SP1 transcriptionally activates HTR2B to aggravate traumatic spinal cord injury by shifting microglial M1/M2 polarization.

BACKGROUND: Spinal cord injury (SCI) can result in structural and functional damage to the spinal cord, which may lead to loss of limb movement and sensation, loss of bowel and bladder control, and other complications. Previous studies have revealed the critical influence of trans-acting transcription factor 1 (SP1) in neurological pathologies, however, its role and mechanism in SCI have not been fully studied. METHODS: The study was performed using mouse microglia BV2 stimulated using lipopolysaccharide (LPS) and male adult mice subjected to spinal hitting. Western blotting was performed to detect protein expression of SP1, 5-hydroxytryptamine (serotonin) receptor 2B (HTR2B), BCL2-associated x protein (Bax), B-cell lymphoma-2 (Bcl-2), inducible nitric oxide synthase (iNOS), clusters of differentiation 86 (CD86), Arginase 1 (Arg-1) and clusters of differentiation 206 (CD206). Cell viability and apoptosis were analyzed by MTT assay and TUNEL assay. mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-4 (IL-4) and tumor necrosis factor-ß (TNF-ß) were quantified by quantitative real-time polymerase chain reaction. The association of SP1 and HTR2B was identified by chromatin immunoprecipitation assay and dual-luciferase reporter assay. HE staining assay was performed to analyze the pathological conditions of spinal cord tissues. RESULTS: LPS treatment induced cell apoptosis and inhibited microglia polarization from M1 to M2 phenotype, accompanied by an increase of Bax protein expression and a decrease of Bcl-2 protein expression, however, these effects were relieved after SP1 silencing. Mechanism assays revealed that SP1 transcriptionally activated HTR2B in BV2 cells, and HTR2B knockdown rescued LPS-induced effects on BV2 cell apoptosis and microglial M1/M2 polarization. Moreover, SP1 absence inhibited BV2 cell apoptosis and promoted microglia polarization from M1 to M2 phenotype by decreasing HTR2B expression. SCI mouse model assay further showed that SP1 downregulation could attenuate spinal hitting-induced promoting effects on cell apoptosis of spinal cord tissues and microglial M1 polarization. CONCLUSION: SP1 transcriptionally activated HTR2B to aggravate traumatic SCI by shifting microglial M1/M2 polarization.

Exosomes derived from umbilical cord-mesenchymal stem cells inhibit the NF-κB/MAPK signaling pathway and reduce the inflammatory response to promote recovery from spinal cord injury.

Spinal cord injury (SCI) is a serious traumatic disease of the central nervous system and leads to incomplete or complete loss of the body's autonomous motor and sensory functions, seriously endangering human health. Recently, exosomes have been proposed as important substances in cell-to-cell interactions. Mesenchymal stem cell (MSC)-derived exosomes exert good therapeutic effects and play a crucial role in neurological damage repair. However, the detailed mechanisms underlying their effects remain unknown. Herein, we found that compared to SCI rats, those subjected to umbilical cord MSC (UC-MSC)-derived exosomes injection showed an improved motor ability. Nevertheless, the transcriptome of BV2 microglia in different treatment groups indicated that the action pathway of exosomes might be the NF-κB/MAPK pathway. Additionally, exosomes from UC-MSCs could inhibit P38, JNK, ERK, and P65 phosphorylation in BV2 microglia and SCI rat tissues. Moreover, exosomes could inhibit apoptosis and inflammatory reaction and reactive oxygen species (ROS) production of BV2 microglia in vitro and in vivo. In conclusion, UC-MSCs-derived exosomes might protect SCI in rats by inhibiting inflammatory response via the NF-κB/MAPK signaling pathway, representing novel treatment targets or approaches for SCI.

The Protein Acetylation after Traumatic Spinal Cord Injury: Mechanisms and Therapeutic Opportunities.

Spinal cord injury (SCI) leads to deficits of various normal functions and is difficult to return to a normal state. Histone and non-histone protein acetylation after SCI is well documented and regulates spinal cord plasticity, axonal growth, and sensory axon regeneration. However, our understanding of protein acetylation after SCI is still limited. In this review, we summarize current research on the role of acetylation of histone and non-histone proteins in regulating neuron growth and axonal regeneration in SCI. Furthermore, we discuss inhibitors and activators targeting acetylation-related enzymes, such as α-tubulin acetyltransferase 1 (αTAT1), histone deacetylase 6 (HDAC6), and sirtuin 2 (SIRT2), to provide promising opportunities for recovery from SCI. In conclusion, a comprehensive understanding of protein acetylation and deacetylation in SCI may contribute to the development of SCI treatment.

P2X7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury.

In contrast to mammals, zebrafish undergo successful neural regeneration following spinal cord injury. Spinal cord ependymo-radial glia (ERG) undergo injury-induced proliferation and neuronal differentiation to replace damaged cells and restore motor function. However, the molecular cues driving these processes remain elusive. Here, we demonstrate that the evolutionarily conserved P2X7 receptors are widely distributed on neurons and ERG within the zebrafish spinal cord. At the protein level, the P2X7 receptor expressed in zebrafish is a truncated splice variant of the full-length variant found in mammals. The protein expression of this 50 kDa isoform was significantly downregulated at 7 days post-injury (dpi) but returned to basal levels at 14 dpi when compared to naïve controls. Pharmacological activation of P2X7 following SCI resulted in a greater number of proliferating cells around the central canal by 7 dpi but did not affect neuronal differentiation at 14 dpi. Our findings suggest that unlike in mammals, P2X7 signaling may not play a maladaptive role following SCI in adult zebrafish and may also work to curb the proliferative response of ERG following injury.

BMSCs-derived exosomes inhibit macrophage/microglia pyroptosis by increasing autophagy through the miR-21a-5p/PELI1 axis in spinal cord injury.

Spinal cord injury (SCI) results in a diverse range of disabilities and lacks effective treatment options. In recent years, exosomes derived from bone mesenchymal stem cells (BMSCs) have emerged as a promising cell-free therapeutic approach for treating ischemic brain injury and other inflammatory conditions. Macrophage/microglial pyroptosis has been identified as a contributing factor to neuroinflammation following SCI. The therapeutic potential of BMSC-derived exosomes in macrophage/microglia pyroptosis-induced neuroinflammation, however, has to be determined. Our findings demonstrate that exosomes derived from BMSCs can enhance motor function recovery and mitigate neuroinflammation subsequent to SCI by upregulating the expression of autophagy-related proteins and inhibiting the activation of NLRP3 inflammasomes in macrophage/microglia. Moreover, miR-21a-5p is markedly increased in BMSCs-derived exosomes, and knocking down miR-21a-5p in BMSCs-derived exosomes eliminates the beneficial effects of administration; upregulation of miR-21a-5p in BMSCs-derived exosomes enhances the beneficial effects of administration. Mechanistically, miR-21a-5p positively regulates the autophagy of macrophage/microglia by reducing PELI1 expression, which in turn inhibits their pyroptosis. This research provides novel evidence that exosomes derived from BMSCs can effectively suppress macrophage/microglia pyroptosis through the miR-21a-5p/PELI1 axis-mediated autophagy pathway, ultimately facilitating functional restoration following SCI. In particular, our constructed miR-21a-5p overexpression exosomes greatly improved the efficacy of BMSCs-derived exosomes in treating spinal cord injury. These results establish a foundation for the prospective utilization of exosomes derived from BMSCs as a novel biological intervention for spinal cord injury.

Multi-platform omics sequencing dissects the atlas of plasma-derived exosomes in rats with or without depression-like behavior after traumatic spinal cord injury.

BACKGROUND: Exosomes can penetrate the blood-brain barrier for material exchange between the peripheral and central nervous systems. Differences in exosome contents could explain the susceptibility of different individuals to depression-like behavior after traumatic spinal cord injury (TSCI). METHODS: Hierarchical clustering was used to integrate multiple depression-related behavioral outcomes in sham and TSCI rats and ultimately identify non-depressed and depressed rats. The difference in plasma exosome contents between non-depressed and depressed rats after TSCI was assessed in 15 random subjects by performing plasma exosome transcriptomics, mass spectroscope-based proteomics, and non-targeted metabolomics analyses. RESULTS: The results revealed that about 27.6% of the rats developed depression-like behavior after TSCI. Totally, 10 differential metabolites, 81 differentially expressed proteins (DEPs), 373 differentially expressed genes (DEGs), and 55 differentially expressed miRNAs (DEmiRNAs) were identified between non-depressed TSCI and sham rats. Meanwhile, 37 differential metabolites, 499 DEPs, 1361 DEGs, and 89 DEmiRNAs were identified between depressed and non-depressed TSCI rats. Enrichment analysis showed that the progression of depression-like behavior after TSCI may be related to amino acid metabolism disorder and dysfunction of multiple signaling pathways, including endocytosis, lipid and atherosclerosis, toll-like receptor, TNF, and PI3K-Akt pathway. CONCLUSION: Overall, our study systematically revealed for the first time the differences in plasma exosome contents between non-depressed and depressed rats after TSCI, which will help broaden our understanding of the complex molecular mechanisms involved in brain functional recombination after TSCI.

Deubiquitinase UCHL1 promotes angiogenesis and blood-spinal cord barrier function recovery after spinal cord injury by stabilizing Sox17.

Improving the function of the blood-spinal cord barrier (BSCB) benefits the functional recovery of mice following spinal cord injury (SCI). The death of endothelial cells and disruption of the BSCB at the injury site contribute to secondary damage, and the ubiquitin-proteasome system is involved in regulating protein function. However, little is known about the regulation of deubiquitinated enzymes in endothelial cells and their effect on BSCB function after SCI. We observed that Sox17 is predominantly localized in endothelial cells and is significantly upregulated after SCI and in LPS-treated brain microvascular endothelial cells. In vitro Sox17 knockdown attenuated endothelial cell proliferation, migration, and tube formation, while in vivo Sox17 knockdown inhibited endothelial regeneration and barrier recovery, leading to poor functional recovery after SCI. Conversely, in vivo overexpression of Sox17 promoted angiogenesis and functional recovery after injury. Additionally, immunoprecipitation-mass spectrometry revealed the interaction between the deubiquitinase UCHL1 and Sox17, which stabilized Sox17 and influenced angiogenesis and BSCB repair following injury. By generating UCHL1 conditional knockout mice and conducting rescue experiments, we further validated that the deubiquitinase UCHL1 promotes angiogenesis and restoration of BSCB function after injury by stabilizing Sox17. Collectively, our findings present a novel therapeutic target for treating SCI by revealing a potential mechanism for endothelial cell regeneration and BSCB repair after SCI.

MicroRNA-133b Dysregulation in a Mouse Model of Cervical Contusion Injury.

Our previous research studies have demonstrated the role of microRNA133b (miR133b) in healing the contused spinal cord when administered either intranasally or intravenously 24 h following an injury. While our data showed beneficial effects of exogenous miR133b delivered within hours of a spinal cord injury (SCI), the kinetics of endogenous miR133b levels in the contused spinal cord and rostral/caudal segments of the injury were not fully investigated. In this study, we examined the miR133b dysregulation in a mouse model of moderate unilateral contusion injury at the fifth cervical (C5) level. Between 30 min and 7 days post-injury, mice were euthanized and tissues were collected from different areas of the spinal cord, ipsilateral and contralateral prefrontal motor cortices, and off-targets such as lung and spleen. The endogenous level of miR133b was determined by RT-qPCR. We found that after SCI, (a) most changes in miR133b level were restricted to the injured area with very limited alterations in the rostral and caudal parts relative to the injury site, (b) acute changes in the endogenous levels were predominantly specific to the lesion site with delayed miR133b changes in the motor cortex, and (c) ipsilateral and contralateral hemispheres responded differently to unilateral SCI. Our results suggest that the therapeutic window for exogenous miR133b therapy begins earlier than 24 h post-injury and potentially lasts longer than 7 days.

Identification of key autophagy-related genes and pathways in spinal cord injury.

Spinal cord injury (SCI) can cause a range of functional impairments, and patients with SCI have limited potential for functional recovery. Previous studies have demonstrated that autophagy plays a role in the pathological process of SCI, but the specific mechanism of autophagy in this context remains unclear. Therefore, we explored the role of autophagy in SCI by identifying key autophagy-related genes and pathways. This study utilized the GSE132242 expression profile dataset, which consists of four control samples and four SCI samples; autophagy-related genes were sourced from GeneCards. R software was used to screen differentially expressed genes (DEGs) in the GSE132242 dataset, which were then intersected with autophagy-related genes to identify autophagy-related DEGs in SCI. Subsequently, the expression levels of these genes were confirmed and analyzed with gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). A protein-protein interaction (PPI) analysis was conducted to identify interaction genes, and the resulting network was visualized with Cytoscape. The MCODE plug-in was used to build gene cluster modules, and the cytoHubba plug-in was applied to screen for hub genes. Finally, the GSE5296 dataset was used to verify the reliability of the hub genes. We screened 129 autophagy-related DEGs, including 126 up-regulated and 3 down-regulated genes. GO and KEGG pathway enrichment analysis showed that these 129 genes were mainly involved in the process of cell apoptosis, angiogenesis, IL-1 production, and inflammatory reactions, the TNF signaling pathway and the p53 signaling pathway. PPI identified 10 hub genes, including CCL2, TGFB1, PTGS2, FN1, HGF, MYC, IGF1, CD44, CXCR4, and SERPINEL1. The GSE5296 dataset revealed that the control group exhibited lower expression levels than the SCI group, although only CD44 and TGFB1 showed significant differences. This study identified 129 autophagy-related genes that might play a role in SCI. CD44 and TGFB1 were identified as potentially important genes in the autophagy process after SCI. These findings provide new targets for future research and offer new perspectives on the pathogenesis of SCI.

Modified ZIF-8 Nanoparticles for Targeted Metabolic Treatment of Acute Spinal Cord Injury.

The activation of proinflammatory M1-type macrophages in the injured lesion accelerates the progression of a spinal cord injury (SCI). However, adverse side effects during systemic treatments targeting M1 macrophages have limited their applications. Nanoplatforms are novel carriers of traditional Chinese medicine because of their great efficiency to deliver and accumulation in the lesion. Herein, we synthesized a modified zeolitic imidazolate framework-8 (ZIF-8) nanoplatform for internalization and accumulation in the injured spinal cord and effective administration for SCI. In vitro and in vivo experiments suggested that Prussian blue and Schisandrin B modified ZIF-8 effectively accumulated in M1 macrophages, inhibited reactive oxygen species (ROS), and polarized the macrophage from proinflammatory M1 to anti-inflammatory M2 for rapid tissue infiltration by reprogramming the metabolic macrophages phenotype. This nanoplatform achieves a synergistic therapeutic effect of immunomodulation and neuroprotection, thereby shedding new light on the application of ZIF-8, and provides great potential for SCI.

Mechanism of Dendrobium Nobile Polysaccharide Inhibition of Ferroptosis in Rats with Spinal Cord Injury.

BACKGROUND: It has been reported that ferroptosis participates in the pathophysiological mechanism of spinal cord injury (SCI). Our preliminary experiments verified that dendrobium nobile polysaccharide (DNP) improved the behavioral function of SCI rats. Therefore, the purpose of this study was to examine the role of DNP on ferroptosis and its neuroprotective mechanism in SCI rats. METHODS: Adult female sprague dawley (SD) rats were exposed to SCI by Allen's method, followed by an intragastric injection of 100 mg/kg DNP per day for 2 weeks. Behavioral features were verified by the Basso-Beattie-Bresnahan (BBB) scale and footprint evaluation. Iron content and glutathione (GSH) were assessed spectrophotometrically. Mitochondrial morphology was examined by transmission electron microscopy. The expression of ferroptosis-related genes, including System Xc- light chain (xCT), G-rich RNA sequence binding Factor 1 (GRSF1) and glutathione peroxidase 4 (Gpx4), was examined by real-time polymerase chain reaction (PCR) and western blot. The spinal cavity was defined using hematoxylin-eosin (HE) staining, and neuronal modifications were detected by immunofluorescence. RESULTS: Compared with the SCI group, the BBB score of rats in the DNP group increased at 7 d, 14 d, 21 d, and 28 d. The differences between the two groups were statistically significant. At 12 h post-injury the iron content began to decrease. At 24 h post-injury the iron content decreased significantly in the DNP group. The morphological changes of the mitochondrial crest and membrane in the DNP group were ameliorated within 24 h. Compared with the sham group, the expression of xCT, GSH, Gpx4, and GRSF1 were significantly reduced after SCI. After DNP treatment, the expression of xCT, Gpx4, and GSH were higher. The tissue cavity area was significantly reduced and the amount of NeuN+ cells was increased in the DNP group at 14 d and 28 d after SCI. CONCLUSIONS: DNP facilitated the post-injury recovery in SCI rats via the inhibition of ferroptosis.

Single-cell RNA sequencing reveals peripheral blood leukocyte responses to spinal cord injury in mice with humanised immune systems.

Next-generation humanised mouse models and single-cell RNA sequencing (scRNAseq) approaches enable in-depth studies into human immune cell biology. Here we used NSG-SGM3 mice engrafted with human umbilical cord haematopoietic stem cells to investigate how human immune cells respond to and/or are changed by traumatic spinal cord injury (SCI). We hypothesised that the use of such mice could help advance our understanding of spinal cord injury-induced immune depression syndrome (SCI-IDS), and also how human leukocytes change as they migrate from the circulation into the lesion site. Our scRNAseq experiments, supplemented by flow cytometry, demonstrate the existence of up to 11 human immune cell (sub-) types and/or states across the blood and injured spinal cord (7 days post-SCI) of humanised NSG-SGM3 mice. Further comparisons of human immune cell transcriptomes between naïve, sham-operated and SCI mice identified a total of 579 differentially expressed genes, 190 of which were 'SCI-specific' (that is, genes regulated only in response to SCI but not sham surgery). Gene ontology analysis showed a prominent downregulation of immune cell function under SCI conditions, including for T cell receptor signalling and antigen presentation, confirming the presence of SCI-IDS and the transcriptional signature of human leukocytes in association with this phenomenon. We also highlight the activating influence of the local spinal cord lesion microenvironment by comparing the transcriptomes of circulating versus infiltrated human immune cells; those isolated from the lesion site were enriched for genes relating to both immune cell activity and function (e.g., oxidative phosphorylation, T cell proliferation and antigen presentation). We lastly applied an integrated bioinformatics approach to determine where immune responses in humanised NSG-SGM3 mice appear congruent to the native responses of human SCI patients, and where they diverge. Collectively, our study provides a valuable resource and methodological framework for the use of these mice in translational research.

Acetyl-11-keto-beta-boswellic acid modulates macrophage polarization and Schwann cell migration to accelerate spinal cord injury repair in rats.

BACKGROUND: Inhibiting secondary inflammatory damage caused by glial cells and creating a stable microenvironment is one of the main strategies to investigate drugs for the treatment of spinal cord injury. Acetyl-11-keto-beta-boswellic acid (AKBA) is the active component of the natural drug boswellia, which has anti-inflammatory and antioxidant effects and offers a possible therapeutic option for spinal cord injury. METHODS: In this study, a spinal cord injury model was established by crushing spinal cord, respectively, to detect the M1 macrophage inflammatory markers: iNOS, TNF-α, IL-1ß, and the M2 macrophage markers CD206, ARG-1, IL-10, and the detection of antioxidant enzymes and MDA. In vitro, macrophages were cultured to verify the main mechanism of the macrophage switch from Nrf2/HO-1 to M2 type by flow cytometry, immunofluorescence, and other techniques. Macrophage and Schwann cell co-culture validated the migration mechanism of Schwann cells promoted by AKBA. RESULTS: AKBA significantly enhanced the antioxidant enzyme activities of CAT, GSH-Px, T-AOC, and SOD, reduced MDA content, and reduced oxidative damage caused by spinal cord injury via the Nrf2/HO-1 signaling pathway; AKBA mediates Nrf2/HO-1/IL-10, converts macrophages from M1 to M2 type, reduces inflammation, and promotes Schwann cell migration, thereby accelerating the repair of spinal cord injury in rats. CONCLUSIONS: Our work demonstrates that AKBA can attenuate oxidative stress as well as the secondary inflammatory injury caused by macrophages after SCI, promote Schwann cell migration to the injury site, and thus accelerate the repair of the injured spinal cord.

The role of foam cells in spinal cord injury: challenges and opportunities for intervention.

Spinal cord injury (SCI) results in a large amount of tissue cell debris in the lesion site, which interacts with various cytokines, including inflammatory factors, and the intrinsic glial environment of the central nervous system (CNS) to form an inhibitory microenvironment that impedes nerve regeneration. The efficient clearance of tissue debris is crucial for the resolution of the inhibitory microenvironment after SCI. Macrophages are the main cells responsible for tissue debris removal after SCI. However, the high lipid content in tissue debris and the dysregulation of lipid metabolism within macrophages lead to their transformation into foamy macrophages during the phagocytic process. This phenotypic shift is associated with a further pro-inflammatory polarization that may aggravate neurological deterioration and hamper nerve repair. In this review, we summarize the phenotype and metabolism of macrophages under inflammatory conditions, as well as the mechanisms and consequences of foam cell formation after SCI. Moreover, we discuss two strategies for foam cell modulation and several potential therapeutic targets that may enhance the treatment of SCI.

Disruption of Neuromuscular Junction Following Spinal Cord Injury and Motor Neuron Diseases.

The neuromuscular junction (NMJ) is a crucial structure that connects the cholinergic motor neurons to the muscle fibers and allows for muscle contraction and movement. Despite the interruption of the supraspinal pathways that occurs in spinal cord injury (SCI), the NMJ, innervated by motor neurons below the injury site, has been found to remain intact. This highlights the importance of studying the NMJ in rodent models of various nervous system disorders, such as amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease (CMT), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA). The NMJ is also involved in myasthenic disorders, such as myasthenia gravis (MG), and is vulnerable to neurotoxin damage. Thus, it is important to analyze the integrity of the NMJ in rodent models during the early stages of the disease, as this may allow for a better understanding of the condition and potential treatment options. The spinal cord also plays a crucial role in the functioning of the NMJ, as the junction relays information from the spinal cord to the muscle fibers, and the integrity of the NMJ could be disrupted by SCI. Therefore, it is vital to study SCI and muscle function when studying NMJ disorders. This review discusses the formation and function of the NMJ after SCI and potential interventions that may reverse or improve NMJ dysfunction, such as exercise, nutrition, and trophic factors.

Inhibition of MST1 ameliorates neuronal apoptosis via GSK3ß/ß-TrCP/NRF2 pathway in spinal cord injury accompanied by diabetes.

AIMS: Spinal cord injury (SCI) is a devastating neurological disease that often results in tremendous loss of motor function. Increasing evidence demonstrates that diabetes worsens outcomes for patients with SCI due to the higher levels of neuronal oxidative stress. Mammalian sterile 20-like kinase (MST1) is a key mediator of oxidative stress in the central nervous system; however, the mechanism of its action in SCI is still not clear. Here, we investigated the role of MST1 activation in induced neuronal oxidative stress in patients with both SCI and diabetes. METHODS: Diabetes was established in mice by diet induction combined with intraperitoneal injection of streptozotocin (STZ). SCI was performed at T10 level through weight dropping. Advanced glycation end products (AGEs) were applied to mimic diabetic conditions in PC12 cell line in vitro. We employed HE, Nissl staining, footprint assessment and Basso mouse scale to evaluate functional recovery after SCI. Moreover, immunoblotting, qPCR, immunofluorescence and protein-protein docking analysis were used to detect the mechanism. RESULTS: Regarding in vivo experiments, diabetes resulted in up-regulation of MST1, excessive neuronal apoptosis and weakened motor function in SCI mice. Furthermore, diabetes impeded NRF2-mediated antioxidant defense of neurons in the damaged spinal cord. Treatment with AAV-siMST1 could restore antioxidant properties of neurons to facilitate reactive oxygen species (ROS) clearance, which subsequently promoted neuronal survival to improve locomotor function recovery. In vitro model found that AGEs worsened mitochondrial dysfunction and increased cellular oxidative stress. While MST1 inhibition through the chemical inhibitor XMU-MP-1 or MST1-shRNA infection restored NRF2 nuclear accumulation and its transcription of downstream antioxidant enzymes, therefore preventing ROS generation. However, these antioxidant effects were reversed by NRF2 knockdown. Our in-depth studies showed that over-activation of MST1 in diabetes directly hindered the neuroprotective AKT1, and subsequently fostered NRF2 ubiquitination and degradation via the GSK3ß/ß-TrCP pathway. CONCLUSION: MST1 inhibition significantly restores neurological function in SCI mice with preexisting diabetes, which is largely attributed to the activation of antioxidant properties via the GSK3ß(Ser 9)/ß-TrCP/NRF2 pathway. MST1 may be a promising pharmacological target for the effective treatment of spinal cord injury patients with diabetes.

Synergistic effects of tetramethylpyrazine and astragaloside IV on spinal cord injury via alteration of astrocyte A1/A2 polarization through the Sirt1-NF-κB pathway.

OBJECTIVE: Reactive astrocytes are hallmarks of traumatic spinal cord injury (T-SCI) and are associated with neuropathic pain (NP). Mediating the functional phenotype of reactive astrocytes helps neural repair and ameliorates NP in T-SCI. Here, we aimed to explore the role of tetramethylpyrazine (TMPZ) and astragaloside IV (AGS-IV) in astrocyte polarization and the underlying molecular mechanism in T-SCI. METHODS: Primary cultured astrocytes from mice were treated with LPS or conditioned medium from "M1" polarized microglia (M1-CM), followed by TMPZ and/or AGS-IV administration. The expression levels of "A1" astrocyte-specific markers (including C3, GBP2, Serping1, iNOS), "A2" astrocyte-specific markers (including S100a10 and PTX3), Sirt1 and NF-κB were detected via western blotting. TNF-α and IL-1ß levels were detected via ELISA. RT-PCR was used to evaluate OIP5-AS1 and miR-34a expression. si-OIP5-AS1 or the Sirt1 inhibitor EX-527 was administered to astrocytes. A spinal cord injury (SCI) model was constructed in Sprague-Dawley (SD) rats. Alterations in astrocytic "A1/A2" polarization in the spinal cord tissues were evaluated. RESULTS: LPS and M1-CM induced "A1" polarization of primary astrocytes. TMPZ and ASG IV could substantially reduce the expression of "A1"-related biomarkers but enhance "A2"-related biomarkers. OIP5-AS1 and Sirt1 levels were reduced in "A1"-polarized astrocytes, while miR-34a and p-NF-κB p65 were elevated. TMPZ and ASG IV enhanced OIP5-AS1 and Sirt1 levels and, in contrast, attenuated the changes in miR-34a and p-NF-κB p65 levels. Notably, the TMPZ and ASG IV combination had stronger effects on astrocyte polarization than the single treatment with TMPZ or ASG IV. OIP5-AS1 knockdown and Sirt1 inhibition both reversed the regulatory effects of TMPZ and ASG IV in astrocytic polarization. According to the in vivo experiments, the expression of "A1"-associated markers was enhanced in the spinal cords of SCI rats. The TMPZ and ASG IV combination reduced astrocytic "A1" polarization and enhanced astrocytic "A2" polarization. The expression of lncRNA OIP5-AS1 and Sirt1 was enhanced by TMPZ and ASG IV, while that of miR-34a and p-NF-κB p65 was inhibited. CONCLUSION: The combination of TMPZ and ASG IV can ameliorate dysregulated astrocytic polarization induced by spinal cord injury by affecting the lncRNA OIP5-AS1-Sirt1-NF-κB pathway.

Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Promote the Recovery of Spinal Cord Injury and Inhibit Ferroptosis by Inactivating IL-17 Pathway.

Mesenchymal stem cell (MSC)-derived exosomes are considered as alternative to cell therapy in various diseases. This study aimed to understand the effect of bone marrow MSC-derived exosomes (BMMSC-exos) on spinal cord injury (SCI) and to unveil its regulatory mechanism on ferroptosis. Exosomes were isolated from BMMSCs and the uptake of BMMSCs-exos by PC12 cells was determined using PKH67 staining. The effect of BMMSC-exos on SCI in rats was studied by evaluating pathological changes of spinal cord tissues, inflammatory cytokines, and ferroptosis-related proteins. Transcriptome sequencing was used to discover the differential expressed genes (DEGs) between SCI rats and BMMSC-exos-treated rats followed by functional enrichment analyses. The effect of BMMSC-exos on ferroptosis and interleukin 17 (IL-17) pathway was evaluated in SCI rats and oxygen-glucose deprivation (OGD)-treated PC12 cells. The results showed that particles extracted from BMMSCs were exosomes that could be taken up by PC12 cells. BMMSC-exos treatment ameliorated injuries of spinal cord, suppressed the accumulation of Fe2+, malondialdehyde (MDA), and reactive oxygen species (ROS), with the elevated glutathione (GSH). Also, BMMSC-exos downregulated the expression of acyl-CoA synthetase long chain family member 4 (ACSL4) and upregulated glutathione peroxidase 4 (GPX4) and cysteine/glutamate antiporter xCT. A total of 110 DEGs were discovered and they were mainly enriched in IL-17 signaling pathway. Further in vitro and in vivo experiments showed that BMMSC-exos inactivated IL-17 pathway. BMMSC-exos promote the recovery of SCI and inhibit ferroptosis by inhibiting the IL-17 pathway, which provides BMMSC-exos as an alternative to the management of SCI.

Extracellular vesicles from UTX-knockout endothelial cells boost neural stem cell differentiation in spinal cord injury.

BACKGROUND: Vascular endothelial cells are pivotal in the pathophysiological progression following spinal cord injury (SCI). The UTX (Ubiquitously Transcribed Tetratripeptide Repeat on Chromosome X) serves as a significant regulator of endothelial cell phenotype. The manipulation of endogenous neural stem cells (NSCs) offers a compelling strategy for the amelioration of SCI. METHODS: Two mouse models were used to investigate SCI: NSCs lineage-traced mice and mice with conditional UTX knockout (UTX KO) in endothelial cells. To study the effects of UTX KO on neural differentiation, we harvested extracellular vesicles (EVs) from both UTX KO spinal cord microvascular endothelial cells (SCMECs) and negative control SCMECs. These EVs were then employed to modulate the differentiation trajectory of endogenous NSCs in the SCI model. RESULTS: In our NSCs lineage-traced mice model of SCI, a marked decrease in neurogenesis was observed post-injury. Notably, NSCs in UTX KO SCMECs mice showed enhanced neuronal differentiation compared to controls. RNA sequencing and western blot analyses revealed an upregulation of L1 cell adhesion molecule (L1CAM), a gene associated with neurogenesis, in UTX KO SCMECs and their secreted EVs. This aligns with the observed promotion of neurogenesis in UTX KO conditions. In vivo administration of L1CAM-rich EVs from UTX KO SCMECs (KO EVs) to the mice significantly enhanced neural differentiation. Similarly, in vitro exposure of NSCs to KO EVs resulted in increased activation of the Akt signaling pathway, further promoting neural differentiation. Conversely, inhibiting Akt phosphorylation or knocking down L1CAM negated the beneficial effects of KO EVs on NSC neuronal differentiation. CONCLUSIONS: In conclusion, our findings substantiate that EVs derived from UTX KO SCMECs can act as facilitators of neural differentiation following SCI. This study not only elucidates a novel mechanism but also opens new horizons for therapeutic interventions in the treatment of SCI. Video Abstract.

Effect of CTLA-4 Inhibition on Inflammation and Apoptosis After Spinal Cord Injury.

Spinal cord injury (SCI) encompasses various pathological processes, notably neuroinflammation and apoptosis, both of which play significant roles. CTLA-4, a well-known immune molecule that suppresses T cell-mediated immune responses, is a key area of research and a focal point for targeted therapy development in treating tumors and autoimmune disorders. Despite its prominence, the impact of CTLA-4 inhibition on inflammation and apoptosis subsequent to SCI remains unexplored. This study aimed to investigate the influence of CTLA-4 on SCI. A weight-drop technique was used to establish a rat model of SCI. To examine the safeguarding effect of CTLA-4 on the restoration of motor function in rats with SCI, the Basso-Beattie-Bresnahan (BBB) scale and inclined plane test were employed to assess locomotion. Neuronal degeneration and apoptosis were assessed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) and Fluoro-Jade B labeling, respectively, and the activity of microglial cells was examined by immunofluorescence. To evaluate the impact of CTLA4 on SCI, the levels of inflammatory markers were measured. After treatment with the CTLA-4 inhibitor ipilimumab, the rats showed worse neurological impairment and more severe neuroinflammation after SCI. Furthermore, the combination therapy with ipilimumab and durvalumab after SCI had more pronounced effects than treatment with either inhibitor alone. These findings indicate that CTLA-4 contributes to neuroinflammation and apoptosis after SCI, presenting a promising new therapeutic target for this traumatic condition.