although in the study of mouse TBI model, we identified that esketamine modulates astrocyte-mediated inflammation by inhibiting the METTL5/c-Myc/PD-L1 pathway. However, the translation of these findings to clinical practice, as well as the potential long-term side effects of esketamine use, including its impact on the nervous system, are important aspects that need to be thoroughly investigated in future study. Conclusion Esketamine can effectively alleviate activated astrocytes and promote the polarization of activated astrocytes toward A2 following TBI by inhibiting the METTL5/c-Myc/PD-L1 axis, demonstrating significant anti-inflammatory and neuroprotective effects. This finding provides new potential drug targets for TBI treatment and lays a theoretical foundation for the application of esketamine in neurological injuries. References Maas A I R, Menon D K, Adelson P D, et al. Traumatic brain injury: integrated approaches to improve prevention, clinical care, and research. Lancet Neurol. 2017;16: 987-1048. doi: 10.1016/S1474-4422(17)30371-X Injury G B D T B, Spinal Cord Injury C. 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Supplementary Files supplementaltable.docx GraphicalAbstract.docx Cite Share Download PDF Status: Published Journal Publication published 13 Mar, 2026 Read the published version in Neurochemical Research → Version 1 posted Editorial decision: Revision requested 02 Nov, 2025 Reviews received at journal 21 Oct, 2025 Reviewers agreed at journal 05 Oct, 2025 Reviewers agreed at journal 20 Aug, 2025 Reviews received at journal 18 Aug, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviewers invited by journal 17 Aug, 2025 Editor assigned by journal 14 Aug, 2025 Submission checks completed at journal 11 Aug, 2025 First submitted to journal 08 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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University","correspondingAuthor":false,"prefix":"","firstName":"Jiali","middleName":"","lastName":"Chen","suffix":""},{"id":503198784,"identity":"103c15ab-745f-4a81-9a62-3728b4c30a18","order_by":11,"name":"Bing Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYBACfmbG9t9/DGwSGCTAfGbCWiTbmw9I8BSkkaDFoOdYggTPh8OkaJHIMTCQMDifZz67O02CocI6sYH97AG8WsyBWhIMDG4Xy9w5u02C4Ux6YgNPXgJeLZYzcgwOJBjcTpwhkbtNgrHtcGKDBI8BfofdyDFsOGBwDqrlHzFazhxLZmwwOADV0kCEFmAgH2NmMEhOnCFzdrNFwrF04zaeHPxagFHZxszwxy5xhnTvxhsfaqxl+9nP4NeCChKAmI0E9aNgFIyCUTAKcAAAJ/JF9R8vES4AAAAASUVORK5CYII=","orcid":"","institution":"Second Affiliated Hospital of Harbin Medical University","correspondingAuthor":true,"prefix":"","firstName":"Bing","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-08-09 03:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7330976/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7330976/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11064-026-04724-8","type":"published","date":"2026-03-13T15:59:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89982299,"identity":"f6fde4f5-c85b-4fe6-8d16-008c63902e07","added_by":"auto","created_at":"2025-08-27 06:28:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":41878616,"visible":true,"origin":"","legend":"\u003cp\u003eEsketamine improves neurological deficits and behavioral outcomes after TBI. (a) The workflow of the study; (b) Schematic of TBI impact point; (c) Neurological deficit were evaluated via mNSS. A significant interaction between group and time was observed (F[9, 108] = 44.65, P \u0026lt; 0.0001).(d) Rotarod test was conducted to determine the motor function. A significant interaction between group and time was observed (F[9, 108] = 12.58, P \u0026lt; 0.0001) (e, g-h) Trajectory map, the total moving distance and center time during the OFT at 7 days after TBI; (f, i) Trajectory map and recognition index in the NOR at 7 days after TBI. \u003csup\u003e\u003cem\u003e**** \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001 compared with the Sham group; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e \u003c/sup\u003e\u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e\u003cem\u003e### \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, \u003csup\u003e\u003cem\u003e#### \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001 compared with the TBI group; Data are expressed as n (%), mean ± SD, or median (interquartile range) (n = 10 per group). TBI: traumatic brain injury; mNSS: the modified Neurological Severity Scoring system; OFT: Open field test; NOR: Novel Object Recognition test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7330976/v1/8365a8400200b48fa7c86ded.png"},{"id":89982303,"identity":"8b2d7a24-f8d0-43c8-8f26-4a438d3328cb","added_by":"auto","created_at":"2025-08-27 06:28:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95524453,"visible":true,"origin":"","legend":"\u003cp\u003eEsketamine significantly reduces neuronal cell death and neuroinflammation following TBI. (a) Nissl staining to assess TBI-induced neural cell death; (b) Number of normal neurons in different groups (n = 4 per group). (c-e) ELISA assay showing the effects of esketamine on IL-1β, TNF-α and IL-10 levels; (f-h) The CCK-8 kit was used to assess the impact of different concentrations of LPS (n = 4 per group) and esketamine on the viability of astrocytes (n = 6 per group). \u003csup\u003e\u003cem\u003e* \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e\u003cem\u003e** \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e\u003cem\u003e*** \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, \u003csup\u003e\u003cem\u003e**** \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001 compared with the Sham group; \u003csup\u003e\u003cem\u003e# \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e\u003cem\u003e## \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e\u003cem\u003e#### \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001 compared with the TBI or LPS group;\u003csup\u003e \u003c/sup\u003e\u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u0026amp;\u0026amp; \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001 compared with the LPS + esketamine group. Data are expressed as n (%) and mean ± SD. TBI: traumatic brain injury.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7330976/v1/e30bb4194df920c1f54b2889.png"},{"id":89982302,"identity":"39533f9e-aa42-408e-9ea9-9625ce1215c5","added_by":"auto","created_at":"2025-08-27 06:28:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":53565490,"visible":true,"origin":"","legend":"\u003cp\u003eEsketamine inhibits METTL5 expression in activated astrocytes and modulates the polarization of activated astrocytes. (a) Specific antibodies are employed to label METTL5, and GFAP labeling identifies activated astrocytes. When compared with the sham group, the TBI group had an increased number of METTL5\u003csup\u003e+\u003c/sup\u003eGFAP\u003csup\u003e+\u003c/sup\u003e astrocytes around the injury region. After esketamine intervention, the number of METTL5\u003csup\u003e+\u003c/sup\u003eGFAP\u003csup\u003e+\u003c/sup\u003e astrocytes decreases. (b-e) In vitro experiments, we utilized specific antibodies against C3, S100A10, and GFAP to label A1 toxic astrocytes, A2 neuroprotective astrocytes, and activated astrocytes respectively. Immunofluorescence techniques were used to analyze the effects of esketamine or METTL5 gene knockdown on the C3 and S100A10 (n = 3 per group). \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, \u003csup\u003e\u003cem\u003e**** \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001 compared with the Sham group; \u003csup\u003e\u003cem\u003e## \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e\u003cem\u003e### \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, \u003csup\u003e\u003cem\u003e#### \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001 compared with the LPS group; \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp; \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u0026amp; \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 compared with the LPS + esketamine group. Data are expressed as the mean ± SD. TBI: traumatic brain injury.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7330976/v1/126f687124697c12bf81c574.png"},{"id":89982300,"identity":"501cee31-d257-4d9a-adcd-db7ff3be3056","added_by":"auto","created_at":"2025-08-27 06:28:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":27432641,"visible":true,"origin":"","legend":"\u003cp\u003eEsketamine suppresses the upregulation of METTL5/c-Myc/PD-L1 after TBI. (a-e) Western blot analysis of METTL5, c-Myc, PD-L1, and GFAP levels after TBI in different groups (n = 5 per group); (f-g) The expression of the METTL5 protein in various plasmids following the successful knockdown of the METTL5 gene (n = 3 per group); (h-l) The effects of esketamine on the protein expression of METTL5, c-Myc, PD-L1, and GFAP in vitro (n = 5 per group); (m-o) qRT-PCR was used to evaluate the effect of esketamine on the mRNA expression of METTL5, c-Myc and PD-L1 in vitro (n = 3 per group). * P \u0026lt; 0.05, ** P \u0026lt; 0.01 *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001 compared with the Sham group; # P \u0026lt; 0.05, ## P \u0026lt; 0.01, ### P \u0026lt; 0.001, #### P \u0026lt; 0.0001 compared with the TBI or LPS group; \u0026amp; P \u0026lt; 0.05, \u0026amp;\u0026amp; P \u0026lt; 0.01, \u0026amp;\u0026amp;\u0026amp; P \u0026lt; 0.001, \u0026amp;\u0026amp;\u0026amp;\u0026amp; P \u0026lt; 0.0001 compared with the LPS + esketamine group. Data are expressed as the mean ± SD. TBI: traumatic brain injury.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7330976/v1/fc7d283d3703907fc5c8221f.png"},{"id":89982297,"identity":"412f867c-7747-49a4-adc1-51a046024a2e","added_by":"auto","created_at":"2025-08-27 06:28:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1818148,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction of METTL5, c-Myc, and PD-L1 in TBI Model. (a) Brain cortical slices from TBI mice were stained with antibodies against METTL5, c-Myc, PD-L1, and GFAP on the 7th day after TBI. The magnified single-cell images are highlighted with a red box to demonstrate the co-localization of METTL5, c-Myc, PD-L1, and GFAP within activated astrocytes. (b) Co-immunoprecipitation was used to indicate the binding of METTL5, c-Myc and PD-L1. TBI: traumatic brain injury.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7330976/v1/9ac73d0612ef18d036b7b811.png"},{"id":89982294,"identity":"26c4766f-b062-4791-a764-a7e42a689006","added_by":"auto","created_at":"2025-08-27 06:28:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":536630,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7330976/v1/c53e12df-a79e-4d39-a864-aaeb9fa9c6cc.pdf"},{"id":89982295,"identity":"07f5fb74-1ecb-4816-a351-da7134dd7df7","added_by":"auto","created_at":"2025-08-27 06:28:51","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":22808,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaltable.docx","url":"https://assets-eu.researchsquare.com/files/rs-7330976/v1/6ad371938d26b17a928e25a8.docx"},{"id":89982296,"identity":"af1151dd-f5ac-4a48-a471-2abfec710207","added_by":"auto","created_at":"2025-08-27 06:28:51","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":170681,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7330976/v1/00b60737d71a4b2cfb1bceeb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Esketamine suppresses astrocyte-driven neuroinflammation in traumatic brain injury via the METTL5/c-Myc/PD-L1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTraumatic brain injury (TBI) is a serious global public health problem that contributes considerably to mortality and disability.\u003csup\u003e1\u003c/sup\u003e It is estimated that 55 million new cases occur each year and this number continues to rise, particularly among young and middle-aged men.\u003csup\u003e1, 2\u003c/sup\u003e TBI is categorized into primary injury caused by direct trauma and secondary injury caused by pathological processes such as excitotoxicity and inflammation. Secondary injury is one of the main causes of poor prognosis in patients, and inhibiting secondary brain injury is the key to treating TBI.\u003csup\u003e3\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Recent studies have demonstrated that N6-methyladenosine (m6A) modification plays an important role in the pathophysiology of TBI.\u003csup\u003e4\u003c/sup\u003e Previous studies on the function of m6A modification in TBI have mainly focused on m6A modification of messenger RNA (mRNA), but recent human genetic studies have indicated that methylation of ribosomal RNA (rRNA m6A) has also been associated with brain development and cognition.\u003csup\u003e4, 5\u003c/sup\u003e However, the specific role of rRNA m6A in TBI remains unclear and further in-depth studies are required.\u003c/p\u003e\n\u003cp\u003eNumerous studies have been conducted on microglia-mediated neuroinflammation after TBI, but the relationship between astrocytes and neuroinflammation after TBI remains controversial, and the mechanisms need to be further revealed. In a recent study, the newly discovered methyltransferase METTL5 together with TRMT112 promoted the m6A methylation of 18S rRNA.\u003csup\u003e6\u003c/sup\u003e In TBI models, the expression of c-Myc gene is significantly upregulated.\u003csup\u003e7\u003c/sup\u003e Knockdown of METTL5 modulates the c-Myc signaling pathway and inhibits PD-L1 production.\u003csup\u003e8\u003c/sup\u003e PD‐L1 is expressed on both microglia and astrocytes and plays an important role in central nervous system inflammation.\u003csup\u003e9\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKetamine is a noncompetitive NMDA receptor antagonist commonly used in clinical practice for anesthetic, analgesia, antidepressant, and anti-anxiety treatment.\u003csup\u003e10\u003c/sup\u003e Research indicates that ketamine may reduce the viability and migration of colon cancer cells by blocking c-Myc.\u003csup\u003e11\u003c/sup\u003e Esketamine, the S-enantiomer of racemic ketamine, has been shown to have higher efficacy and plays a neuroprotective role in neurological deficits and cerebral edema caused by TBI.\u003csup\u003e12\u003c/sup\u003e However, the effect of esketamine on astrocyte-driven neuroinflammation after TBI is still unknown, and more research is needed to reveal the impact of esketamine on TBI induced astrocyte activation and whether the mechanism is related to regulating rRNA methylation through the METTL5/c-Myc/PD-L1 signaling pathway to exert neuroprotective effects.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1.\u0026nbsp;\u003c/strong\u003eAnimals\u003c/p\u003e\n\u003cp\u003eMale C57BL/6J mice (22-25\u0026nbsp;g in weight, 8-12 weeks old) were ordered from the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University. Mice were maintained at 22\u0026nbsp;\u0026plusmn;\u0026nbsp;1\u0026nbsp;\u0026deg;C, 50\u0026nbsp;\u0026plusmn;\u0026nbsp;1% relative humidity, 12/12 light/dark cycle, and free access to water and food. The experimental procedures were conducted in accordance with the ethical guidelines set by the Institutional Review Board of the Second Affiliated Hospital of Harbin Medical University (Reference No.:YJSDW2024-115), which followed the ARRIVE guidelines and AVMA guidelines for the euthanasia of animals in the experiment. Mice were randomly divided into four groups (n = 10 per group): (1) Sham + 0.9% Nacl, (2) TBI + 0.9% Nacl, (3) TBI + 4 mg kg\u003csup\u003e-1\u003c/sup\u003e esketamine, and (4) TBI + 8 mg kg\u003csup\u003e-1\u003c/sup\u003e esketamine. A researcher who was blinded to the experimental conditions and treatment collected the data. All reagents used in the experiment are listed in Table S1.\u003c/p\u003e\n\u003cp\u003e2.2. TBI Model\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our study, we used the Flierl et al. weight-drop model for TBI.\u003csup\u003e13\u003c/sup\u003e Mice were anesthetized with 50 mg kg\u003csup\u003e-1\u003c/sup\u003e pentobarbital sodium, and their skulls were shaved and disinfected. Anesthesia depth was assessed by monitoring respiratory rate, pedal withdrawal reflexes, and maintaining body temperature at 37\u0026deg;C to ensure optimal conditions prior to surgery. Local anesthesia with 0.2% ropivacaine (1 mg/kg) was administered along the incision line at least 5 minutes before skin incision and reapplied postoperatively. A 4 mm bone window was made on the right side of the skull, 1.5 mm behind the bregma and beside the central sulcus (Figure 1b). A 20 g weight was dropped from a distance of 20 cm to cause a 3 mm diameter, 2 mm deep cortical injury. The mice convulsed and stopped breathing, but they recovered spontaneously after a few seconds. The sham group received identical procedures to the TBI group, excluding the 20-gram weight-drop impact. They were sutured and recovered from anesthesia on a warming pad. All mice were given an intraperitoneal injection of either vehicle or esketamine for 7 consecutive days. The first treatment begins at 2 hours after TBI. Postoperative analgesic criteria, aligned with national and international guidelines, were predefined to include signs of severe pain or discomfort (e.g., aggression, piloerection, hunched posture, or rapid shallow breathing). All animals in both the TBI and sham groups were monitored for pain multiple times daily for 7 consecutive days post-surgery. No animals required postoperative analgesic treatment during this period. By the 7th day, mice were deeply anesthetized with pentobarbital (70 mg/kg, i.p.) and then euthanized via cervical dislocation upon loss of consciousness and absence of pain reflexes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.3. Neurobehavioral evaluation\u003c/p\u003e\n\u003cp\u003eThe modified Neurological Severity Scoring System (mNSS) is employed to assess and grade various aspects of neurological function, including motor abilities, sensory responses, and reflex actions.\u003csup\u003e14\u003c/sup\u003e A score of 10 to 14 indicates severe injury; 5 to 9 indicates moderate injury; and 1 to 4 indicates mild injury. The maximum possible score is 14. The Table S2 provides a detailed scoring guideline.\u003c/p\u003e\n\u003cp\u003e2.4. Rotarod test\u003c/p\u003e\n\u003cp\u003eMotor coordination and balance were determined by the rotarod test.\u003csup\u003e15\u003c/sup\u003e Before TBI, the mice were introduced to the rotarod apparatus, starting at a slow speed of 4 r min\u003csup\u003e-1\u003c/sup\u003e, and gradually increasing to 40 r min\u003csup\u003e-1\u003c/sup\u003e. The mice were required to remain their balance on the rotarod for at least 150 seconds. Data were collected at 1, 3, 5 and 7 days after TBI. Each trial ended when the mouse fell off the rod or after a maximum of 5 minutes. All animals were tested 3 times with a break of at least 10 minutes between tests. Data were presented as the mean of 3 tests.\u003c/p\u003e\n\u003cp\u003e2.5. Open field test\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOpen field test (OFT) was used to evaluate the anxiety levels of the mice on the 7th day after TBI.\u003csup\u003e16\u003c/sup\u003e Mice were acclimated for 10 minutes in an opaque cage of 40 \u0026times; 40 \u0026times; 40 cm the day before the experiment. Before each experiment, mouse excrement was removed, and the field was wiped with 75% alcohol and left to air dry naturally. During the experiment, there should be no other sounds and no unintended interference. The mouse was placed in the center of the field, and its free movement was captured with a camera during the next five minutes. The center time and total moving distance of each mouse were analyzed by the automated behavioral tracking system.\u003c/p\u003e\n\u003cp\u003e2.6. Novel Object Recognition test\u003c/p\u003e\n\u003cp\u003eOn the 7th day post-TBI, the Novel Object Recognition (NOR) test was used to assess mice\u0026apos;s learning and memory recovery.\u003csup\u003e17\u003c/sup\u003e Two 4 cm diameter spherical wooden objects were placed at the box\u0026apos;s bottom corners. The mouse was positioned at the bottom facing the wall, and its movements were recorded for 5 minutes. After 1 hour, one sphere was replaced with a cubic object of equal size, and the mouse\u0026apos;s interactions were recorded for another 5 minutes. The results were expressed using the novel object recognition index, which was calculated as follows: Novel Object Recognition Index = Time spent exploring the novel object / (Time spent exploring the novel object + Time spent exploring the familiar object).\u003c/p\u003e\n\u003cp\u003e2.7. Western blot\u003c/p\u003e\n\u003cp\u003eCortical tissue surrounding the lesion or cell lysate (n = 5 per group) were lysed, and total protein was assessed by the BCA method. Proteins were separated using SDS‐PAGE and then transferred to 0.45\u0026thinsp;\u0026mu;m PVDF membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 2\u0026thinsp;hours before incubating with primary antibodies overnight. The membranes were incubated with secondary antibodies for 1\u0026thinsp;h at room temperature the next day. Enhanced chemiluminescence (ECL) kits were used to observe immunoblots and for imaging on a GenoSens 2250 instrument (Shanghai, China), and the results were quantified using ImageJ software (National Institutes of Health, MD, USA).\u003c/p\u003e\n\u003cp\u003e2.8. Enzyme‐linked immunosorbent assay\u003c/p\u003e\n\u003cp\u003eThe levels of IL‐1\u0026beta;, TNF‐\u0026alpha;, and IL‐10 in cortical tissue were quantified using Enzyme‐linked immunosorbent assay (ELISA) kits according to the manufacturer\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003e2.9. Nissl staining\u003c/p\u003e\n\u003cp\u003eNissl staining was performed according to the manufacturer\u0026rsquo;s instructions. After deparaffinizing and rehydrating 20 \u0026mu;m coronal sections, the slides were stained with Nissl Staining Solution at 37\u0026thinsp;\u0026deg;C for 5\u0026thinsp;minutes. A typical neuron has a large cell body, plenty of cytoplasm, and significant Nissl body levels. Other cell types, such as a shrunken cell body, condensed nuclei, and decreased or absent Nissl bodies, indicate damaged cells. Three randomly selected fields in the surrounding ipsilateral injured cortex were examined. Cell counting was performed by using the ImageJ software, and 4 mice from each group were quantified.\u003c/p\u003e\n\u003cp\u003e2.10. Immunofluorescence and confocal microscopy analysis\u003c/p\u003e\n\u003cp\u003eFix cells or tissue samples with 4% paraformaldehyde for 10 minutes. Then, wash the samples three times with PBS for 5 minutes each. To enhance antibody penetration, treat the samples with 0.3% Triton X-100 for 10 minutes. After that, block the samples with 2% BSA at room temperature for 2 hours. Add the diluted primary antibody solution and incubate overnight at 4 \u0026deg;C. The next day, wash the samples three times with PBS. Incubate with the fluorescently labeled secondary antibody at room temperature for 1 hour. Wash the samples three times with PBS again. Stain the second target antibody with the same way, and then mount the samples with an anti-fade reagent. Finally, use a confocal microscope to observe and record the fluorescence signals.\u003c/p\u003e\n\u003cp\u003e2.11. Cell culture\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe murine astrocyte cell lines, C8-D1A, were obtained from Procell Life Technology (Wuhan, China). The cells were routinely maintained in C8-D1A-specific culture medium and incubated in a humidified incubator at 37 \u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The astrocyte cell was pretreated with 1\u0026mu;g ml\u003csup\u003e-1\u003c/sup\u003e of lipopolysaccharide (LPS) for 24 h and then treated with esketamine for 24 h.\u003c/p\u003e\n\u003cp\u003e2.12. Plasmid transfection\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All of the small interfering RNAs (siRNAs), including METTL5-1, METTL5-2, METTL5-3 as well as the negative controls (NC siRNA) and positive controls (GAPDH siRNA) were purchased from Genecreate biological engineering (Wuhan, China). The astrocytes were transfected using Lipo2000 reagent (Invitrogen, USA), according to the manufacturer\u0026rsquo;s instructions. Briefly, 9 \u0026mu;l Lipo2000 reagent and 5 \u0026mu;l plasmid were diluted into 50 \u0026mu;l Opti-MEM and incubated at room temperature for 5 minutes, then mix and incubate for an additional 20 minutes. After 24 hours of transfection, cells were used for follow-up experiments.\u003c/p\u003e\n\u003cp\u003e2.13. The viability of astrocytes\u003c/p\u003e\n\u003cp\u003eThe Cell Counting Kit-8 (CCK-8) assay was used to evaluate the viability of astrocytes. Astrocytes were cultured in 96-well plates at a density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well. After adding 10 \u0026mu;l of the CCK-8 solution to each well, and the cells were incubated at 37 \u0026deg;C for 1 hour. The absorbance was measured at 450 nm using a Thermo Scientific microplate reader.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.14. Quantitative reverse transcription‐polymerase chain reaction (qRT-PCR)\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cells using Trizol according to the manufacturer\u0026rsquo;s instructions (Invitrogen, California, USA). cDNA was synthesized using the Reverse Transcription kit (Servicebio, Wuhan, China), and quantitative polymerase chain reaction was performed using SYBR Green (Servicebio, Wuhan, China). All results were normalized to the mRNA expression level of GAPDH. Gene-specific primers were shown in Table S3.\u003c/p\u003e\n\u003cp\u003e2.15. Co-immunoprecipitation\u003c/p\u003e\n\u003cp\u003eIncubate 20 \u0026mu;l of magnetic beads with 4.0 \u0026mu;g METTL5-specific primary antibody or 2 \u0026mu;g mouse IgG control antibody for 2 hours at 4 \u0026deg;C. Subsequently, add the protease inhibitor-treated cell lysate to the antibody-magnetic bead complex and incubate overnight at 4 \u0026deg;C. After incubation, place the antigen-antibody-magnetic bead complex on a magnetic stand to facilitate separation, and carefully discard the supernatant. To eliminate any unattached proteins, wash the magnetic beads with PBS three times. Finally, add 20 \u0026mu;l of 1 \u0026times; SDS loading buffer to the beads, boil for 5 minutes to denature the proteins, and proceed with the Western blot analysis.\u003c/p\u003e\n\u003cp\u003e2.16. Statistical Analysis\u003c/p\u003e\n\u003cp\u003eData are presented as n (%), mean \u0026plusmn; standard deviation (SD), median (interquartile range), or mean \u0026plusmn; standard error of the mean (SEM), as appropriate. All statistical analyses were performed using GraphPad Prism 10.0. The normality of continuous data was assessed using the Shapiro-Wilk or Kolmogorov-Smirnov test. For data satisfying the assumptions of normal distribution and homogeneity of variance, one-way analysis of variance (ANOVA) followed by Tukey\u0026apos;s post hoc test was used for multiple comparisons. Non-normally distributed data were analyzed using the Kruskal-Wallis test, with Mann-Whitney U test for pairwise comparisons. For longitudinal data involving repeated measures, a two-way repeated measures ANOVA was applied to account for within-subject correlations and the interaction between group and time, with Bonferroni correction used for post hoc analyses. A P-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEsketamine improves neurological deficits and behavioral outcomes after TBI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the effects of different doses of esketamine on neurological function after TBI, we performed the mNSS, rotarod test, OFT, and NOR. These tests were administered in accordance with the research procedure (Figure 1a) to evaluate sensory-motor, cognitive, and anxiety-related behaviors. There were no significant differences among the groups before the TBI (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). On the first day following TBI, both the TBI group and esketamine-treated groups (4 mg kg\u003csup\u003e-1\u003c/sup\u003e and 8 mg kg\u003csup\u003e-1\u003c/sup\u003e) exhibited severe neurological deficits, as evidenced by significantly elevated modified Neurological Severity Score (mNSS) (P \u0026lt; 0.0001), and demonstrated marked motor coordination impairments, indicated by a significantly reduced latency to fall in the rotarod test (P \u0026lt; 0.0001). However, on subsequent days, neurological deficits and motor coordination deficits started to recover in all the groups. By the 7th day, the esketamine-treated group demonstrated a significantly greater reduction in neurological deficit scores (P \u0026lt; 0.0001) (Figure 1c) and a more pronounced improvement in motor coordination (P \u0026lt; 0.0001) (Figure 1d) compared to the TBI group. Besides, mice treated with esketamine displayed significantly increased total distance moved (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001) and time in the center of the open field (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01), suggesting improved activity and exploration (Figure 1e, 1g-h). In comparison to the TBI group, there was a significant difference in exploration time between novel and familiar objects (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001), suggesting improved cognitive function and memory (Figure 1f, 1i). However, no significant difference was observed between the 4 mg kg\u003csup\u003e-1\u003c/sup\u003e and 8 mg kg\u003csup\u003e-1\u003c/sup\u003e esketamine groups (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003eIn summary, esketamine has significantly promoted the recovery of neurological function after TBI, manifested by increased sensory and motor coordination, spatial learning and short-term memory, and a decrease in anxiety-related behaviors. The experimental data indicate that the therapeutic efficacy is optimum at a dose of 4 mg kg\u003csup\u003e-1\u003c/sup\u003e for esketamine. Consequently, we decided to utilize the 4 mg kg\u003csup\u003e-1\u003c/sup\u003e of esketamine in the subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEsketamine significantly reduces neuronal cell death and neuroinflammation following TBI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to evaluate the neuroprotective potential of esketamine, Nissl staining was employed on the mouse brain tissue seven days following TBI. The findings revealed that the brain tissue morphology of the Sham group mice remained unaffected, characterized by a contiguous and intact cortex. However, both the TBI group and the esketamine group exhibited severe parietal cortex damage and cortical morphology disruption. Microscopic examination revealed that neurons in the Sham group were neatly arranged with uniform, round shapes and centrally located, large, circular nuclei, surrounded by many Nissl bodies. In stark contrast, the TBI group displayed injured neurons with disorganized arrangements, irregular shapes, smaller sizes, and condensed chromatin. Nissl bodies were notably decreased, fragmented, and sometimes absent, indicating severe cellular damage. Notably, the intervention with esketamine significantly decreased the number of damaged neurons in the TBI group (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05), indicating its potential to mitigate injury and promote recovery in TBI-damaged brain tissue (Figure 2a-b). The in vitro CCK8 experiment revealed treatment with esketamine significantly enhanced the viability of astrocytes and effectively suppressed cell death induced by LPS (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001) (Figure 2f-h). This finding is consistent with the results observed through Nissl staining in vivo.\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of esketamine on inflammation in mice, levels of pro-inflammatory cytokines IL-1\u0026beta; and TNF-\u0026alpha;, as well as the anti-inflammatory cytokine IL-10, were evaluated. Mice with TBI showed significantly higher levels of pro-inflammatory cytokines including IL-1\u0026beta; (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) and TNF-\u0026alpha;, (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Concurrently, there was a decrease in the levels of the anti-inflammatory cytokine IL-10 (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05). Esketamine delivery decreased IL-1\u0026beta; and TNF-\u0026alpha;, levels while increasing IL-10 levels (Figure 2c-e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEsketamine inhibits METTL5 expression in activated astrocytes and modulates the polarization of activated astrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn physiological conditions, astrocytes in the adult human brain are typically in a non-migratory and quiescent state. However, under pathological conditions such as trauma and inflammation, astrocytes can be activated, characterized by hypertrophy, adoption of a fibroblast-like amoeboid morphology, increased migratory potential, and a concomitant increase in the expression of GFAP.\u003csup\u003e18\u003c/sup\u003e In this study, we employed specific antibodies to label METTL5 and GFAP for the identification of activated astrocytes. We observed an increase in METTL5 and GFAP double positive (METTL5\u003csup\u003e+\u003c/sup\u003eGFAP\u003csup\u003e+\u003c/sup\u003e) astrocytes surrounding the damage region in the TBI group. After treatment with esketamine, the number of METTL5\u003csup\u003e+\u003c/sup\u003eGFAP\u003csup\u003e+\u003c/sup\u003e astrocytes decreased (Figure 3a). The Western blot analysis demonstrated that, compared to the sham group, GFAP and METTL5 expression increased in the injury area in the TBI group, but decreased after esketamine intervention (Figure 4e, 4l). Therefore, esketamine may suppress neuroinflammation by inhibiting the activated astrocytes.\u003c/p\u003e\n\u003cp\u003eAstrocytes can be activated into two polarization states: the neurotoxic or pro-inflammatory phenotype (A1) marked by C3 and the neuroprotective or anti-inflammatory phenotype (A2) marked by S100A10 in response to pathological conditions such as trauma and inflammation.\u003csup\u003e19\u003c/sup\u003e In vitro, we used specific antibodies against C3, S100A10, and GFAP to mark A1 astrocytes, A2 astrocytes, and activated astrocytes. In the LPS group, the average fluorescence intensity of C3 increased while S100A10 decreased. esketamine treatment inhibited both the increase in C3 expression and the decrease in S100A10 expression (Figure 3b-e). These results suggest that esketamine reduces the activation of A1 astrocytes while increasing the formation of A2 astrocytes, which might be the mechanism behind its protective effect.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEsketamine suppresses the upregulation of METTL5/c-Myc/PD-L1 after TBI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of the METTL5/c-Myc/PD-L1 pathway in the neuroprotection of esketamine, western blot analysis was performed on cortical protein samples from the wounded region seven days after TBI. Our findings showed a considerable increase in METTL5, c-Myc, and PD-L1 protein levels in the TBI group versus sham surgery. This suggests that TBI may activate the METTL5/c-Myc/PD-L1 signaling pathway. The expression levels of these three proteins were dramatically reduced after esketamine treatment (Figure 4a-d), indicating that esketamine suppresses the METTL5/c-Myc/PD-L1 signaling pathway following TBI.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;To further understand the mechanism of action of esketamine, we conducted in vitro experiments using LPS. The in vitro results were comparable to those of the in vivo experiments, showing that esketamine administration significantly suppressed the expression of METTL5, c-Myc, and PD-L1 (Figure 4h-k). This result is consistent with the results from in vivo experiments.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; In subsequent in vitro experiments, we employed plasmid transfection technology to knock down the expression of the METTL5 gene. Firstly, a plasmid, METTL5-1 siRNA, which effectively knocked down the expression of METTL5 protein, was selected for use in following investigations through Western blot analysis (Figure 4f-g). We then assessed the transcriptional and protein levels of METTL5, c-Myc, and PD-L1. The results showed that after knocking down METTL5, the expression levels of c-Myc and PD-L1 were significantly reduced, which was consistent with the effects of esketamine administration (Figure 4h-k, 4m-o). This provides strong evidence that esketamine can inhibit the METTL5/c-Myc/PD-L1 signaling pathway after TBI.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.\u0026nbsp;\u003c/strong\u003e \u003cstrong\u003eInteraction of METTL\u003c/strong\u003e\u003cstrong\u003e5, c-Myc, and PD-L1 in TBI Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the TBI model, we utilized specific antibodies to label METTL5, c-Myc, and PD-L1 individually, while concurrently employing the GFAP antibody to identify activated astrocytes. We observed colocalization of METTL5, c-Myc, and PD-L1 within astrocytes, suggesting a potential interaction between METTL5 and c-Myc, PD-L1 (Figure 5a). To further validate the interaction between METTL5 and c-Myc, PD-L1, we conducted Co-IP analysis in vitro. Using a METTL5-specific antibody for the immunoprecipitation, the experimental results confirmed the existence of an actual interaction between METTL5 and c-Myc, as well as PD-L1 (Figure 5b).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe activated astrocytes are strongly linked to the development of the neuroinflammatory cascade. Hence, efficient management of the neuroinflammatory cascade triggered by activated astrocytes is crucial for improving the prognosis of TBI patients.\u003csup\u003e20\u003c/sup\u003e In the current work, we found that esketamine exerted a protective effect on neurological function after TBI by blocking the METTL5/c-Myc/PD-L1 signaling axis, promoting the polarization of activated astrocytes toward A2, and significantly reducing astrocyte-mediated neuroinflammation.\u003c/p\u003e\n\u003cp\u003eFor a long time, it has been believed that ketamine may increase in intracranial pressure in TBI patients, leading to severe complications and limiting its use in TBI treatment.\u003csup\u003e21\u003c/sup\u003e However, a recent systematic review of 11 studies found that ketamine use does not cause sustained increases in intracranial pressure. Only one study in this analysis reported an intermittent rise of ICP while cerebral perfusion pressure remaining constant. More importantly, in these studies, there was no evidence of to suggest that ketamine causes any harm.\u003csup\u003e22\u003c/sup\u003e Besides, the multiple advantages of ketamine, including its excellent anesthetic and analgesic effects, antidepressant and antiepileptic properties, prevention of glutamate-induced neuronal necrosis, anti-inflammatory effects, as well as positive impact on airway resistance and hemodynamics, have rekindled interest in its use in patients with TBI.\u003csup\u003e10\u003c/sup\u003e The latest research shows that in the treatment of subarachnoid hemorrhage patients, esketamine has been confirmed to effectively and long-term inhibit the key process that leads to the worsening of brain injury, known as spreading depolarizations.\u003csup\u003e23\u003c/sup\u003e In addition, esketamine has neuroprotective effects against TBI by modulating autophagy and oxidative stress via AMPK/mTOR-dependent TFEB nuclear translocation.\u003csup\u003e12\u003c/sup\u003e In both in vivo and in vitro experiments, we observed significant improvements in neurological function and an increase in the number of surviving neurons in TBI mice treated with esketamine. In addition, the levels of inflammatory factors IL-1\u0026beta; and TNF-\u0026alpha; in the mice\u0026apos;s cortical tissue were significantly decreased, while the levels of the anti-inflammatory factor IL-10 increased. Our in vitro findings further revealed that treatment with esketamine significantly increased the viability of astrocytes. These findings confirm esketamine\u0026rsquo;s capacity to improve neuronal cell survival and neurological outcome by inhibiting the neuroinflammation after TBI, which enhances its potential for treatment in TBI.\u003c/p\u003e\n\u003cp\u003eAs the most dynamic and reversible chemical modification on ribonucleic acids, the m6A modification is found not only in mRNA but also in a wide range of non-coding RNAs (ncRNA).\u003csup\u003e4\u003c/sup\u003e m6A methylation plays an important role in disease mechanisms and therapy. The dynamic modulation of m6A methylation is achieved through the coordinated action of methyltransferases, demethylases, and RNA-binding proteins.\u003csup\u003e24\u003c/sup\u003e This intricate interplay ensures accurate regulation of RNA processing, such as splicing, translation, and degradation, which ultimately affects the stability and biological function of RNA molecules.\u003csup\u003e25\u003c/sup\u003e The m6A modification is well-documented for its roles in processes such as tumor immune responses\u003csup\u003e26\u003c/sup\u003e and metabolic reprogramming of tumor cells.\u003csup\u003e27\u003c/sup\u003e Emerging evidence suggests that the modulation of m6A modification can provide neuroprotective effects in central nervous system injuries. The neuroprotective effects of m6A modification have been attributed to its effects on improving neurological injury, suppressing inflammation, inhibiting apoptosis, reducing pyroptosis, and attenuating ferroptosis.\u003csup\u003e4\u003c/sup\u003e In Wu et al.\u0026apos;s study, acute brain injury resulted in upregulation of METTL3 protein expression in microglia. The increased METTL3 enhances the stability of BATF mRNA through the m6A-IGF2BP2-dependent pathway, which promotes BATF-dependent pathogenic genes expression in microglia and triggers both local and peripheral immune responses.\u003csup\u003e28\u003c/sup\u003e In a recent study, the newly discovered methyltransferase METTL5, in conjunction with TRMT112, facilitates the m6A methylation of 18S rRNA.\u003csup\u003e6\u003c/sup\u003e This modification not only improves rRNA metabolic stability, but also has a significant impact on ribosomal activity and cellular development processes. The dysregulation of METTL5 expression is notably linked to intellectual disability, cancer, and congenital dysplasia.\u003csup\u003e5\u003c/sup\u003e However, the exact role of METTL5 in TBI has yet to be fully elucidated. In both in vivo and in vitro experiments, we demonstrated for the first time that esketamine could inhibit the expression of METTL5 and improve the neurological function after TBI, which further reveals the mechanism of esketamine\u0026apos;s protective effects against TBI.\u003c/p\u003e\n\u003cp\u003ec-Myc is a common transcription factor that regulates the expression of multiple gene products involved in cell proliferation, growth, differentiation, and apoptosis.\u003csup\u003e29\u003c/sup\u003e Previous reports indicate that c-Myc can directly target the PD-L1 promoter, thereby regulating PD-L1 levels.\u003csup\u003e30\u003c/sup\u003e In a TBI model, reducing PD-L1 levels or deleting PD-1 signaling can ameliorate TBI-induced learning and memory deficits.\u003csup\u003e31\u003c/sup\u003e The expression of PD-L1 in senescent cells facilitates immune evasion, leading to the accumulation of senescent cells and enhanced inflammatory signaling.\u003csup\u003e32\u003c/sup\u003e In this study, we found that TBI leads to an increase in METTL5 protein expression in astrocytes, which enhances the stability of c-Myc rRNA and promotes the expression of c-Myc. Through plasmid-mediated knockdown of METTL5 or treatment with esketamine, we observed a reduction in METTL5 expression levels, along with a decrease in the expression of c-Myc and PD-L1. In addition, we verified the interaction relationship between METTL5, c-Myc, and PD-L1 through methods such as Co-IP and immunofluorescence colocalization. These findings suggest that the protective effect of esketamine after TBI may be achieved by suppressing METTL5/c-Myc/PD-L1.\u003c/p\u003e\n\u003cp\u003eNeuroinflammation plays an important role in secondary brain injury after TBI. After TBI, acute and intense inflammatory cascade reactions occur, and the massive release of inflammatory mediators and cytokines can lead to secondary neuronal cell death and neurological dysfunction. Microglia have typically been the primary focus when exploring neuroinflammation after TBI.\u003csup\u003e33\u003c/sup\u003e However, with the increasing research on TBI in recent years, researchers have discovered that astrocytes also undergo significant structural changes in response to central nervous system injuries and diseases, a phenomenon known as astrocytes activation.\u003csup\u003e20\u003c/sup\u003e Although the role of activated astrocytes in TBI remains controversial, many clinical and experimental studies provide convincing evidence that activated astrocytes can exert a detrimental effect on central nervous system cells and tissues by producing inflammatory mediators and chemokines to exacerbate inflammation, hampering synapse sprouting or axon growth, the release of excitatory glutamate, and the disruption of the blood-brain barrier.\u003csup\u003e34-36\u003c/sup\u003e It has been now found that astrocytes can be activated into two polarization states: the neurotoxic or pro-inflammatory phenotype (A1) and the neuroprotective or anti-inflammatory phenotype (A2). A1 astrocytes lose their normal astrocyte functions of maintaining synapses due to the overexpression of complement cascade genes, and can exacerbate the progression of the disease by secretion of soluble neurotoxins to kill a portion of neurons and mature oligodendrocytes. In contrast, A2 astrocytes demonstrate a potential neuroprotective role by upregulating neurotrophic and anti-inflammatory genes, promoting neuronal survival and growth, and improving the reparative process.\u003csup\u003e19\u003c/sup\u003e In this study, we found that the expression of GFAP was significantly increased in the brain tissue surrounding the injury in mice after TBI, whereas esketamine treatment inhibited TBI-induced astrocyte activation. In vitro cellular experiments also confirmed that knockdown of METTL5 or treatment with esketamine produced similar results with reduced GFAP expression. Additionally, by using immunofluorescence labeling to detect subtype changes in activated astrocytes, we found that knockdown of METTL5 or treatment with esketamine suppressed the expression of A1 pro-inflammatory astrocytes and promoted the expression of A2 anti-inflammatory astrocytes. Taken together, we conclude that esketamine may exert a protective effect through the inhibition of activated astrocytes and the modulation of their polarization state.\u003c/p\u003e\n\u003cp\u003eOur study has several limitations. First, in our experiments, we only used a single astrocyte without establishing a co-culture system, which prevented us from observing the interactions between astrocytes and other neuroimmune cells such as microglia. Second, although in the study of mouse TBI model, we identified that esketamine modulates astrocyte-mediated inflammation by inhibiting the METTL5/c-Myc/PD-L1 pathway. However, the translation of these findings to clinical practice, as well as the potential long-term side effects of esketamine use, including its impact on the nervous system, are important aspects that need to be thoroughly investigated in future study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eEsketamine can effectively alleviate activated astrocytes and promote the polarization of activated astrocytes toward A2 following TBI by inhibiting the METTL5/c-Myc/PD-L1 axis, demonstrating significant anti-inflammatory and neuroprotective effects. This finding provides new potential drug targets for TBI treatment and lays a theoretical foundation for the application of esketamine in neurological injuries.\u0026nbsp;\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMaas A I R, Menon D K, Adelson P D, et al. Traumatic brain injury: integrated approaches to improve prevention, clinical care, and research. 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Exosomal MicroRNAs Released by Activated Astrocytes as Potential Neuroinflammatory Biomarkers. Int J Mol Sci. 2020;21. doi: 10.3390/ijms21072312\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Esketamine, traumatic brain injury, neuroinflammation, activated astrocyte, A1/A2 astrocytes, METTL5/c-Myc/PD-L1","lastPublishedDoi":"10.21203/rs.3.rs-7330976/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7330976/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\n\u003cp\u003eTraumatic brain injury (TBI) is a major global health issue leading to high mortality and disability.\u003c/p\u003e\n\u003cp\u003eActivated astrocytes are one of the pivotal driving factors in the neuroinflammatory cascade following TBI. Our study investigates the role of esketamine in modulating astrocyte-induced neuroinflammation, a key factor in enhancing TBI patient outcomes.\u003c/p\u003e\n\u003cp\u003eMethod\u003c/p\u003e\n\u003cp\u003eMice received a mouse weight-drop cortical impact or sham surgery and TBI mice were treated with either vehicle or esketamine at 2 h post-injury for 7 consecutive days. The modified Neurological Severity Scoring system, Rotarod test, Open Field test and Novel Object Recognition test were used to assess the neurological function after TBI. And cortical tissues surrounding focal trauma were obtained for Nissl staining, immunofluorescence, ELISA assay and western blotting. In vitro, astrocytes were induced with LPS, followed by the addition of esketamine to the culture medium. After a 24-hour exposure, the astrocytes were collected for CCK-8 assay, qRT-PCR, western blotting, immunofluorescence and Co-IP analysis.\u003c/p\u003e\n\u003cp\u003eResults\u003c/p\u003e\n\u003cp\u003eEsketamine dramatically improved the neurological outcome of mice and reduced neuronal cell death (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05) and neuroinflammation after TBI. Its anti-inflammatory benefits stem from its ability to suppress astrocyte activation (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), inhibit pro-inflammatory A1 astrocyte differentiation (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01), and promote the formation of protective A2 astrocytes (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Esketamine exerts its effects by inhibiting the METTL5/c-Myc/PD-L1 signaling pathway.\u003c/p\u003e\n\u003cp\u003eConclusions\u003c/p\u003e\n\u003cp\u003eEsketamine can effectively alleviate activated astrocytes and promote the polarization of activated astrocytes toward A2 following TBI by inhibiting the METTL5/c-Myc/PD-L1, demonstrating significant anti-inflammatory and neuroprotective effects.\u003c/p\u003e","manuscriptTitle":"Esketamine suppresses astrocyte-driven neuroinflammation in traumatic brain injury via the METTL5/c-Myc/PD-L1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 06:28:46","doi":"10.21203/rs.3.rs-7330976/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-03T01:09:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T14:58:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"209275672624270296099710803443093267550","date":"2025-10-05T10:19:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5939749277082022481930554698738838285","date":"2025-08-20T14:47:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-18T04:18:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"131694248910732760443474266050431112361","date":"2025-08-18T04:03:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-18T03:11:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-14T20:06:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-11T10:48:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neurochemical Research","date":"2025-08-09T03:09:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a58acf71-0f57-4357-b2eb-73364bfcf9d3","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:08:06+00:00","versionOfRecord":{"articleIdentity":"rs-7330976","link":"https://doi.org/10.1007/s11064-026-04724-8","journal":{"identity":"neurochemical-research","isVorOnly":false,"title":"Neurochemical Research"},"publishedOn":"2026-03-13 15:59:53","publishedOnDateReadable":"March 13th, 2026"},"versionCreatedAt":"2025-08-27 06:28:46","video":"","vorDoi":"10.1007/s11064-026-04724-8","vorDoiUrl":"https://doi.org/10.1007/s11064-026-04724-8","workflowStages":[]},"version":"v1","identity":"rs-7330976","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7330976","identity":"rs-7330976","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}