Pantothenic acid-mediated inhibition of microglial inflammation via the JAK2/STAT3 pathway enhances motor function recovery after Spinal cord injury

Pantothenic acid-mediated inhibition of microglial inflammation via the JAK2/STAT3 pathway enhances motor function recovery after Spinal cord injury | 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 Pantothenic acid-mediated inhibition of microglial inflammation via the JAK2/STAT3 pathway enhances motor function recovery after Spinal cord injury Yuepeng Fang, Ce Zhang, Zhijie Yang, Xiangrui Zhao, yongcheng Yin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5648324/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study employed transcriptome sequencing and targeted metabolomics to delve into the molecular alterations in mouse spinal cords following spinal cord injury (SCI). Notably, a significant depletion of pantothenic acid (PA) was observed in the injured spinal cord, exhibiting an inverse correlation with microglial inflammation and activation. To further elucidate this relationship, experimental interventions using PA were conducted in SCI mouse models. The results demonstrated that PA administration effectively inhibited microglial inflammation via modulation of the JAK2/STAT3 signaling pathway. This inhibition not only mitigated the neuroinflammatory milieu but also fostered an environment conducive to axonal growth and neuronal regeneration. Consequently, SCI mice treated with PA exhibited improved motor function recovery compared to untreated controls. Our findings not only deepen the understanding of the relationship between PA and neuroinflammatory processes in SCI but also highlight the therapeutic potential of PA in promoting neuronal regeneration and functional recovery. spinal cord injury pantothenic acid microglial inflammation JAK2/STAT3 signaling pathway neuronal regeneration motor function recovery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights Integrated metabolomics and transcriptomics analysis post-SCI PA ameliorates inflammatory response and promotes axonal growth following SCI PA inhibits microglial inflammation via JAK2/STAT3 signaling pathway Introduction Despite advancements in medical care, SCI leading to permanent disability, remains without an effective treatment[ 1 ]. The pathophysiological process of SCI involves a complex cascade of events, with neuroinflammation playing a pivotal role in neuronal dysfunction and SCI progression[ 2 – 4 ]. Microglial activation, the cornerstone of neuroinflammation, triggers the release of inflammatory cytokines and chemokines, creating a hostile environment for neuronal survival and regeneration. This exacerbates the injury site and perpetuates disability[ 5 , 6 ]. Within this context, alterations in local metabolite levels arise and function as crucial regulators within the microenvironment[ 7 – 10 ]. They influence cellular activity and neuronal regeneration post-injury and play a crucial role in modulating inflammatory responses[ 7 , 9 , 11 , 12 ]. However, this area remains underappreciated and not fully understood, highlighting the need for further research to elucidate the mechanisms underlying the interactions between local metabolites and neuroinflammatory processes in SCI[ 13 ]. In this study, we utilized transcriptome sequencing and targeted metabolomics to explore the molecular changes in mouse spinal cords following SCI. Notably, we discovered a significant depletion of PA in the injured spinal cord, which exhibited an inverse correlation with microglial inflammation and activation[ 14 ]. This finding reveals a previously unknown connection between PA and neuroinflammatory processes in SCI. PA may indirectly aid in reducing inflammation by maintaining a healthy cellular environment, thereby potentially mitigating the inflammatory response associated with SCI[ 15 ]. However, direct evidence linking PA to these benefits in the context of SCI remains limited and warrants further investigation. To further elucidate this relationship, we conducted experimental interventions using PA in SCI mouse models. Our results demonstrated that PA administration effectively inhibited microglial inflammation via modulation of the JAK2/STAT3 signaling pathway. This inhibition not only mitigated the neuroinflammatory milieu but also created a permissive environment conducive to axonal growth and neuronal regeneration[ 13 , 16 , 17 ]. Consequently, PA-treated SCI mice exhibited improved motor function recovery compared to untreated controls. Our findings not only deepen our understanding of the relationship between spinal cord local metabolites and neuroinflammatory processes in SCI but also highlight the therapeutic potential of PA in promoting neuronal regeneration and functional recovery[ 18 ]. By inhibiting microglial inflammation and fostering an environment conducive to axonal growth, PA offers a promising new approach to treating SCI and mitigating its devastating consequences[ 19 , 20 ]. Materials and Methods Animals Adult male C57BL/6 mice, aged 7–8 weeks, obtained from Vital River Laboratories in Beijing, China, were selected for the establishment of SCI models and the execution of sham surgeries. The animal procedures utilized in this study were approved by the Animal Ethics Committee of Shandong University, Jinan, China (approval number: KYLL-JNCHIACUC-202114), ensuring strict adherence to the ethical standards and principles of animal welfare. The procedure for establishing a SCI model Mice were anesthetized with 2% pentobarbital and underwent a laminectomy at the T8-T10 vertebral levels to expose the spinal cord. Subsequently, a spinal cord impactor (model 68100, manufactured by RWD, Shenzhen, China) was utilized to induce SCI, adhering to the manufacturer's instructions. Specifically, the impactor was set to deliver a controlled compression force at a depth of 2 mm and a velocity of 1 m/s, targeting the exposed spinal cord segment. Successful induction of SCI was confirmed by a clear and discernible impact site on the spinal cord, accompanied by immediate, involuntary limb twitching and tail flicking, indicative of spinal cord trauma. Following the injury, the surgical site was meticulously sutured in anatomical layers to ensure proper healing and prevent infection. The mice were then returned to their individual cages to recover from anesthesia. To aid in urinary function recovery post-SCI, manual bladder expression was performed two times daily for each mouse until spontaneous voiding was reestablished. In contrast, mice in the sham group underwent a similar surgical procedure, including laminectomy, but without the application of the spinal cord impactor. All other postoperative care and monitoring procedures were identical to those performed in the SCI group to ensure comparability between the two experimental cohorts. Western-blotting, immunofluorescence, ELISA and quantitative realtime PCR analysis For Western blotting, BV2 cell lysates or tissue samples (spinal cord segments) were prepared by homogenizing in RIPA buffer containing protease inhibitors (Sigma-aldrich Cat NO. R0278). The lysates were then separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk and incubated overnight at 4°C with specific primary antibodies targeting the proteins of interest. After washing, the membrane was incubated with appropriate secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (Yeasen, Cat NO.36208ES60) and analyzed using ImageJ software. For immunofluorescence staining, BV2 cells grown on glass coverslips or tissue sections were fixed with 4% paraformaldehyde (Sigma-aldrich Cat NO. P6148) and permeabilized with 0.1% Triton X-100 (Sigma-aldrich Cat NO. X100). The samples were blocked with 10% normal goat serum and incubated overnight at 4°C with primary antibodies. After washing, the samples were incubated with secondary antibodies labeled with fluorescent dyes for 1 hour at room temperature. Nuclei were stained with DAPI (Beyotime Cat NO. C1002), and the stained samples were mounted with anti-fade mounting medium and visualized under a fluorescence microscope. ELISA (Abclonal Cat NO. RM17860 and Abclonal Cat NO. RK 000008) was performed to quantify the levels of specific analytes in cell culture supernatants or serum samples. Cell supernatants were collected from BV2 cell cultures, while serum was obtained from mouse blood samples by centrifugation. The samples were diluted appropriately and incubated in microplate wells coated with specific capture antibodies. After washing, bound analytes were detected with biotinylated detection antibodies followed by streptavidin-HRP conjugate. The color reaction was developed with TMB substrate and stopped with sulfuric acid. The optical density was measured spectrophotometrically at 450 nm, and analyte concentrations were calculated using a standard curve. For PCR analysis, RNA was extracted from BV2 cells or tissue samples using TRIzol reagent (Thermo Fisher Cat NO.15596018CN). The RNA was reverse-transcribed into cDNA using a reverse transcriptase kit. The cDNA was amplified in a qPCR reaction mixture containing specific primers and SYBR Green dye on a thermocycler. The amplification was monitored in real-time, and the cycle threshold (Ct) values were determined. Gene expression levels were calculated using the 2 ^−ΔΔCt method, with expression normalized to a GAPDH. T2-weighted magnetic resonance imaging The mice underwent MRI evaluations at 14 and 35 days post-injection using a high-performance 9.4-Tesla small animal MRI scanner manufactured by Bruker (Bruker 9.4T Biospec, Bruker BioSpin, Germany). This scanner was equipped with a four-channel surface coil designed for maximum signal reception efficiency. Prior to the imaging procedure, the mice were anesthetized through inhalation of 1.5% isoflurane (sourced from RWD, Shenzhen, China) using a specialized anesthesia apparatus compatible with MRI systems. The animals were then carefully positioned within a tailored fixation device. The imaging protocol utilized T2-weighted sequences, featuring a matrix dimension of 320 × 320 pixels, a slice thickness of 0.3 mm, an echo time (TE) of 24 milliseconds, a repetition time (TR) of 1200 milliseconds, and a flip angle of 90 degrees. These parameters enabled the acquisition of high-resolution T2-weighted images in the sagittal plane, executed through the Bruker ParaVision 6.0 software (Bruker, Ettlingen, Germany). After the imaging session, the mice were moved to a heated pad for recovery and subsequent post-procedural care. CATWALK gait analysis, BMS scoring, and the inclined plane test In our study, gait analysis was conducted using the CATWALK system to evaluate detailed gait characteristics of the animals. Motor function recovery was assessed using the BMS locomotor rating scale. Additionally, the inclined plane test was employed to further evaluate hindlimb weight-bearing ability and motor function recovery post-SCI. For the CATWALK analysis (Noldus XT, Netherlands), paw prints were captured and analyzed to derive gait parameters. The BMS scale is a well-established method for assessing hindlimb locomotor function in SCI Mice. Animals were observed and scored based on their ability to move, with higher scores indicating better motor function. The scoring criteria included various aspects of hindlimb movement, such as joint movement, stepping ability, coordination, and trunk stability. This method provided an objective and quantitative measure of motor recovery. In the inclined plane test, mice were placed on an adjustable inclined board and the maximum stable angle they could maintain without slipping or falling was recorded. These methods, employed in a precise and systematic manner, allowed for an objective and quantitative evaluation of gait, motor function, and hindlimb strength in the animal models. Integrated transcriptome sequencing and targeted metabolomics analysis To perform an in-depth comparison of mouse spinal cord tissue in sham-operated and SCI groups, we utilized a combination of transcriptome sequencing and targeted quantitative metabolomics. For transcriptome sequencing, RNA was extracted from spinal cord tissues of both groups and subjected to high-throughput sequencing to generate comprehensive gene expression profiles. These profiles were then analyzed using bioinformatic tools to identify genes with differential expression patterns between sham and SCI tissues. In parallel, for targeted metabolomics, spinal cord tissues were extracted and analyzed using advanced chromatography-mass spectrometry (LC-MS/GC-MS) techniques to quantify a wide range of small molecule metabolites. The resulting metabolomic data were processed and normalized to identify metabolic alterations associated with SCI. Finally, both transcriptomic and metabolomic datasets were integrated through bioinformatic and statistical approaches to uncover correlations between gene expression changes and metabolic perturbations, providing a holistic view of the biological responses to SCI. Intraperitoneal injection method of PA in mouse models of SCI In mouse models of SCI, the intraperitoneal injection method for administering PA involves first preparing a sterile solution of PA in Phosphate-Buffered Saline (PBS) at a concentration of 20 mg/mL, ensuring it is free of contaminants through sterile filtration. The mouse's abdominal area is then gently shaved and disinfected with an antiseptic solution to prevent infection. Using a sterile syringe and needle, the prepared PA solution is drawn up and injected slowly into the intraperitoneal cavity of the mouse at an appropriate angle and depth, avoiding vital organs and blood vessels, while the mouse is handled gently to minimize stress. The injection dose is calculated based on 20 mg/kg of the mouse's body weight. Following the injection, the needle is withdrawn carefully, and the injection site is gently pressed to prevent leakage or bleeding. The mouse is then placed in a recovery cage and closely monitored until it fully recovers from any potential anesthesia or discomfort associated with the procedure. Statistical analysis In the current study, statistical analyses were meticulously conducted using R version 4.2.1 and GraphPad Prism 9, adhering strictly to established principles. Prior to test selection, we evaluated data normality and variance homogeneity to ascertain the appropriateness of parametric or non-parametric methods. Parametric data were analyzed using ANOVA and t-tests, while non-parametric data were handled with appropriate non-parametric techniques. Gene expression data underwent normalization to mitigate technical biases and facilitate precise differential expression analysis, followed by statistical testing to identify significant changes. Immunohistochemical data were quantitatively analyzed using ImageJ, with statistical evaluations focusing on staining intensity and distribution. Behavioral assessments employed repeated measures ANOVA to discern meaningful changes over time, controlling for inter-individual variability. Statistical significance was set at p < 0.05, ensuring a rigorous and conservative approach to data interpretation. Results Integrated transcriptomic and metabolomic analysis reveals a correlation between decreased PA and microglial activation and inflammation following SCI To investigate the metabolic alterations associated with SCI-induced neuroinflammation and identify potential key metabolites, we conducted an integrated analysis of transcriptomic and metabolomic sequencing in mice (n = 12 per group) from both the sham and SCI groups (14 days post-SCI) within the same cohort ( Fig. 1 A, B ) . The 14-day post-SCI timepoint was chosen to capture a stabilized glial scar and a localized inflammatory microenvironment, characterized by persistent microglial inflammation[ 2 , 21 ]. By analyzing metabolic signatures at this critical stage, we aimed to elucidate the mechanisms underlying microglial activation and inflammation, potentially uncovering novel therapeutic targets for SCI recovery. Our results revealed a significant downregulation of PA in tissues[ 22 ] post-SCI (Fig. 1 C). Using criteria including variable importance in projection (VIP), p-value, and fold change, we selectively analyzed four metabolites, with PA being a focal point, for their correlation with upregulated inflammatory genes and microglial activation genes following SCI (Fig. 1 D, E). Notably, PA emerged as the most significant metabolite, demonstrating a strong and robust correlation with multiple microglial inflammatory activation genes, including CD68, CD86, CCL2, CCL3, and IL1α ( Fig. 1 E ) . This underscores the prominent role of PA among the metabolites analyzed[ 3 , 10 ]. Furthermore, transcriptome sequencing analysis showed a substantial upregulation of inflammatory cytokines, chemokines, and neurotoxic factors such as IL1β, LCN2, CCL3, CCL5, and TNF following SCI (Fig. 1 F). GO functional annotation and KEGG pathway analysis revealed significant enrichment of inflammatory responses, intercellular interactions, and the activation of JAK-STAT and NF-κB signaling pathways at the 14-day post-SCI (Fig. 1 G, H). These findings highlight the prevalent inflammatory landscape and the activation of key inflammatory pathways during the acute phase of SCI, aligning with current research in SCI pathophysiology[ 23 – 25 ]. Suppression of microglial inflammation and activation by PA: enhancing neuronal survival and ameliorating histopathological changes after SCI To validate the direct role of PA as a metabolite in regulating microglial inflammation and activation at the molecular level following SCI, we conducted a study using intraperitoneal injection of PA as a supplement (Fig. 2 A). Our aim was to explore its impact on the inflammatory response and subsequent histopathological changes in the spinal cord tissue post-SCI. We comprehensively assessed the histopathological changes through Hematoxylin and Eosin (HE) staining, Luxol Fast Blue (LFB) staining, and Nissl staining in comparison between the PA-treated group and its control group. Our findings revealed that a significant reduction in inflammatory cell infiltration, preservation of myelin integrity, and enhanced neuronal viability in the PA-treated group (Fig. 2 B-D, G-I). These observations underscore the therapeutic potential of PA in mitigating the histopathological consequences of SCI. Subsequently, using immunofluorescence staining with markers such as IBA-1 and INOS, we observed a significant downregulation of microglial activation and inflammatory responses[ 26 , 27 ]. This was further corroborated by PCR analysis of tissue lysates and ELISA of serum samples, which both demonstrated decreased levels of IL-1β and IL-6 following PA intervention. The mRNA and protein levels in the tissue similarly revealed the inhibitory effect of PA on microglial inflammatory marker NOS2 following SCI (Fig. 2 F and Figure S1 A, B ). MRI results provided specific insights into the reduction of inflammation. Specifically, T2-weighted images revealed a decrease in hyperintensity within the lesion site, suggesting a reduction in edema and inflammation, indicating better tissue integrity and reduced inflammatory-induced disruption. These MRI findings, in conjunction with the biochemical data, suggest that PA treatment effectively dampens the inflammatory responses associated with SCI, promoting a more favorable healing environment. Transcriptome sequencing reveals PA-mediated inhibition of microglial inflammation via the JAK-STAT Signaling Pathway Microglia are primary contributors to inflammation following SCI, directly participating as immune cells in the release of neurotoxic factors such as inflammatory cytokines and chemokines[ 21 , 28 , 29 ]. To elucidate the specific effects of PA on microglia and the underlying mechanisms, we conducted an in vitro study on the BV2 cell line, dividing it into three groups: a control group, a TNFα-treated group, and a co-intervention group exposed to both TNFα and PA (Fig. 3 A). RNA sequencing was then performed to investigate these effects. PCA analysis revealed a clear segregation of the three cellular groups based on their gene expression profiles, indicating distinct molecular signatures associated with each group that are likely reflective of differential responses to the experimental conditions (Fig. 3 B). The volcano plot illustrates that the majority of differential genes are upregulated in response to the inflammatory stimulus induced by TNFα alone. Through functional enrichment analysis using GO and KEGG, we found that TNFα stimulation also triggers the activation of microglial immune responses, the release of chemokines, and the activation of inflammation-related pathways ( Figure S2 A-C ). Notably, the primary differential genes between the TNFα and TNFα + PA groups are downregulated genes (Fig. 3 C, D). We investigated these downregulated differential genes and found that PA significantly inhibited the upregulation of microglial inflammatory activation markers (NOS2, CD68, CD86, Aif1), inflammatory cytokines (IL1β, IL6), and chemokines (CCL2, CCL3). Functional enrichment analysis using GO and KEGG revealed that PA primarily modulates microglial immune responses and inflammatory reactions. Notably, the JAK-STAT signaling pathway was significantly downregulated upon PA intervention (Fig. 3 D-F). Consistent results were also observed in Gene Set Enrichment Analysis (Fig. 3 G, H). Similarly, these transcriptome sequencing results are consistent with the functional outcomes observed in mice following SCI. The JAK-STAT signaling pathway plays a pivotal role in microglial activation and inflammation, regulating the production of inflammatory cytokines and chemokines. In the context of SCI, activation of this pathway contributes to neuroinflammation and secondary damage. Based on the aforementioned results, we hypothesize that PA may inhibit microglial inflammation by suppressing the JAK-STAT signaling pathway, with potential implications for neuroprotection in the context of SCI and other neurological disorders[ 16 , 30 ]. PA inhibits microglial activation and inflammation by suppressing the JAK2/STAT3 signaling pathway Next, we validated the transcriptome sequencing results by examining the inflammatory state of BV2 cells. To establish the optimal intervention concentration of PA, we performed a CCK8 assay across a range of concentrations. The results demonstrated that PA concentrations up to 10µmol/ml exerted no significant effect on the proliferation of BV2 cells. Consequently, we chose 10µmol/ml as the working concentration of PA to investigate its modulatory effects on inflammatory responses (Fig. 4 A). Our study further revealed that PA intervention significantly downregulated the expression of microglial inflammatory markers, IBA-1 and INOS, under TNFα stimulation[ 28 , 31 , 32 ]. Notably, the percentage of BV2 cells co-expressing IBA-1 and INOS decreased substantially (Fig. 4 B, C). Similarly, through PCR, Western blot (WB) analysis, and ELISA detection of cell culture supernatants, we confirmed that PA indeed reduced the expression of inflammatory markers (NOS2 and COX2) and release of cytokines, and chemokines in TNFα-stimulated BV2 cells (Fig. 4 D-G ). Upon analyzing the transcriptome sequencing results, we discovered that PA suppressed the JAK/STAT pathway in microglia stimulated by TNFα. Considering the pivotal role of activated JAK2/STAT3 signaling in enhancing inflammatory responses in microglia, we subsequently conducted further validation[ 33 – 35 ]. We subsequently conducted WB analysis to investigate the effects of PA on JAK2 and STAT3 phosphorylation. The findings were consistent with our expectations, demonstrating that PA inhibited both JAK2 and STAT3 phosphorylation[ 36 , 37 ]. Furthermore, the use of the JAK2/STAT3 signaling pathway inhibitor WP1066 revealed an additive effect with PA in influencing the JAK2/STAT3 pathway ( Fig. 5 A-D ) . These results contribute to our understanding of PA's potential therapeutic mechanisms in neuroinflammatory conditions, particularly in the context of SCI[ 17 ]. Given that phosphorylated STAT3 (p-STAT3) translocates to the nucleus to regulate transcription of inflammation-related factors, we next conducted immunofluorescence experiments to investigate the effect of PA on p-STAT3 nuclear translocation[ 38 , 39 ]. Our findings revealed that, under TNFα stimulation, there was a significant increase in p-STAT3 nuclear translocation. However, when PA was co-administered, p-STAT3 nuclear translocation was markedly reduced. Notably, the use of the JAK/STAT signaling pathway inhibitor WP1066[ 35 ] exhibited an additive effect with PA in reducing p-STAT3 nuclear translocation (Fig. 5 E, F). Concurrently, through the assessment of the inflammatory state of BV2 cells, we found that the anti-inflammatory effects of PA on BV2 cells were indeed mediated through the inhibition of the JAK2/STAT3 signaling pathway (Fig. 5 G, H and Figure S3 A ). Subsequently, we discovered that PA also inhibits the JAK2/STAT3 signaling pathway in injured spinal cord tissues, suggesting that PA may exert neuroprotective and anti-inflammatory effects through this mechanism (Fig. 5 I, J). PA enhances neural growth and promotes motor functional recovery in mice with SCI. Given the proven inhibitory effect of PA on inflammation subsequent to SCI, we administered PA via intraperitoneal injection to investigate whether its anti-inflammatory properties confer beneficial effects on neuronal axon growth and functional restoration in the hindlimbs of mice[ 40 – 42 ]. Our objective was to elucidate the therapeutic potential of PA in facilitating neuroregeneration and enhancing functional recovery (Fig. 6 A). Through immunofluorescence staining for NEUN and NFH, we observed notable enhancements in the retention of mature neurons and axon growth in mice with SCI after PA treatment[ 43 , 44 ]. Specifically, the axons of neurons in the PA-administered group were capable of growing into the injury core region, accompanied by a significantly higher number of viable mature neurons surrounding the injury site compared to the SCI control group (Fig. 6 B-D). To assess the recovery of hindlimb locomotor function in mice, the catwalk test was employed. Results indicated that the majority of mice in the SCI group were unable to achieve paw or dorsum plantar contact with the ground. In contrast, mice subjected to PA intervention demonstrated a significant capacity for plantar standing. This was manifested by a marked increase in the maximum intensity of hindlimb movement in the PA-treated group compared to the SCI group (Fig. 6 E, F). Subsequently, the mice were euthanized, and their spinal cord tissues were harvested. Total proteins were extracted and subjected to WB analysis. The results demonstrated a significant upregulation of TUJ1 and MAP2 expression in the PA intervention group compared to the SCI group. This finding suggests that PA intervention promote neuronal regeneration within the spinal cord (Fig. 6 G, H). To conduct a comprehensive assessment of the recovery of motor function in mice, a battery of tests including the Basso Mouse Scale (BMS) score, the inclined plane test, and the catwalk analysis were employed to evaluate the motor restoration and coordination of the hindlimbs. The BMS scores indicated significant functional recovery improvement in the PA intervention group compared to the SCI group at 28 days post-injury. Specifically, at 35 days post-injury, only 2 out of 6 mice in the SCI group could occasionally or consistently achieve dorsal paw placement without plantar support, whereas 3 out of 6 mice in the PA intervention group demonstrated occasional plantar paw placement, and the remaining 3 out of 6 mice could occasionally or consistently achieve dorsal paw standing (Fig. 7 A). Similarly, at 35 days post-SCI, mice in the PA intervention group exhibited a significantly increased tolerance to inclined plane angles compared to those in the SCI control group. Specifically, all 6 mice (6/6) in the PA intervention group could withstand angles of 35 degrees or higher, whereas none of the mice (0/6) in the SCI group could achieve this level of functional recovery at 35 days post-SCI (Fig. 7 B). The footprint analysis further demonstrated superior recovery in mice from the PA group compared to those in the SCI group, with significantly higher coordination levels observed in the PA group (Fig. 7 C-E). The swing speed, as well as the length and intensity of hindlimb footprints, were also significantly improved in the PA group compared to mice in the SCI group (Fig. 7 F-H). Discussion Our study presents novel insights into the therapeutic potential of PA in SCI. By leveraging transcriptome sequencing and targeted metabolomics, we have illuminated the molecular changes occurring in mouse spinal cords following SCI, particularly highlighting the depletion of PA and its inverse correlation with microglial inflammation and activation. The integration of transcriptomic and metabolomic data revealed a significant downregulation of PA in spinal cord tissues post-SCI. This downregulation was robustly correlated with the upregulation of multiple microglial inflammatory activation genes. These findings underscore the pivotal role of PA in the neuroinflammatory processes following SCI, a connection previously unknown[ 45 ]. Furthermore, transcriptome sequencing analysis demonstrated a substantial upregulation of inflammatory cytokines, chemokines, and neurotoxic factors, aligning with the activation of key inflammatory pathways such as JAK-STAT. This inflammatory landscape is characteristic of the acute phase of SCI and highlights the need for therapeutic interventions capable of dampening these responses[ 46 ]. Our experimental interventions using PA in SCI mouse models provided compelling evidence for its neuroprotective and anti-inflammatory effects[ 47 – 49 ]. PA administration effectively inhibited microglial inflammation via modulation of the JAK2/STAT3 signaling pathway. This inhibition not only mitigated the neuroinflammatory milieu but also created a permissive environment conducive to axonal growth and neuronal regeneration[ 50 , 51 ]. Consequently, PA-treated SCI mice exhibited improved motor function recovery compared to untreated controls. These results were further corroborated by MRI findings, which revealed a reduction in edema and inflammation within the lesion site, suggesting better tissue integrity and reduced inflammatory-induced disruption[ 52 , 53 ]. Our in vitro studies on the BV2 cell line further elucidated the specific effects of PA on microglia and the underlying mechanisms. PA significantly inhibited the upregulation of microglial inflammatory activation markers, inflammatory cytokines, and chemokines[ 54 ]. Functional enrichment analysis revealed that PA primarily modulates microglial immune responses and inflammatory reactions, with a particular focus on the JAK-STAT signaling pathway[ 55 ].The observed inhibitory effect of PA on JAK2/STAT3 phosphorylation further solidified its potential therapeutic mechanisms in neuroinflammatory conditions. The use of the JAK/STAT signaling pathway inhibitor WP1066 revealed an additive effect with PA in influencing the JAK/STAT pathway, suggesting a synergistic interaction between the two[ 56 , 57 ]. Immunofluorescence experiments demonstrated that PA reduced p-STAT3 nuclear translocation, indicating its ability to regulate the transcription of inflammation-related factors[ 58 , 59 ]. In conclusion, our findings deepen our understanding of the relationship between PA and neuroinflammatory processes in SCI and highlight its therapeutic potential. Future studies are needed to further elucidate the mechanisms underlying PA's neuroprotective effects and to explore its potential as a therapeutic agent in clinical settings. The identification of PA as a key metabolite involved in SCI-induced neuroinflammation opens new avenues for research and provides a foundation for the development of novel therapeutic interventions. Conclusions Our study unveils a novel therapeutic potential of PA in SCI by elucidating its anti-inflammatory and neuroprotective mechanisms. Through integrated transcriptome sequencing and metabolomic analysis, we identified a significant depletion of PA in injured spinal cord tissues, which inversely correlated with microglial inflammation and activation. Experimental interventions using PA in SCI mouse models demonstrated its efficacy in inhibiting microglial inflammation via modulation of the JAK2/STAT3 signaling pathway, thereby fostering an environment conducive to axonal growth and neuronal regeneration. Consequently, PA-treated SCI mice exhibited improved motor function recovery. These findings deepen our understanding of the relationship between PA and neuroinflammatory processes in SCI and pave the way for future research exploring PA as a therapeutic agent in clinical settings. Abbreviations SCI: Spinal Cord Injury PA: Pantothenic Acid JAK2: Janus Kinase 2 STAT3: Signal Transducer and Activator of Transcription 3 PBS: Phosphate-Buffered Saline RNA: Ribonucleic Acid IL-1β: Interleukin-1β IL-6: Interleukin-6 iNOS: Inducible Nitric Oxide Synthase IBA-1: Ionized Calcium Binding Adaptor Molecule 1 LFB: Luxol Fast Blue NISSL: Nissl staining HE: Hematoxylin and Eosin DAPI: 4',6-Diamidino-2-Phenylindole NEUN: Neuronal Nuclei NFH: Neurofilament Heavy WB: Western Blotting ELISA: Enzyme-Linked Immunosorbent Assay BMS: Basso, Beattie, and Bresnahan locomotor rating scale GO: Gene Ontology KEGG: Kyoto Encyclopedia of Genes and Genomes MRI: Magnetic Resonance Imaging Declarations Author contributions Fang Y. conducted experiments, analyzed data, and interpreted results concerning PA and neuroinflammatory processes in SCI mouse models. Zhang C. designed and executed experiments, with a focus on transcriptome sequencing and metabolomics analyses post-SCI. Yang Z., Zhao X., Yin Y., Jin Z., and Zhu P. supported various research aspects such as data collection, experimental setup, and coordination. Ning B. served as the lead author, overseeing the study design, implementation, and interpretation, and drafted the manuscript. All authors reviewed and approved the final manuscript. Declaration of interests The authors declare that they have no conflict of interest. ACKNOWLEDGEMENTS This work was comprehensively supported by multiple funding sources, including grants from the National Natural Science Fund of China (grant numbers 82071383 and 82371392), the Natural Science Foundation of Shandong Province (Key Project) with grant number ZR2020KH007, and the "Taishan Scholar Distinguished Expert Program" of Shandong Province, under grant number tstp20231257, all of which contributed towards the research endeavors of Bin Ning. The cell model in the picture comes from the Servier Medical Art and BioRender APP. In this study, AI-assisted tools were employed under human supervision to enhance the English writing process, ensuring academic rigor while expediting the drafting of our manuscript. However, the final content was meticulously crafted and validated by human authors, ensuring the originality and scientific accuracy of the presented findings. Availability of data and materials Data supporting the present study are available from the corresponding author upon reasonable request. References Jug M, Komadina R, Wendt K, Pape HC, Bloemers F, Nau C. Thoracolumbar spinal cord injury: management, techniques, timing. Eur J Trauma Emerg Surg. 2024;50:1969–75. Hu X, Xu W, Ren Y, Wang Z, He X, Huang R, Ma B, Zhao J, Zhu R, Cheng L. Spinal cord injury: molecular mechanisms and therapeutic interventions. Signal Transduct Target Ther. 2023;8:245. Fang YP, Qin ZH, Zhang Y, Ning B. Implications of microglial heterogeneity in spinal cord injury progression and therapy. Exp Neurol. 2023;359:114239. Li C, Wu Z, Zhou L, Shao J, Hu X, Xu W, Ren Y, Zhu X, Ge W, Zhang K, et al. Temporal and spatial cellular and molecular pathological alterations with single-cell resolution in the adult spinal cord after injury. Signal Transduct Target Ther. 2022;7:65. Hoogland IC, Houbolt C, van Westerloo DJ, van Gool WA, van de Beek D. Systemic inflammation and microglial activation: systematic review of animal experiments. J Neuroinflammation. 2015;12:114. Sun S, Li J, Wang S, Li J, Ren J, Bao Z, Sun L, Ma X, Zheng F, Ma S, et al. CHIT1-positive microglia drive motor neuron ageing in the primate spinal cord. Nature. 2023;624:611–20. Kong G, Zhang W, Zhang S, Chen J, He K, Zhang C, Yuan X, Xie B. The gut microbiota and metabolite profiles are altered in patients with spinal cord injury. Mol Brain. 2023;16:26. Hamilton AM, Blackmer-Raynolds L, Li Y, Kelly SD, Kebede N, Williams AE, Chang J, Garraway SM, Srinivasan S, Sampson TR. Diet-microbiome interactions promote enteric nervous system resilience following spinal cord injury. NPJ Biofilms Microbiomes. 2024;10:75. Jing Y, Yang D, Bai F, Wang Q, Zhang C, Yan Y, Li Z, Li Y, Chen Z, Li J, Yu Y. Spinal cord injury-induced gut dysbiosis influences neurological recovery partly through short-chain fatty acids. NPJ Biofilms Microbiomes. 2023;9:99. Wang X, Wang Z, Cao J, Dong Y, Chen Y. Gut microbiota-derived metabolites mediate the neuroprotective effect of melatonin in cognitive impairment induced by sleep deprivation. Microbiome. 2023;11:17. Li F, Sami A, Noristani HN, Slattery K, Qiu J, Groves T, Wang S, Veerasammy K, Chen YX, Morales J, et al. Glial Metabolic Rewiring Promotes Axon Regeneration and Functional Recovery in the Central Nervous System. Cell Metab. 2020;32:767–e785767. Pfyffer D, Wyss PO, Huber E, Curt A, Henning A, Freund P. Metabolites of neuroinflammation relate to neuropathic pain after spinal cord injury. Neurology. 2020;95:e805–14. O'Shea TM, Burda JE, Sofroniew MV. Cell biology of spinal cord injury and repair. J Clin Invest. 2017;127:3259–70. Liu W, Rong Y, Wang J, Zhou Z, Ge X, Ji C, Jiang D, Gong F, Li L, Chen J, et al. Exosome-shuttled miR-216a-5p from hypoxic preconditioned mesenchymal stem cells repair traumatic spinal cord injury by shifting microglial M1/M2 polarization. J Neuroinflammation. 2020;17:47. Zha X, Zheng G, Skutella T, Kiening K, Unterberg A, Younsi A. Microglia: a promising therapeutic target in spinal cord injury. Neural Regen Res. 2025;20:454–63. Zheng B, Tuszynski MH. Regulation of axonal regeneration after mammalian spinal cord injury. Nat Rev Mol Cell Biol. 2023;24:396–413. Yang Z, Liang Z, Rao J, Xie H, Zhou M, Xu X, Lin Y, Lin F, Wang C, Chen C. Hypoxic-preconditioned mesenchymal stem cell-derived small extracellular vesicles promote the recovery of spinal cord injury by affecting the phenotype of astrocytes through the miR-21/JAK2/STAT3 pathway. CNS Neurosci Ther. 2024;30:e14428. Wang C, Wang Q, Lou Y, Xu J, Feng Z, Chen Y, Tang Q, Zheng G, Zhang Z, Wu Y, et al. Salidroside attenuates neuroinflammation and improves functional recovery after spinal cord injury through microglia polarization regulation. J Cell Mol Med. 2018;22:1148–66. Squair JW, Milano M, de Coucy A, Gautier M, Skinnider MA, James ND, Cho N, Lasne A, Kathe C, Hutson TH, et al. Recovery of walking after paralysis by regenerating characterized neurons to their natural target region. Science. 2023;381:1338–45. Clifford T, Finkel Z, Rodriguez B, Joseph A, Cai L. Current Advancements in Spinal Cord Injury Research-Glial Scar Formation and Neural Regeneration. Cells 2023, 12. Hellenbrand DJ, Quinn CM, Piper ZJ, Morehouse CN, Fixel JA, Hanna AS. Inflammation after spinal cord injury: a review of the critical timeline of signaling cues and cellular infiltration. J Neuroinflammation. 2021;18:284. Liu H, Zhang J, Xu X, Lu S, Yang D, Xie C, Jia M, Zhang W, Jin L, Wang X, et al. SARM1 promotes neuroinflammation and inhibits neural regeneration after spinal cord injury through NF-κB signaling. Theranostics. 2021;11:4187–206. Milich LM, Choi JS, Ryan C, Cerqueira SR, Benavides S, Yahn SL, Tsoulfas P, Lee JK. Single-cell analysis of the cellular heterogeneity and interactions in the injured mouse spinal cord. J Exp Med 2021, 218. Pang QM, Chen SY, Xu QJ, Fu SP, Yang YC, Zou WH, Zhang M, Liu J, Wan WH, Peng JC, Zhang T. Neuroinflammation and Scarring After Spinal Cord Injury: Therapeutic Roles of MSCs on Inflammation and Glial Scar. Front Immunol. 2021;12:751021. Ma H, Wang C, Han L, Kong F, Liu Z, Zhang B, Chu W, Wang H, Wang L, Li Q, et al. Tofacitinib Promotes Functional Recovery after Spinal Cord Injury by Regulating Microglial Polarization via JAK/STAT Signaling Pathway. Int J Biol Sci. 2023;19:4865–82. Wang G, Li X, Li N, Wang X, He S, Li W, Fan W, Li R, Liu J, Hou S. Icariin alleviates uveitis by targeting peroxiredoxin 3 to modulate retinal microglia M1/M2 phenotypic polarization. Redox Biol. 2022;52:102297. Yin Z, Han Z, Hu T, Zhang S, Ge X, Huang S, Wang L, Yu J, Li W, Wang Y, et al. Neuron-derived exosomes with high miR-21-5p expression promoted polarization of M1 microglia in culture. Brain Behav Immun. 2020;83:270–82. Brennan FH, Li Y, Wang C, Ma A, Guo Q, Li Y, Pukos N, Campbell WA, Witcher KG, Guan Z, et al. Microglia coordinate cellular interactions during spinal cord repair in mice. Nat Commun. 2022;13:4096. Green-Fulgham SM, Ball JB, Kwilasz AJ, Harland ME, Frank MG, Dragavon JM, Grace PM, Watkins LR. Interleukin-1beta and inflammasome expression in spinal cord following chronic constriction injury in male and female rats. Brain Behav Immun. 2024;115:157–68. Devanney NA, Stewart AN, Gensel JC. Microglia and macrophage metabolism in CNS injury and disease: The role of immunometabolism in neurodegeneration and neurotrauma. Exp Neurol. 2020;329:113310. Zou Z, Liu R, Wang Y, Tan H, An G, Zhang B, Wang Y, Dong D. Protein arginine methyltransferase 8 regulates ferroptosis and macrophage polarization in spinal cord injury via glial cell-derived neurotrophic factor. CNS Neurosci Ther. 2023;29:2145–61. Ren J, Zhu B, Gu G, Zhang W, Li J, Wang H, Wang M, Song X, Wei Z, Feng S. Schwann cell-derived exosomes containing MFG-E8 modify macrophage/microglial polarization for attenuating inflammation via the SOCS3/STAT3 pathway after spinal cord injury. Cell Death Dis. 2023;14:70. Lee JY, Park CS, Seo KJ, Kim IY, Han S, Youn I, Yune TY. IL-6/JAK2/STAT3 axis mediates neuropathic pain by regulating astrocyte and microglia activation after spinal cord injury. Exp Neurol. 2023;370:114576. Wang X, Li X, Zuo X, Liang Z, Ding T, Li K, Ma Y, Li P, Zhu Z, Ju C, et al. Photobiomodulation inhibits the activation of neurotoxic microglia and astrocytes by inhibiting Lcn2/JAK2-STAT3 crosstalk after spinal cord injury in male rats. J Neuroinflammation. 2021;18:256. Xiao S, Zhang Y, Liu Z, Li A, Tong W, Xiong X, Nie J, Zhong N, Zhu G, Liu J, Liu Z. Alpinetin inhibits neuroinflammation and neuronal apoptosis via targeting the JAK2/STAT3 signaling pathway in spinal cord injury. CNS Neurosci Ther. 2023;29:1094–108. Yu M, Wang F, Han K. Silencing of SH3BP2 Inhibits Microglia Activation Via the JAK/STAT Signaling in Spinal Cord Injury Models. Inflammation 2024. Tao L, Yu W, Liu Z, Zhao D, Lin S, Szalóki D, Kicsák M, Kurtán T, Zhang H. JE-133 Suppresses LPS-Induced Neuroinflammation Associated with the Regulation of JAK/STAT and Nrf2 Signaling Pathways. ACS Chem Neurosci. 2024;15:258–67. Zhang M, Zhou L, Xu Y, Yang M, Xu Y, Komaniecki GP, Kosciuk T, Chen X, Lu X, Zou X, et al. A STAT3 palmitoylation cycle promotes T(H)17 differentiation and colitis. Nature. 2020;586:434–9. Liu Y, Che X, Yu X, Shang H, Cui P, Fu X, Lu X, Liu Y, Wu C, Yang J. Phosphorylation of STAT3 at Tyr705 contributes to TFEB-mediated autophagy-lysosomal pathway dysfunction and leads to ischemic injury in rats. Cell Mol Life Sci. 2023;80:160. Li Y, Ritzel RM, Khan N, Cao T, He J, Lei Z, Matyas JJ, Sabirzhanov B, Liu S, Li H, et al. Delayed microglial depletion after spinal cord injury reduces chronic inflammation and neurodegeneration in the brain and improves neurological recovery in male mice. Theranostics. 2020;10:11376–403. Gensel JC, Zhang B. Macrophage activation and its role in repair and pathology after spinal cord injury. Brain Res. 2015;1619:1–11. Ge X, Ye W, Zhu Y, Cui M, Zhou J, Xiao C, Jiang D, Tang P, Wang J, Wang Z, et al. USP1/UAF1-Stabilized METTL3 Promotes Reactive Astrogliosis and Improves Functional Recovery after Spinal Cord Injury through m(6)A Modification of YAP1 mRNA. J Neurosci. 2023;43:1456–74. Wang R, Wu X, Tian Z, Hu T, Cai C, Wu G, Jiang GB, Liu B. Sustained release of hydrogen sulfide from anisotropic ferrofluid hydrogel for the repair of spinal cord injury. Bioact Mater. 2023;23:118–28. Martínez-Rojas B, Giraldo E, Grillo-Risco R, Hidalgo MR, López-Mocholi E, Alastrue-Agudo A, García-García F, Moreno-Manzano V. NPC transplantation rescues sci-driven cAMP/EPAC2 alterations, leading to neuroprotection and microglial modulation. Cell Mol Life Sci. 2022;79:455. Cui Y, Liu J, Lei X, Liu S, Chen H, Wei Z, Li H, Yang Y, Zheng C, Li Z. Dual-directional regulation of spinal cord injury and the gut microbiota. Neural Regen Res. 2024;19:548–56. Freyermuth-Trujillo X, Segura-Uribe JJ, Salgado-Ceballos H, Orozco-Barrios CE, Coyoy-Salgado A. Inflammation: A Target for Treatment in Spinal Cord Injury. Cells 2022, 11. Chiang MC, Tsai TY, Wang CJ. The Potential Benefits of Quercetin for Brain Health: A Review of Anti-Inflammatory and Neuroprotective Mechanisms. Int J Mol Sci 2023, 24. Wu L, Qin Y, Yuan H, Zhu Y, Hu A. Anti-inflammatory and neuroprotective effects of insulin-like growth factor-1 overexpression in pentylenetetrazole (PTZ)-induced mouse model of chronic epilepsy. Brain Res. 2022;1785:147881. He Z, Hu Y, Zhang Y, Xie J, Niu Z, Yang G, Zhang J, Zhao Z, Wei S, Wu H, Hu W. Asiaticoside exerts neuroprotection through targeting NLRP3 inflammasome activation. Phytomedicine. 2024;127:155494. Fan L, Liu C, Chen X, Zheng L, Zou Y, Wen H, Guan P, Lu F, Luo Y, Tan G, et al. Exosomes-Loaded Electroconductive Hydrogel Synergistically Promotes Tissue Repair after Spinal Cord Injury via Immunoregulation and Enhancement of Myelinated Axon Growth. Adv Sci (Weinh). 2022;9:e2105586. Gao X, Han Z, Huang C, Lei H, Li G, Chen L, Feng D, Zhou Z, Shi Q, Cheng L, Zhou X. An anti-inflammatory and neuroprotective biomimetic nanoplatform for repairing spinal cord injury. Bioact Mater. 2022;18:569–82. David G, Mohammadi S, Martin AR, Cohen-Adad J, Weiskopf N, Thompson A, Freund P. Traumatic and nontraumatic spinal cord injury: pathological insights from neuroimaging. Nat Rev Neurol. 2019;15:718–31. Freund P, Seif M, Weiskopf N, Friston K, Fehlings MG, Thompson AJ, Curt A. MRI in traumatic spinal cord injury: from clinical assessment to neuroimaging biomarkers. Lancet Neurol. 2019;18:1123–35. Zhang Y, Zhao L, Wang X, Ma W, Lazere A, Qian HH, Zhang J, Abu-Asab M, Fariss RN, Roger JE, Wong WT. Repopulating retinal microglia restore endogenous organization and function under CX3CL1-CX3CR1 regulation. Sci Adv. 2018;4:eaap8492. Guo X, Ji J, Zhang J, Hou X, Fu X, Luo Y, Mei Z, Feng Z. Anti-inflammatory and osteoprotective effects of Chikusetsusaponin Ⅳa on rheumatoid arthritis via the JAK/STAT signaling pathway. Phytomedicine. 2021;93:153801. Yang NN, Yang JW, Ye Y, Huang J, Wang L, Wang Y, Su XT, Lin Y, Yu FT, Ma SM, et al. Electroacupuncture ameliorates intestinal inflammation by activating α7nAChR-mediated JAK2/STAT3 signaling pathway in postoperative ileus. Theranostics. 2021;11:4078–89. Yang J, Li N, Zhao X, Guo W, Wu Y, Nie C, Yuan Z. WP1066, a small molecule inhibitor of STAT3, chemosensitizes paclitaxel-resistant ovarian cancer cells to paclitaxel by simultaneously inhibiting the activity of STAT3 and the interaction of STAT3 with Stathmin. Biochem Pharmacol. 2024;221:116040. Wang S, Cao M, Xu S, Shi J, Mao X, Yao X, Liu C. Luteolin Alters Macrophage Polarization to Inhibit Inflammation. Inflammation. 2020;43:95–108. Gao W, McCormick J, Connolly M, Balogh E, Veale DJ, Fearon U. Hypoxia and STAT3 signalling interactions regulate pro-inflammatory pathways in rheumatoid arthritis. Ann Rheum Dis. 2015;74:1275–83. Additional Declarations No competing interests reported. Supplementary Files FigureS1.jpg FigureS2.jpg FigureS3.jpg Westernblotimages.pdf figurelegendofsupplementaryFigures.docx Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5648324","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":391607582,"identity":"6350d06f-aad4-4ceb-a4ff-cc0764803aaf","order_by":0,"name":"Yuepeng Fang","email":"","orcid":"","institution":"Jinan Central Hospital, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yuepeng","middleName":"","lastName":"Fang","suffix":""},{"id":391607583,"identity":"13821941-3c7c-4e1d-8366-e1d34fb917bf","order_by":1,"name":"Ce Zhang","email":"","orcid":"","institution":"Jinan Central Hospital, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Ce","middleName":"","lastName":"Zhang","suffix":""},{"id":391607584,"identity":"d879618a-7469-4fb8-bd74-9f3c889b9e19","order_by":2,"name":"Zhijie Yang","email":"","orcid":"","institution":"Central Hospital affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhijie","middleName":"","lastName":"Yang","suffix":""},{"id":391607585,"identity":"7f9d0dff-f4bb-448f-8caa-f667abd75df4","order_by":3,"name":"Xiangrui Zhao","email":"","orcid":"","institution":"Shandong Second Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiangrui","middleName":"","lastName":"Zhao","suffix":""},{"id":391607586,"identity":"8ce288f8-4f53-4637-91df-7a6143892a18","order_by":4,"name":"yongcheng Yin","email":"","orcid":"","institution":"Shandong Second Medical University","correspondingAuthor":false,"prefix":"","firstName":"yongcheng","middleName":"","lastName":"Yin","suffix":""},{"id":391607587,"identity":"b6751394-df0e-4dfd-b02b-f42e77372400","order_by":5,"name":"zhengxin Jin","email":"","orcid":"","institution":"Jinan Central Hospital, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"zhengxin","middleName":"","lastName":"Jin","suffix":""},{"id":391607588,"identity":"2eedbc38-2dba-4d8c-9f6a-d25815260610","order_by":6,"name":"Pengchong Zhu","email":"","orcid":"","institution":"Jinan Central Hospital, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Pengchong","middleName":"","lastName":"Zhu","suffix":""},{"id":391607591,"identity":"5642a9e8-eb9a-4d37-bb3c-74d743f98dfb","order_by":7,"name":"Bin Ning","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYDACZhAyYJBjYEgA8thI0GJMghaILobEBqK1mLPzHn5dUHAnfX57jgHDh7LDDPyzG/BrsWzmS7OeYfAsd8OZNwaMM84dZpC4cwC/FoPDPGbGPAaHczdI5Bgw87YdZjCQSCBOS7r8DKCWv0RqMX4M1JLAcAOohZFYW5hnGBw23HDmWcHBnnPpPBI3CGk5f8b4c8Gfw/Ly7ckbH/wos5bjn0FACxCwScBYB4CYh6B6IGD+QIyqUTAKRsEoGMEAAHlYQFHHbuY8AAAAAElFTkSuQmCC","orcid":"","institution":"Jinan Central Hospital, Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Bin","middleName":"","lastName":"Ning","suffix":""}],"badges":[],"createdAt":"2024-12-15 15:53:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5648324/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5648324/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71877430,"identity":"c3048384-413a-4ce8-87f6-018429ea322a","added_by":"auto","created_at":"2024-12-19 11:08:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16508378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between PA Levels and microglial activation and inflammation after SCI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic Experimental Design: This panel illustrates the procedure to obtain spinal cord tissues from both Sham-operated and SCI mice. These tissues were subjected to an integrated analysis combining transcriptome sequencing and quantitative targeted metabolomics. (B) HE stained histological sections of mouse spinal cords from both sham-operated controls and SCI groups, scale bar = 1mm. (C) Quantification of PA Levels in mouse spinal cord tissue: comparison between Sham and SCI groups utilizing the t-Test with statistical significance indicated by Asterisks (****), p \u0026lt; 0.0001. (D) Top differential metabolites in spinal cord tissues, annotated with P-value (indicating statistical significance of concentration differences), VIP (reflecting metabolite's importance in group distinction), and Fold Change (showing magnitude of concentration changes between groups). (E) Correlation analysis between metabolites and inflammatory cytokines or microglial inflammatory activation markers in Transcriptome. (F) Differential expression heatmap of inflammation-related genes between Sham and SCI group. (G) GO enrichment analysis of differentially expressed genes between Sham and SCI Groups. (H) KEGG pathway enrichment analysis of differentially expressed genes between Sham and SCI Groups.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/32c5ad71731c3ed43856e97d.png"},{"id":71876677,"identity":"fdb27f85-cbd7-4d19-8c3f-edf039c39166","added_by":"auto","created_at":"2024-12-19 11:00:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18885997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePA Treatment ameliorates microglial inflammatory activation and cytokine release following SCI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental design and timeline. (B-D) Representative sagittal sections of spinal cord tissue stained with LFB (B), NISSL (C), and HE (D) in SCI and SCI+PA Groups, Scale bar = 1mm, the enlarged image has a scale of 10μm. (E) Representative sagittal immunohistochemical images of spinal cord tissue in SCI and SCI+PA Groups,Scale bar = 1mm, the enlarged image has a scale of 10μm.The images show immunostaining for IBA-1 (green), a marker of microglia/macrophages, iNOS (red), an indicator of nitric oxide production, and DAPI (blue), which stains nuclei. (F) RT-PCR Analysis of IL-1β, IL-6, and iNOS gene expression in spinal cord tissue, compared using the t-test (p \u0026lt; 0.05 *, p \u0026lt; 0.001 ***). (G-I) Demylination area (G), Nissl body (H) and Lesion length analysis (I) of SCI and SCI+PA group. (J) Representative sagittal and axial T2-weighted MRI images of spinal cord in SCI and SCI+PA Groups, compared using the t-test ( p \u0026lt; 0.05 *). (K) ELISA analysis of IL-1β and IL-6 Levels in Serum of SCI and SCI+PA groups, compared using the t-test (p \u0026lt; 0.05 *).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/ef60cf7cb41534f8dd865974.png"},{"id":71874999,"identity":"ce8eaa71-3fa8-4fb9-92f4-7db30308188e","added_by":"auto","created_at":"2024-12-19 10:52:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8864121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome sequencing indicates PA suppresses inflammatory response in BV2 Cells via JAK-STAT Signaling Pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Schematic diagram outlines the experimental design and grouping of samples, detailing the interventions with PA at a concentration of 10μM/mL and TNF-α at a concentration of 20 ng/mL. (B) PCA Analysis of three groups of cellular samples. (C) Differential gene Volcano Plot for TNFα and TNFα+PA Groups. (D) Heatmap that illustrates the differential gene expression between TNFα and TNFα+PA Groups, including NOS2, CD68, CD86, and AIF1. (E)Go Functional analysis of differential genes in TNFα and TNFα+PA Groups (Biological Processes). (F) KEGG pathway analysis of differential genes in TNFα and TNFα+PA Groups. (G) GSEA analysis of inflammatory response in TNFα and TNFα+PA Groups, as indicated by P-values (measuring significance) and NES scores (standardized enrichment metric). (H) GSEA analysis of JAK-STAT signaling pathway in TNFα and TNFα+PA Groups, as indicated by P-values (measuring significance) and NES scores (standardized enrichment metric).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/d226c1e64ccfc8a3a3e64907.png"},{"id":71875026,"identity":"de6db8a7-5bc1-49e0-b660-c53d3f7d0232","added_by":"auto","created_at":"2024-12-19 10:52:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18885997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePA Inhibits Inflammatory Response in BV2 Cells Induced by TNFα in virtro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Assessment of BV2 cell proliferation rates across various PA concentrations using the CCK8 Assay, with statistical significance determined by Two-Way ANOVA, (p \u0026lt; 0.01 **, p \u0026lt; 0.0001 ****). (B) BV2 cells on coverslips, were immunostained to display INOS in red, IBA-1 in green, and nuclei stained with DAPI in blue; the Merge image represents a superimposition of these three fluorescent signals, Scale bar=100μm. (C) A statistical analysis of immunofluorescence data reveals the percentage of IBA-1 and INOS double-positive cells, with statistical significance determined by One-Way ANOVA, (p \u0026lt; 0.05 *, p \u0026lt; 0.001 ***). (D) WB experiment was conducted to assess the expression levels of INOS and COX2 in total protein extracts from BV2 cells across various groups, with GAPDH serving as the internal reference protein. (E) Statistical analysis of grayscale values in Figure 4D, conducted using ImageJ software, employed the One-Way ANOVA method, ( p \u0026lt; 0.05 *, p \u0026lt; 0.01 **, p \u0026lt; 0.0001 ****). (F) RT-PCR Analysis of IL-1β, IL-6, NOS2 and CCL2 gene expression in BV2 cell, compared using the One-Way ANOVA, (p \u0026lt; 0.05 *, p \u0026lt; 0.01 **, p \u0026lt; 0.001 ***). (G) ELISA detection of IL-1β and IL-6 in the supernatants of cultured BV2 cells, (p \u0026lt; 0.01 **, p \u0026lt; 0.001 ***).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/ea56dbcded30fdfae4e42288.png"},{"id":71878849,"identity":"9e76a82b-ba76-4c9e-943e-8201a9f9cd13","added_by":"auto","created_at":"2024-12-19 11:16:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10388672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePA inhibits microglial inflammation by suppressing the JAK2/STAT3 signaling pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative WB images showing the expression levels of p-JAK2, JAK2, p-STAT3, and STAT3 proteins in BV2 cells, with GAPDH serving as the internal control protein. (B) Statistical analysis of grayscale values for proteins in Figure 5A, showing ratios of pJAK2/JAK2 and pSTAT3/STAT3, using the One-Way ANOVA, ( p \u0026lt; 0.05 *, p \u0026lt; 0.01 **, p \u0026lt; 0.001 ***). (C) Representative WB images depicting the expression levels of p-JAK2, JAK2, p-STAT3, and STAT3 proteins in BV2 cells, utilizing GAPDH as the internal control protein, following treatment with WP1066 at a concentration of 10µM. (D) Statistical analysis of grayscale values for proteins in Figure 5C, showing ratios of pJAK2/JAK2 and pSTAT3/STAT3, using the One-Way ANOVA, (p \u0026lt; 0.05 *, p \u0026lt; 0.01 **, p \u0026lt; 0.001 ***). (E) Immunofluorescence image of BV2 cells stained for pSTAT3 (red) and DAPI (blue), with scale bars indicating 75 µm for the main image and 10µm for the enlarged inset. (F) PA inhibits the nuclear translocation of pSTAT3 by suppressing the JAK2/STAT3 signaling pathway, thereby attenuating microglial inflammatory responses. (G) Representative Western blot images showing the expression levels of COX2 and iNOS proteins in BV2 cells, with GAPDH serving as the internal control. (H) Statistical analysis of grayscale values for COX2 and iNOS proteins in Figure 5H, conducted using One-Way ANOVA, with significance levels indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. (I) Representative WB images illustrating the expression levels of p-JAK2, JAK2, p-STAT3, and STAT3 proteins in injured mouse spinal cords treated with PA versus untreated controls, with GAPDH serving as the internal control protein. (J) Statistical analysis of grayscale values for proteins in Figure 5i, showing ratios of pJAK2/JAK2 and pSTAT3/STAT3, using the One-Way ANOVA, ( p \u0026lt; 0.05 *, p \u0026lt; 0.01 **, p \u0026lt; 0.001 ***).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/fd7bb234aec421b7adddec41.png"},{"id":71875018,"identity":"c1057bae-3c88-4c8f-8a0d-95ca256d2c41","added_by":"auto","created_at":"2024-12-19 10:52:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":20067985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSystematic evaluation of neuronal survival, axonal growth, and motor function recovery in SCI mice following PA treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental design and timeline. (B) Immunofluorescence images of sagittal sections of spinal cord tissue from SCI mice and SCI+PA mice at 35 days post-injury, stained for NEUN (green), NFH (red), and DAPI (blue), with a scale bar indicating 1 mm. (C) Magnified view of Figure 6B with a scale bar of 10 micrometers. (D) Statistical analysis of relative fluorescence intensity (normalized to SCI group) of NFH+ and NEUN+ areas between SCI and SCI+PA immunofluorescence images, using t-test; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. (E) Comparative analysis of max intensity in left and right hindlimbs between SCI and SCI+PA groups using t-test, *p \u0026lt; 0.05, **p \u0026lt; 0.01. (F) 3D reconstruction of the pressure on mice paws contacting the ceiling. (G) Representative WB images of MAP2 and TUJ1 protein expression in total protein extracts from spinal cord tissue of SCI and SCI+PA mice. (H) Statistical analysis of grayscale values in Figure 6G, conducted using ImageJ software, employed the One-Way ANOVA method, ( p \u0026lt; 0.05 *, p \u0026lt; 0.01 **).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/d1a2c79e2589f43a664d9988.png"},{"id":71875014,"identity":"5c3acec4-9a2b-4eac-8561-f11fe6355a95","added_by":"auto","created_at":"2024-12-19 10:52:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7861494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePA treatment promotes functional recovery in SCI mouse.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)BMS scores for SCI and SCI+PA mouse groups, illustrating functional recovery outcomes. (B) Inclined plane test for SCI and SCI+PA mouse groups (C) Typical diagrams of Catwalk test in SCI and SCI+PA mice. (D) Step sequence analysis of catwalk gait in SCI and SCI+PA mice, (Regularity index). (E) Representative footprint patterns in SCI and SCI+PA mice. (F) Comparative analysis of Swing speed in left and right hindlimbs between SCI and SCI+PA groups, employed the t-test method, (p \u0026lt; 0.01 **). (G) Print intensity analysisof SCI and SCI+PA groups. (H) Comparative analysis of print length in left and right hindlimbs between SCI and SCI+PA groups, employed the t-test method, (p \u0026lt; 0.001 ***).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/f59e8822b3a81718d787a187.png"},{"id":72840381,"identity":"3a7446c2-0833-42e2-93e5-5ca22224699a","added_by":"auto","created_at":"2025-01-02 18:02:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":93438948,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/f9c64d91-0960-4915-bd1e-460b5d4bfd22.pdf"},{"id":71876670,"identity":"3cfc828b-8e98-4683-a9ff-2b59bbe0a3ca","added_by":"auto","created_at":"2024-12-19 11:00:46","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":597318,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/b015c9dd73df80afa204e7f9.jpg"},{"id":71877427,"identity":"06815393-037b-416d-b298-761ac2ae0e43","added_by":"auto","created_at":"2024-12-19 11:08:46","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1135632,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/9607d2d05a759ace93f2edce.jpg"},{"id":71875002,"identity":"90f0c54c-7423-4caa-b521-2221c80df862","added_by":"auto","created_at":"2024-12-19 10:52:46","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":731435,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/0a7f7311c795e4ab089ea93e.jpg"},{"id":71876674,"identity":"0db7f9e3-5565-4d60-a03f-653a0aba82db","added_by":"auto","created_at":"2024-12-19 11:00:46","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":417773,"visible":true,"origin":"","legend":"","description":"","filename":"Westernblotimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/49c67bca0868eb3885ff4f95.pdf"},{"id":71877437,"identity":"7065abd5-5d8d-48df-b447-3c5a8beba001","added_by":"auto","created_at":"2024-12-19 11:08:46","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1326862,"visible":true,"origin":"","legend":"","description":"","filename":"figurelegendofsupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-5648324/v1/46d7278baa9258e1a179a89e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pantothenic acid-mediated inhibition of microglial inflammation via the JAK2/STAT3 pathway enhances motor function recovery after Spinal cord injury","fulltext":[{"header":"Highlights","content":"\u003cp\u003eIntegrated metabolomics and transcriptomics analysis post-SCI\u003c/p\u003e\n\u003cp\u003ePA ameliorates inflammatory response and promotes axonal growth following SCI\u003c/p\u003e\n\u003cp\u003ePA inhibits microglial inflammation via JAK2/STAT3 signaling pathway\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eDespite advancements in medical care, SCI leading to permanent disability, remains without an effective treatment[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The pathophysiological process of SCI involves a complex cascade of events, with neuroinflammation playing a pivotal role in neuronal dysfunction and SCI progression[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Microglial activation, the cornerstone of neuroinflammation, triggers the release of inflammatory cytokines and chemokines, creating a hostile environment for neuronal survival and regeneration. This exacerbates the injury site and perpetuates disability[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Within this context, alterations in local metabolite levels arise and function as crucial regulators within the microenvironment[\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. They influence cellular activity and neuronal regeneration post-injury and play a crucial role in modulating inflammatory responses[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, this area remains underappreciated and not fully understood, highlighting the need for further research to elucidate the mechanisms underlying the interactions between local metabolites and neuroinflammatory processes in SCI[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we utilized transcriptome sequencing and targeted metabolomics to explore the molecular changes in mouse spinal cords following SCI. Notably, we discovered a significant depletion of PA in the injured spinal cord, which exhibited an inverse correlation with microglial inflammation and activation[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This finding reveals a previously unknown connection between PA and neuroinflammatory processes in SCI. PA may indirectly aid in reducing inflammation by maintaining a healthy cellular environment, thereby potentially mitigating the inflammatory response associated with SCI[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, direct evidence linking PA to these benefits in the context of SCI remains limited and warrants further investigation.\u003c/p\u003e \u003cp\u003eTo further elucidate this relationship, we conducted experimental interventions using PA in SCI mouse models. Our results demonstrated that PA administration effectively inhibited microglial inflammation via modulation of the JAK2/STAT3 signaling pathway. This inhibition not only mitigated the neuroinflammatory milieu but also created a permissive environment conducive to axonal growth and neuronal regeneration[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Consequently, PA-treated SCI mice exhibited improved motor function recovery compared to untreated controls.\u003c/p\u003e \u003cp\u003eOur findings not only deepen our understanding of the relationship between spinal cord local metabolites and neuroinflammatory processes in SCI but also highlight the therapeutic potential of PA in promoting neuronal regeneration and functional recovery[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. By inhibiting microglial inflammation and fostering an environment conducive to axonal growth, PA offers a promising new approach to treating SCI and mitigating its devastating consequences[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAdult male C57BL/6 mice, aged 7\u0026ndash;8 weeks, obtained from Vital River Laboratories in Beijing, China, were selected for the establishment of SCI models and the execution of sham surgeries. The animal procedures utilized in this study were approved by the Animal Ethics Committee of Shandong University, Jinan, China (approval number: KYLL-JNCHIACUC-202114), ensuring strict adherence to the ethical standards and principles of animal welfare.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe procedure for establishing a SCI model\u003c/h3\u003e\n\u003cp\u003eMice were anesthetized with 2% pentobarbital and underwent a laminectomy at the T8-T10 vertebral levels to expose the spinal cord. Subsequently, a spinal cord impactor (model 68100, manufactured by RWD, Shenzhen, China) was utilized to induce SCI, adhering to the manufacturer's instructions. Specifically, the impactor was set to deliver a controlled compression force at a depth of 2 mm and a velocity of 1 m/s, targeting the exposed spinal cord segment. Successful induction of SCI was confirmed by a clear and discernible impact site on the spinal cord, accompanied by immediate, involuntary limb twitching and tail flicking, indicative of spinal cord trauma. Following the injury, the surgical site was meticulously sutured in anatomical layers to ensure proper healing and prevent infection. The mice were then returned to their individual cages to recover from anesthesia. To aid in urinary function recovery post-SCI, manual bladder expression was performed two times daily for each mouse until spontaneous voiding was reestablished. In contrast, mice in the sham group underwent a similar surgical procedure, including laminectomy, but without the application of the spinal cord impactor. All other postoperative care and monitoring procedures were identical to those performed in the SCI group to ensure comparability between the two experimental cohorts.\u003c/p\u003e\n\u003ch3\u003eWestern-blotting, immunofluorescence, ELISA and quantitative realtime PCR analysis\u003c/h3\u003e\n\u003cp\u003eFor Western blotting, BV2 cell lysates or tissue samples (spinal cord segments) were prepared by homogenizing in RIPA buffer containing protease inhibitors (Sigma-aldrich Cat NO. R0278). The lysates were then separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk and incubated overnight at 4\u0026deg;C with specific primary antibodies targeting the proteins of interest. After washing, the membrane was incubated with appropriate secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (Yeasen, Cat NO.36208ES60) and analyzed using ImageJ software. For immunofluorescence staining, BV2 cells grown on glass coverslips or tissue sections were fixed with 4% paraformaldehyde (Sigma-aldrich Cat NO. P6148) and permeabilized with 0.1% Triton X-100 (Sigma-aldrich Cat NO. X100). The samples were blocked with 10% normal goat serum and incubated overnight at 4\u0026deg;C with primary antibodies. After washing, the samples were incubated with secondary antibodies labeled with fluorescent dyes for 1 hour at room temperature. Nuclei were stained with DAPI (Beyotime Cat NO. C1002), and the stained samples were mounted with anti-fade mounting medium and visualized under a fluorescence microscope. ELISA (Abclonal Cat NO. RM17860 and Abclonal Cat NO. RK 000008) was performed to quantify the levels of specific analytes in cell culture supernatants or serum samples. Cell supernatants were collected from BV2 cell cultures, while serum was obtained from mouse blood samples by centrifugation. The samples were diluted appropriately and incubated in microplate wells coated with specific capture antibodies. After washing, bound analytes were detected with biotinylated detection antibodies followed by streptavidin-HRP conjugate. The color reaction was developed with TMB substrate and stopped with sulfuric acid. The optical density was measured spectrophotometrically at 450 nm, and analyte concentrations were calculated using a standard curve. For PCR analysis, RNA was extracted from BV2 cells or tissue samples using TRIzol reagent (Thermo Fisher Cat NO.15596018CN). The RNA was reverse-transcribed into cDNA using a reverse transcriptase kit. The cDNA was amplified in a qPCR reaction mixture containing specific primers and SYBR Green dye on a thermocycler. The amplification was monitored in real-time, and the cycle threshold (Ct) values were determined. Gene expression levels were calculated using the 2\u003csup\u003e^\u0026minus;ΔΔCt\u003c/sup\u003e method, with expression normalized to a GAPDH.\u003c/p\u003e\n\u003ch3\u003eT2-weighted magnetic resonance imaging\u003c/h3\u003e\n\u003cp\u003eThe mice underwent MRI evaluations at 14 and 35 days post-injection using a high-performance 9.4-Tesla small animal MRI scanner manufactured by Bruker (Bruker 9.4T Biospec, Bruker BioSpin, Germany). This scanner was equipped with a four-channel surface coil designed for maximum signal reception efficiency. Prior to the imaging procedure, the mice were anesthetized through inhalation of 1.5% isoflurane (sourced from RWD, Shenzhen, China) using a specialized anesthesia apparatus compatible with MRI systems. The animals were then carefully positioned within a tailored fixation device.\u003c/p\u003e \u003cp\u003eThe imaging protocol utilized T2-weighted sequences, featuring a matrix dimension of 320 \u0026times; 320 pixels, a slice thickness of 0.3 mm, an echo time (TE) of 24 milliseconds, a repetition time (TR) of 1200 milliseconds, and a flip angle of 90 degrees. These parameters enabled the acquisition of high-resolution T2-weighted images in the sagittal plane, executed through the Bruker ParaVision 6.0 software (Bruker, Ettlingen, Germany). After the imaging session, the mice were moved to a heated pad for recovery and subsequent post-procedural care.\u003c/p\u003e\n\u003ch3\u003eCATWALK gait analysis, BMS scoring, and the inclined plane test\u003c/h3\u003e\n\u003cp\u003eIn our study, gait analysis was conducted using the CATWALK system to evaluate detailed gait characteristics of the animals. Motor function recovery was assessed using the BMS locomotor rating scale. Additionally, the inclined plane test was employed to further evaluate hindlimb weight-bearing ability and motor function recovery post-SCI. For the CATWALK analysis (Noldus XT, Netherlands), paw prints were captured and analyzed to derive gait parameters. The BMS scale is a well-established method for assessing hindlimb locomotor function in SCI Mice. Animals were observed and scored based on their ability to move, with higher scores indicating better motor function. The scoring criteria included various aspects of hindlimb movement, such as joint movement, stepping ability, coordination, and trunk stability. This method provided an objective and quantitative measure of motor recovery. In the inclined plane test, mice were placed on an adjustable inclined board and the maximum stable angle they could maintain without slipping or falling was recorded. These methods, employed in a precise and systematic manner, allowed for an objective and quantitative evaluation of gait, motor function, and hindlimb strength in the animal models.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIntegrated transcriptome sequencing and targeted metabolomics analysis\u003c/h2\u003e \u003cp\u003eTo perform an in-depth comparison of mouse spinal cord tissue in sham-operated and SCI groups, we utilized a combination of transcriptome sequencing and targeted quantitative metabolomics. For transcriptome sequencing, RNA was extracted from spinal cord tissues of both groups and subjected to high-throughput sequencing to generate comprehensive gene expression profiles. These profiles were then analyzed using bioinformatic tools to identify genes with differential expression patterns between sham and SCI tissues. In parallel, for targeted metabolomics, spinal cord tissues were extracted and analyzed using advanced chromatography-mass spectrometry (LC-MS/GC-MS) techniques to quantify a wide range of small molecule metabolites. The resulting metabolomic data were processed and normalized to identify metabolic alterations associated with SCI. Finally, both transcriptomic and metabolomic datasets were integrated through bioinformatic and statistical approaches to uncover correlations between gene expression changes and metabolic perturbations, providing a holistic view of the biological responses to SCI.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIntraperitoneal injection method of PA in mouse models of SCI\u003c/h3\u003e\n\u003cp\u003eIn mouse models of SCI, the intraperitoneal injection method for administering PA involves first preparing a sterile solution of PA in Phosphate-Buffered Saline (PBS) at a concentration of 20 mg/mL, ensuring it is free of contaminants through sterile filtration. The mouse's abdominal area is then gently shaved and disinfected with an antiseptic solution to prevent infection. Using a sterile syringe and needle, the prepared PA solution is drawn up and injected slowly into the intraperitoneal cavity of the mouse at an appropriate angle and depth, avoiding vital organs and blood vessels, while the mouse is handled gently to minimize stress. The injection dose is calculated based on 20 mg/kg of the mouse's body weight. Following the injection, the needle is withdrawn carefully, and the injection site is gently pressed to prevent leakage or bleeding. The mouse is then placed in a recovery cage and closely monitored until it fully recovers from any potential anesthesia or discomfort associated with the procedure.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIn the current study, statistical analyses were meticulously conducted using R version 4.2.1 and GraphPad Prism 9, adhering strictly to established principles. Prior to test selection, we evaluated data normality and variance homogeneity to ascertain the appropriateness of parametric or non-parametric methods. Parametric data were analyzed using ANOVA and t-tests, while non-parametric data were handled with appropriate non-parametric techniques. Gene expression data underwent normalization to mitigate technical biases and facilitate precise differential expression analysis, followed by statistical testing to identify significant changes. Immunohistochemical data were quantitatively analyzed using ImageJ, with statistical evaluations focusing on staining intensity and distribution. Behavioral assessments employed repeated measures ANOVA to discern meaningful changes over time, controlling for inter-individual variability. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ensuring a rigorous and conservative approach to data interpretation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIntegrated transcriptomic and metabolomic analysis reveals a correlation between decreased PA and microglial activation and inflammation following SCI\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the metabolic alterations associated with SCI-induced neuroinflammation and identify potential key metabolites, we conducted an integrated analysis of transcriptomic and metabolomic sequencing in mice (n\u0026thinsp;=\u0026thinsp;12 per group) from both the sham and SCI groups (14 days post-SCI) within the same cohort \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. The 14-day post-SCI timepoint was chosen to capture a stabilized glial scar and a localized inflammatory microenvironment, characterized by persistent microglial inflammation[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. By analyzing metabolic signatures at this critical stage, we aimed to elucidate the mechanisms underlying microglial activation and inflammation, potentially uncovering novel therapeutic targets for SCI recovery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur results revealed a significant downregulation of PA in tissues[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] post-SCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Using criteria including variable importance in projection (VIP), p-value, and fold change, we selectively analyzed four metabolites, with PA being a focal point, for their correlation with upregulated inflammatory genes and microglial activation genes following SCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Notably, PA emerged as the most significant metabolite, demonstrating a strong and robust correlation with multiple microglial inflammatory activation genes, including CD68, CD86, CCL2, CCL3, and IL1α \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. This underscores the prominent role of PA among the metabolites analyzed[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, transcriptome sequencing analysis showed a substantial upregulation of inflammatory cytokines, chemokines, and neurotoxic factors such as IL1β, LCN2, CCL3, CCL5, and TNF following SCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). GO functional annotation and KEGG pathway analysis revealed significant enrichment of inflammatory responses, intercellular interactions, and the activation of JAK-STAT and NF-κB signaling pathways at the 14-day post-SCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, H). These findings highlight the prevalent inflammatory landscape and the activation of key inflammatory pathways during the acute phase of SCI, aligning with current research in SCI pathophysiology[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eSuppression of microglial inflammation and activation by PA: enhancing neuronal survival and ameliorating histopathological changes after SCI\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo validate the direct role of PA as a metabolite in regulating microglial inflammation and activation at the molecular level following SCI, we conducted a study using intraperitoneal injection of PA as a supplement (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Our aim was to explore its impact on the inflammatory response and subsequent histopathological changes in the spinal cord tissue post-SCI. We comprehensively assessed the histopathological changes through Hematoxylin and Eosin (HE) staining, Luxol Fast Blue (LFB) staining, and Nissl staining in comparison between the PA-treated group and its control group. Our findings revealed that a significant reduction in inflammatory cell infiltration, preservation of myelin integrity, and enhanced neuronal viability in the PA-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D, G-I). These observations underscore the therapeutic potential of PA in mitigating the histopathological consequences of SCI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, using immunofluorescence staining with markers such as IBA-1 and INOS, we observed a significant downregulation of microglial activation and inflammatory responses[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This was further corroborated by PCR analysis of tissue lysates and ELISA of serum samples, which both demonstrated decreased levels of IL-1β and IL-6 following PA intervention. The mRNA and protein levels in the tissue similarly revealed the inhibitory effect of PA on microglial inflammatory marker NOS2 following SCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF \u003cb\u003eand Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e A, B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eMRI results provided specific insights into the reduction of inflammation. Specifically, T2-weighted images revealed a decrease in hyperintensity within the lesion site, suggesting a reduction in edema and inflammation, indicating better tissue integrity and reduced inflammatory-induced disruption. These MRI findings, in conjunction with the biochemical data, suggest that PA treatment effectively dampens the inflammatory responses associated with SCI, promoting a more favorable healing environment.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome sequencing reveals PA-mediated inhibition of microglial inflammation via the JAK-STAT Signaling Pathway\u003c/h2\u003e \u003cp\u003eMicroglia are primary contributors to inflammation following SCI, directly participating as immune cells in the release of neurotoxic factors such as inflammatory cytokines and chemokines[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. To elucidate the specific effects of PA on microglia and the underlying mechanisms, we conducted an in vitro study on the BV2 cell line, dividing it into three groups: a control group, a TNFα-treated group, and a co-intervention group exposed to both TNFα and PA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). RNA sequencing was then performed to investigate these effects. PCA analysis revealed a clear segregation of the three cellular groups based on their gene expression profiles, indicating distinct molecular signatures associated with each group that are likely reflective of differential responses to the experimental conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The volcano plot illustrates that the majority of differential genes are upregulated in response to the inflammatory stimulus induced by TNFα alone. Through functional enrichment analysis using GO and KEGG, we found that TNFα stimulation also triggers the activation of microglial immune responses, the release of chemokines, and the activation of inflammation-related pathways (\u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-C\u003c/b\u003e). Notably, the primary differential genes between the TNFα and TNFα\u0026thinsp;+\u0026thinsp;PA groups are downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). We investigated these downregulated differential genes and found that PA significantly inhibited the upregulation of microglial inflammatory activation markers (NOS2, CD68, CD86, Aif1), inflammatory cytokines (IL1β, IL6), and chemokines (CCL2, CCL3). Functional enrichment analysis using GO and KEGG revealed that PA primarily modulates microglial immune responses and inflammatory reactions. Notably, the JAK-STAT signaling pathway was significantly downregulated upon PA intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). Consistent results were also observed in Gene Set Enrichment Analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H). Similarly, these transcriptome sequencing results are consistent with the functional outcomes observed in mice following SCI. The JAK-STAT signaling pathway plays a pivotal role in microglial activation and inflammation, regulating the production of inflammatory cytokines and chemokines. In the context of SCI, activation of this pathway contributes to neuroinflammation and secondary damage. Based on the aforementioned results, we hypothesize that PA may inhibit microglial inflammation by suppressing the JAK-STAT signaling pathway, with potential implications for neuroprotection in the context of SCI and other neurological disorders[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePA inhibits microglial activation and inflammation by suppressing the JAK2/STAT3 signaling pathway\u003c/h2\u003e \u003cp\u003eNext, we validated the transcriptome sequencing results by examining the inflammatory state of BV2 cells. To establish the optimal intervention concentration of PA, we performed a CCK8 assay across a range of concentrations. The results demonstrated that PA concentrations up to 10\u0026micro;mol/ml exerted no significant effect on the proliferation of BV2 cells. Consequently, we chose 10\u0026micro;mol/ml as the working concentration of PA to investigate its modulatory effects on inflammatory responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Our study further revealed that PA intervention significantly downregulated the expression of microglial inflammatory markers, IBA-1 and INOS, under TNFα stimulation[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Notably, the percentage of BV2 cells co-expressing IBA-1 and INOS decreased substantially (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Similarly, through PCR, Western blot (WB) analysis, and ELISA detection of cell culture supernatants, we confirmed that PA indeed reduced the expression of inflammatory markers (NOS2 and COX2) and release of cytokines, and chemokines in TNFα-stimulated BV2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-G\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon analyzing the transcriptome sequencing results, we discovered that PA suppressed the JAK/STAT pathway in microglia stimulated by TNFα. Considering the pivotal role of activated JAK2/STAT3 signaling in enhancing inflammatory responses in microglia, we subsequently conducted further validation[\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. We subsequently conducted WB analysis to investigate the effects of PA on JAK2 and STAT3 phosphorylation. The findings were consistent with our expectations, demonstrating that PA inhibited both JAK2 and STAT3 phosphorylation[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Furthermore, the use of the JAK2/STAT3 signaling pathway inhibitor WP1066 revealed an additive effect with PA in influencing the JAK2/STAT3 pathway \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D\u003cb\u003e)\u003c/b\u003e. These results contribute to our understanding of PA's potential therapeutic mechanisms in neuroinflammatory conditions, particularly in the context of SCI[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Given that phosphorylated STAT3 (p-STAT3) translocates to the nucleus to regulate transcription of inflammation-related factors, we next conducted immunofluorescence experiments to investigate the effect of PA on p-STAT3 nuclear translocation[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Our findings revealed that, under TNFα stimulation, there was a significant increase in p-STAT3 nuclear translocation. However, when PA was co-administered, p-STAT3 nuclear translocation was markedly reduced. Notably, the use of the JAK/STAT signaling pathway inhibitor WP1066[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] exhibited an additive effect with PA in reducing p-STAT3 nuclear translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). Concurrently, through the assessment of the inflammatory state of BV2 cells, we found that the anti-inflammatory effects of PA on BV2 cells were indeed mediated through the inhibition of the JAK2/STAT3 signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H \u003cb\u003eand Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA\u003c/b\u003e). Subsequently, we discovered that PA also inhibits the JAK2/STAT3 signaling pathway in injured spinal cord tissues, suggesting that PA may exert neuroprotective and anti-inflammatory effects through this mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, J).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePA enhances neural growth and promotes motor functional recovery in mice with SCI.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven the proven inhibitory effect of PA on inflammation subsequent to SCI, we administered PA via intraperitoneal injection to investigate whether its anti-inflammatory properties confer beneficial effects on neuronal axon growth and functional restoration in the hindlimbs of mice[\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our objective was to elucidate the therapeutic potential of PA in facilitating neuroregeneration and enhancing functional recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Through immunofluorescence staining for NEUN and NFH, we observed notable enhancements in the retention of mature neurons and axon growth in mice with SCI after PA treatment[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Specifically, the axons of neurons in the PA-administered group were capable of growing into the injury core region, accompanied by a significantly higher number of viable mature neurons surrounding the injury site compared to the SCI control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D). To assess the recovery of hindlimb locomotor function in mice, the catwalk test was employed. Results indicated that the majority of mice in the SCI group were unable to achieve paw or dorsum plantar contact with the ground. In contrast, mice subjected to PA intervention demonstrated a significant capacity for plantar standing. This was manifested by a marked increase in the maximum intensity of hindlimb movement in the PA-treated group compared to the SCI group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). Subsequently, the mice were euthanized, and their spinal cord tissues were harvested. Total proteins were extracted and subjected to WB analysis. The results demonstrated a significant upregulation of TUJ1 and MAP2 expression in the PA intervention group compared to the SCI group. This finding suggests that PA intervention promote neuronal regeneration within the spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo conduct a comprehensive assessment of the recovery of motor function in mice, a battery of tests including the Basso Mouse Scale (BMS) score, the inclined plane test, and the catwalk analysis were employed to evaluate the motor restoration and coordination of the hindlimbs. The BMS scores indicated significant functional recovery improvement in the PA intervention group compared to the SCI group at 28 days post-injury. Specifically, at 35 days post-injury, only 2 out of 6 mice in the SCI group could occasionally or consistently achieve dorsal paw placement without plantar support, whereas 3 out of 6 mice in the PA intervention group demonstrated occasional plantar paw placement, and the remaining 3 out of 6 mice could occasionally or consistently achieve dorsal paw standing (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Similarly, at 35 days post-SCI, mice in the PA intervention group exhibited a significantly increased tolerance to inclined plane angles compared to those in the SCI control group. Specifically, all 6 mice (6/6) in the PA intervention group could withstand angles of 35 degrees or higher, whereas none of the mice (0/6) in the SCI group could achieve this level of functional recovery at 35 days post-SCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The footprint analysis further demonstrated superior recovery in mice from the PA group compared to those in the SCI group, with significantly higher coordination levels observed in the PA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-E). The swing speed, as well as the length and intensity of hindlimb footprints, were also significantly improved in the PA group compared to mice in the SCI group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study presents novel insights into the therapeutic potential of PA in SCI. By leveraging transcriptome sequencing and targeted metabolomics, we have illuminated the molecular changes occurring in mouse spinal cords following SCI, particularly highlighting the depletion of PA and its inverse correlation with microglial inflammation and activation.\u003c/p\u003e \u003cp\u003eThe integration of transcriptomic and metabolomic data revealed a significant downregulation of PA in spinal cord tissues post-SCI. This downregulation was robustly correlated with the upregulation of multiple microglial inflammatory activation genes. These findings underscore the pivotal role of PA in the neuroinflammatory processes following SCI, a connection previously unknown[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Furthermore, transcriptome sequencing analysis demonstrated a substantial upregulation of inflammatory cytokines, chemokines, and neurotoxic factors, aligning with the activation of key inflammatory pathways such as JAK-STAT. This inflammatory landscape is characteristic of the acute phase of SCI and highlights the need for therapeutic interventions capable of dampening these responses[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur experimental interventions using PA in SCI mouse models provided compelling evidence for its neuroprotective and anti-inflammatory effects[\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. PA administration effectively inhibited microglial inflammation via modulation of the JAK2/STAT3 signaling pathway. This inhibition not only mitigated the neuroinflammatory milieu but also created a permissive environment conducive to axonal growth and neuronal regeneration[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Consequently, PA-treated SCI mice exhibited improved motor function recovery compared to untreated controls. These results were further corroborated by MRI findings, which revealed a reduction in edema and inflammation within the lesion site, suggesting better tissue integrity and reduced inflammatory-induced disruption[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Our in vitro studies on the BV2 cell line further elucidated the specific effects of PA on microglia and the underlying mechanisms. PA significantly inhibited the upregulation of microglial inflammatory activation markers, inflammatory cytokines, and chemokines[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Functional enrichment analysis revealed that PA primarily modulates microglial immune responses and inflammatory reactions, with a particular focus on the JAK-STAT signaling pathway[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].The observed inhibitory effect of PA on JAK2/STAT3 phosphorylation further solidified its potential therapeutic mechanisms in neuroinflammatory conditions. The use of the JAK/STAT signaling pathway inhibitor WP1066 revealed an additive effect with PA in influencing the JAK/STAT pathway, suggesting a synergistic interaction between the two[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Immunofluorescence experiments demonstrated that PA reduced p-STAT3 nuclear translocation, indicating its ability to regulate the transcription of inflammation-related factors[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, our findings deepen our understanding of the relationship between PA and neuroinflammatory processes in SCI and highlight its therapeutic potential. Future studies are needed to further elucidate the mechanisms underlying PA's neuroprotective effects and to explore its potential as a therapeutic agent in clinical settings. The identification of PA as a key metabolite involved in SCI-induced neuroinflammation opens new avenues for research and provides a foundation for the development of novel therapeutic interventions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study unveils a novel therapeutic potential of PA in SCI by elucidating its anti-inflammatory and neuroprotective mechanisms. Through integrated transcriptome sequencing and metabolomic analysis, we identified a significant depletion of PA in injured spinal cord tissues, which inversely correlated with microglial inflammation and activation. Experimental interventions using PA in SCI mouse models demonstrated its efficacy in inhibiting microglial inflammation via modulation of the JAK2/STAT3 signaling pathway, thereby fostering an environment conducive to axonal growth and neuronal regeneration. Consequently, PA-treated SCI mice exhibited improved motor function recovery. These findings deepen our understanding of the relationship between PA and neuroinflammatory processes in SCI and pave the way for future research exploring PA as a therapeutic agent in clinical settings.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSCI: Spinal Cord Injury\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePA: Pantothenic Acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJAK2: Janus Kinase 2\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSTAT3: Signal Transducer and Activator of Transcription 3\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePBS: Phosphate-Buffered Saline\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRNA: Ribonucleic Acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIL-1β: Interleukin-1β\u003c/p\u003e\n\u003cp\u003eIL-6: Interleukin-6\u003c/p\u003e\n\u003cp\u003eiNOS: Inducible Nitric Oxide Synthase\u003c/p\u003e\n\u003cp\u003eIBA-1: Ionized Calcium Binding Adaptor Molecule 1\u003c/p\u003e\n\u003cp\u003eLFB: Luxol Fast Blue\u003c/p\u003e\n\u003cp\u003eNISSL: Nissl staining\u003c/p\u003e\n\u003cp\u003eHE: Hematoxylin and Eosin\u003c/p\u003e\n\u003cp\u003eDAPI: 4',6-Diamidino-2-Phenylindole\u003c/p\u003e\n\u003cp\u003eNEUN: Neuronal Nuclei\u003c/p\u003e\n\u003cp\u003eNFH: Neurofilament Heavy\u003c/p\u003e\n\u003cp\u003eWB: Western Blotting\u003c/p\u003e\n\u003cp\u003eELISA: Enzyme-Linked Immunosorbent Assay\u003c/p\u003e\n\u003cp\u003eBMS: Basso, Beattie, and Bresnahan locomotor rating scale\u003c/p\u003e\n\u003cp\u003eGO: Gene Ontology\u003c/p\u003e\n\u003cp\u003eKEGG: Kyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\n\u003cp\u003eMRI: Magnetic Resonance Imaging\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFang Y. conducted experiments, analyzed data, and interpreted results concerning PA and neuroinflammatory processes in SCI mouse models. Zhang C. designed and executed experiments, with a focus on transcriptome sequencing and metabolomics analyses post-SCI. Yang Z., Zhao X., Yin Y., Jin Z., and Zhu P. supported various research aspects such as data collection, experimental setup, and coordination. Ning B. served as the lead author, overseeing the study design, implementation, and interpretation, and drafted the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was comprehensively supported by multiple funding sources, including grants from the National Natural Science Fund of China (grant numbers 82071383 and 82371392), the Natural Science Foundation of Shandong Province (Key Project) with grant number ZR2020KH007, and the \"Taishan Scholar Distinguished Expert Program\" of Shandong Province, under grant number tstp20231257, all of which contributed towards the research endeavors of Bin Ning. The cell model in the picture comes from the Servier Medical Art and BioRender APP. In this study, AI-assisted tools were employed under human supervision to enhance the English writing process, ensuring academic rigor while expediting the drafting of our manuscript. However, the final content was meticulously crafted and validated by human authors, ensuring the originality and scientific accuracy of the presented findings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJug M, Komadina R, Wendt K, Pape HC, Bloemers F, Nau C. Thoracolumbar spinal cord injury: management, techniques, timing. Eur J Trauma Emerg Surg. 2024;50:1969\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu X, Xu W, Ren Y, Wang Z, He X, Huang R, Ma B, Zhao J, Zhu R, Cheng L. Spinal cord injury: molecular mechanisms and therapeutic interventions. Signal Transduct Target Ther. 2023;8:245.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang YP, Qin ZH, Zhang Y, Ning B. Implications of microglial heterogeneity in spinal cord injury progression and therapy. Exp Neurol. 2023;359:114239.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Wu Z, Zhou L, Shao J, Hu X, Xu W, Ren Y, Zhu X, Ge W, Zhang K, et al. Temporal and spatial cellular and molecular pathological alterations with single-cell resolution in the adult spinal cord after injury. Signal Transduct Target Ther. 2022;7:65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoogland IC, Houbolt C, van Westerloo DJ, van Gool WA, van de Beek D. Systemic inflammation and microglial activation: systematic review of animal experiments. J Neuroinflammation. 2015;12:114.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun S, Li J, Wang S, Li J, Ren J, Bao Z, Sun L, Ma X, Zheng F, Ma S, et al. CHIT1-positive microglia drive motor neuron ageing in the primate spinal cord. Nature. 2023;624:611\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong G, Zhang W, Zhang S, Chen J, He K, Zhang C, Yuan X, Xie B. The gut microbiota and metabolite profiles are altered in patients with spinal cord injury. Mol Brain. 2023;16:26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamilton AM, Blackmer-Raynolds L, Li Y, Kelly SD, Kebede N, Williams AE, Chang J, Garraway SM, Srinivasan S, Sampson TR. Diet-microbiome interactions promote enteric nervous system resilience following spinal cord injury. NPJ Biofilms Microbiomes. 2024;10:75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJing Y, Yang D, Bai F, Wang Q, Zhang C, Yan Y, Li Z, Li Y, Chen Z, Li J, Yu Y. Spinal cord injury-induced gut dysbiosis influences neurological recovery partly through short-chain fatty acids. NPJ Biofilms Microbiomes. 2023;9:99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Wang Z, Cao J, Dong Y, Chen Y. Gut microbiota-derived metabolites mediate the neuroprotective effect of melatonin in cognitive impairment induced by sleep deprivation. Microbiome. 2023;11:17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi F, Sami A, Noristani HN, Slattery K, Qiu J, Groves T, Wang S, Veerasammy K, Chen YX, Morales J, et al. Glial Metabolic Rewiring Promotes Axon Regeneration and Functional Recovery in the Central Nervous System. Cell Metab. 2020;32:767\u0026ndash;e785767.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePfyffer D, Wyss PO, Huber E, Curt A, Henning A, Freund P. Metabolites of neuroinflammation relate to neuropathic pain after spinal cord injury. Neurology. 2020;95:e805\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'Shea TM, Burda JE, Sofroniew MV. Cell biology of spinal cord injury and repair. J Clin Invest. 2017;127:3259\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu W, Rong Y, Wang J, Zhou Z, Ge X, Ji C, Jiang D, Gong F, Li L, Chen J, et al. Exosome-shuttled miR-216a-5p from hypoxic preconditioned mesenchymal stem cells repair traumatic spinal cord injury by shifting microglial M1/M2 polarization. J Neuroinflammation. 2020;17:47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZha X, Zheng G, Skutella T, Kiening K, Unterberg A, Younsi A. Microglia: a promising therapeutic target in spinal cord injury. Neural Regen Res. 2025;20:454\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng B, Tuszynski MH. Regulation of axonal regeneration after mammalian spinal cord injury. Nat Rev Mol Cell Biol. 2023;24:396\u0026ndash;413.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Z, Liang Z, Rao J, Xie H, Zhou M, Xu X, Lin Y, Lin F, Wang C, Chen C. Hypoxic-preconditioned mesenchymal stem cell-derived small extracellular vesicles promote the recovery of spinal cord injury by affecting the phenotype of astrocytes through the miR-21/JAK2/STAT3 pathway. CNS Neurosci Ther. 2024;30:e14428.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang C, Wang Q, Lou Y, Xu J, Feng Z, Chen Y, Tang Q, Zheng G, Zhang Z, Wu Y, et al. Salidroside attenuates neuroinflammation and improves functional recovery after spinal cord injury through microglia polarization regulation. J Cell Mol Med. 2018;22:1148\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSquair JW, Milano M, de Coucy A, Gautier M, Skinnider MA, James ND, Cho N, Lasne A, Kathe C, Hutson TH, et al. Recovery of walking after paralysis by regenerating characterized neurons to their natural target region. Science. 2023;381:1338\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClifford T, Finkel Z, Rodriguez B, Joseph A, Cai L. Current Advancements in Spinal Cord Injury Research-Glial Scar Formation and Neural Regeneration. Cells 2023, 12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHellenbrand DJ, Quinn CM, Piper ZJ, Morehouse CN, Fixel JA, Hanna AS. Inflammation after spinal cord injury: a review of the critical timeline of signaling cues and cellular infiltration. J Neuroinflammation. 2021;18:284.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu H, Zhang J, Xu X, Lu S, Yang D, Xie C, Jia M, Zhang W, Jin L, Wang X, et al. SARM1 promotes neuroinflammation and inhibits neural regeneration after spinal cord injury through NF-κB signaling. Theranostics. 2021;11:4187\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMilich LM, Choi JS, Ryan C, Cerqueira SR, Benavides S, Yahn SL, Tsoulfas P, Lee JK. Single-cell analysis of the cellular heterogeneity and interactions in the injured mouse spinal cord. J Exp Med 2021, 218.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePang QM, Chen SY, Xu QJ, Fu SP, Yang YC, Zou WH, Zhang M, Liu J, Wan WH, Peng JC, Zhang T. Neuroinflammation and Scarring After Spinal Cord Injury: Therapeutic Roles of MSCs on Inflammation and Glial Scar. Front Immunol. 2021;12:751021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa H, Wang C, Han L, Kong F, Liu Z, Zhang B, Chu W, Wang H, Wang L, Li Q, et al. Tofacitinib Promotes Functional Recovery after Spinal Cord Injury by Regulating Microglial Polarization via JAK/STAT Signaling Pathway. Int J Biol Sci. 2023;19:4865\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang G, Li X, Li N, Wang X, He S, Li W, Fan W, Li R, Liu J, Hou S. Icariin alleviates uveitis by targeting peroxiredoxin 3 to modulate retinal microglia M1/M2 phenotypic polarization. Redox Biol. 2022;52:102297.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin Z, Han Z, Hu T, Zhang S, Ge X, Huang S, Wang L, Yu J, Li W, Wang Y, et al. Neuron-derived exosomes with high miR-21-5p expression promoted polarization of M1 microglia in culture. Brain Behav Immun. 2020;83:270\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrennan FH, Li Y, Wang C, Ma A, Guo Q, Li Y, Pukos N, Campbell WA, Witcher KG, Guan Z, et al. Microglia coordinate cellular interactions during spinal cord repair in mice. Nat Commun. 2022;13:4096.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreen-Fulgham SM, Ball JB, Kwilasz AJ, Harland ME, Frank MG, Dragavon JM, Grace PM, Watkins LR. Interleukin-1beta and inflammasome expression in spinal cord following chronic constriction injury in male and female rats. Brain Behav Immun. 2024;115:157\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDevanney NA, Stewart AN, Gensel JC. Microglia and macrophage metabolism in CNS injury and disease: The role of immunometabolism in neurodegeneration and neurotrauma. Exp Neurol. 2020;329:113310.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou Z, Liu R, Wang Y, Tan H, An G, Zhang B, Wang Y, Dong D. Protein arginine methyltransferase 8 regulates ferroptosis and macrophage polarization in spinal cord injury via glial cell-derived neurotrophic factor. CNS Neurosci Ther. 2023;29:2145\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen J, Zhu B, Gu G, Zhang W, Li J, Wang H, Wang M, Song X, Wei Z, Feng S. Schwann cell-derived exosomes containing MFG-E8 modify macrophage/microglial polarization for attenuating inflammation via the SOCS3/STAT3 pathway after spinal cord injury. Cell Death Dis. 2023;14:70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee JY, Park CS, Seo KJ, Kim IY, Han S, Youn I, Yune TY. IL-6/JAK2/STAT3 axis mediates neuropathic pain by regulating astrocyte and microglia activation after spinal cord injury. Exp Neurol. 2023;370:114576.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Li X, Zuo X, Liang Z, Ding T, Li K, Ma Y, Li P, Zhu Z, Ju C, et al. Photobiomodulation inhibits the activation of neurotoxic microglia and astrocytes by inhibiting Lcn2/JAK2-STAT3 crosstalk after spinal cord injury in male rats. J Neuroinflammation. 2021;18:256.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao S, Zhang Y, Liu Z, Li A, Tong W, Xiong X, Nie J, Zhong N, Zhu G, Liu J, Liu Z. Alpinetin inhibits neuroinflammation and neuronal apoptosis via targeting the JAK2/STAT3 signaling pathway in spinal cord injury. CNS Neurosci Ther. 2023;29:1094\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu M, Wang F, Han K. Silencing of SH3BP2 Inhibits Microglia Activation Via the JAK/STAT Signaling in Spinal Cord Injury Models. \u003cem\u003eInflammation\u003c/em\u003e 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTao L, Yu W, Liu Z, Zhao D, Lin S, Szal\u0026oacute;ki D, Kics\u0026aacute;k M, Kurt\u0026aacute;n T, Zhang H. JE-133 Suppresses LPS-Induced Neuroinflammation Associated with the Regulation of JAK/STAT and Nrf2 Signaling Pathways. ACS Chem Neurosci. 2024;15:258\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang M, Zhou L, Xu Y, Yang M, Xu Y, Komaniecki GP, Kosciuk T, Chen X, Lu X, Zou X, et al. A STAT3 palmitoylation cycle promotes T(H)17 differentiation and colitis. Nature. 2020;586:434\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Che X, Yu X, Shang H, Cui P, Fu X, Lu X, Liu Y, Wu C, Yang J. Phosphorylation of STAT3 at Tyr705 contributes to TFEB-mediated autophagy-lysosomal pathway dysfunction and leads to ischemic injury in rats. Cell Mol Life Sci. 2023;80:160.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Ritzel RM, Khan N, Cao T, He J, Lei Z, Matyas JJ, Sabirzhanov B, Liu S, Li H, et al. Delayed microglial depletion after spinal cord injury reduces chronic inflammation and neurodegeneration in the brain and improves neurological recovery in male mice. Theranostics. 2020;10:11376\u0026ndash;403.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGensel JC, Zhang B. Macrophage activation and its role in repair and pathology after spinal cord injury. Brain Res. 2015;1619:1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGe X, Ye W, Zhu Y, Cui M, Zhou J, Xiao C, Jiang D, Tang P, Wang J, Wang Z, et al. USP1/UAF1-Stabilized METTL3 Promotes Reactive Astrogliosis and Improves Functional Recovery after Spinal Cord Injury through m(6)A Modification of YAP1 mRNA. J Neurosci. 2023;43:1456\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang R, Wu X, Tian Z, Hu T, Cai C, Wu G, Jiang GB, Liu B. Sustained release of hydrogen sulfide from anisotropic ferrofluid hydrogel for the repair of spinal cord injury. Bioact Mater. 2023;23:118\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez-Rojas B, Giraldo E, Grillo-Risco R, Hidalgo MR, L\u0026oacute;pez-Mocholi E, Alastrue-Agudo A, Garc\u0026iacute;a-Garc\u0026iacute;a F, Moreno-Manzano V. NPC transplantation rescues sci-driven cAMP/EPAC2 alterations, leading to neuroprotection and microglial modulation. Cell Mol Life Sci. 2022;79:455.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui Y, Liu J, Lei X, Liu S, Chen H, Wei Z, Li H, Yang Y, Zheng C, Li Z. Dual-directional regulation of spinal cord injury and the gut microbiota. Neural Regen Res. 2024;19:548\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFreyermuth-Trujillo X, Segura-Uribe JJ, Salgado-Ceballos H, Orozco-Barrios CE, Coyoy-Salgado A. Inflammation: A Target for Treatment in Spinal Cord Injury. Cells 2022, 11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChiang MC, Tsai TY, Wang CJ. The Potential Benefits of Quercetin for Brain Health: A Review of Anti-Inflammatory and Neuroprotective Mechanisms. Int J Mol Sci 2023, 24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu L, Qin Y, Yuan H, Zhu Y, Hu A. Anti-inflammatory and neuroprotective effects of insulin-like growth factor-1 overexpression in pentylenetetrazole (PTZ)-induced mouse model of chronic epilepsy. Brain Res. 2022;1785:147881.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe Z, Hu Y, Zhang Y, Xie J, Niu Z, Yang G, Zhang J, Zhao Z, Wei S, Wu H, Hu W. Asiaticoside exerts neuroprotection through targeting NLRP3 inflammasome activation. Phytomedicine. 2024;127:155494.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan L, Liu C, Chen X, Zheng L, Zou Y, Wen H, Guan P, Lu F, Luo Y, Tan G, et al. Exosomes-Loaded Electroconductive Hydrogel Synergistically Promotes Tissue Repair after Spinal Cord Injury via Immunoregulation and Enhancement of Myelinated Axon Growth. Adv Sci (Weinh). 2022;9:e2105586.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao X, Han Z, Huang C, Lei H, Li G, Chen L, Feng D, Zhou Z, Shi Q, Cheng L, Zhou X. An anti-inflammatory and neuroprotective biomimetic nanoplatform for repairing spinal cord injury. Bioact Mater. 2022;18:569\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavid G, Mohammadi S, Martin AR, Cohen-Adad J, Weiskopf N, Thompson A, Freund P. Traumatic and nontraumatic spinal cord injury: pathological insights from neuroimaging. Nat Rev Neurol. 2019;15:718\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFreund P, Seif M, Weiskopf N, Friston K, Fehlings MG, Thompson AJ, Curt A. MRI in traumatic spinal cord injury: from clinical assessment to neuroimaging biomarkers. Lancet Neurol. 2019;18:1123\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Zhao L, Wang X, Ma W, Lazere A, Qian HH, Zhang J, Abu-Asab M, Fariss RN, Roger JE, Wong WT. Repopulating retinal microglia restore endogenous organization and function under CX3CL1-CX3CR1 regulation. Sci Adv. 2018;4:eaap8492.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo X, Ji J, Zhang J, Hou X, Fu X, Luo Y, Mei Z, Feng Z. Anti-inflammatory and osteoprotective effects of Chikusetsusaponin Ⅳa on rheumatoid arthritis via the JAK/STAT signaling pathway. Phytomedicine. 2021;93:153801.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang NN, Yang JW, Ye Y, Huang J, Wang L, Wang Y, Su XT, Lin Y, Yu FT, Ma SM, et al. Electroacupuncture ameliorates intestinal inflammation by activating α7nAChR-mediated JAK2/STAT3 signaling pathway in postoperative ileus. Theranostics. 2021;11:4078\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Li N, Zhao X, Guo W, Wu Y, Nie C, Yuan Z. WP1066, a small molecule inhibitor of STAT3, chemosensitizes paclitaxel-resistant ovarian cancer cells to paclitaxel by simultaneously inhibiting the activity of STAT3 and the interaction of STAT3 with Stathmin. Biochem Pharmacol. 2024;221:116040.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Cao M, Xu S, Shi J, Mao X, Yao X, Liu C. Luteolin Alters Macrophage Polarization to Inhibit Inflammation. Inflammation. 2020;43:95\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao W, McCormick J, Connolly M, Balogh E, Veale DJ, Fearon U. Hypoxia and STAT3 signalling interactions regulate pro-inflammatory pathways in rheumatoid arthritis. Ann Rheum Dis. 2015;74:1275\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"spinal cord injury, pantothenic acid, microglial inflammation, JAK2/STAT3 signaling pathway, neuronal regeneration, motor function recovery","lastPublishedDoi":"10.21203/rs.3.rs-5648324/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5648324/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study employed transcriptome sequencing and targeted metabolomics to delve into the molecular alterations in mouse spinal cords following spinal cord injury (SCI). Notably, a significant depletion of pantothenic acid (PA) was observed in the injured spinal cord, exhibiting an inverse correlation with microglial inflammation and activation. To further elucidate this relationship, experimental interventions using PA were conducted in SCI mouse models. The results demonstrated that PA administration effectively inhibited microglial inflammation via modulation of the JAK2/STAT3 signaling pathway. This inhibition not only mitigated the neuroinflammatory milieu but also fostered an environment conducive to axonal growth and neuronal regeneration. Consequently, SCI mice treated with PA exhibited improved motor function recovery compared to untreated controls. Our findings not only deepen the understanding of the relationship between PA and neuroinflammatory processes in SCI but also highlight the therapeutic potential of PA in promoting neuronal regeneration and functional recovery.\u003c/p\u003e","manuscriptTitle":"Pantothenic acid-mediated inhibition of microglial inflammation via the JAK2/STAT3 pathway enhances motor function recovery after Spinal cord injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 10:52:41","doi":"10.21203/rs.3.rs-5648324/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c0231585-cc41-458f-874c-019af300b1a1","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-02T17:53:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-19 10:52:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5648324","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5648324","identity":"rs-5648324","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}