Exosome-deferoxamine loaded bioengineered neural stem cell microfibers inhibited ferroptosis after severe spinal cord injury via H4K12 lactylation

Exosome-deferoxamine loaded bioengineered neural stem cell microfibers inhibited ferroptosis after severe spinal cord injury via H4K12 lactylation | 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 Exosome-deferoxamine loaded bioengineered neural stem cell microfibers inhibited ferroptosis after severe spinal cord injury via H4K12 lactylation Jin Zhang, Chao Xu, Huan Xiong, Lili Guo, Wenqiao Qiu, Yangyang Wang, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8521282/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 Ferroptosis occurs after spinal cord injury (SCI), leading to neuronal loss and impairment of neural regeneration. Therefore, combining the beneficial effects of existing neural stem cell (NSC) scaffolds with ferroptosis inhibition strategies might lead to better therapeutic outcomes. This study proposes a combinatorial approach integrating NSC microfibers, umbilical cord mesenchymal stem cell-derived exosomes (ucMSC-exos), and deferoxamine (DFO) for SCI repair. In vitro experiments demonstrated that the combined application of ucMSC-exos and DFO successfully inhibited ferroptosis by clearing iron ions and reactive oxygen species (ROS). Furthermore, ucMSC-exos and DFO promoted cellular lactylation by increasing lactate and lactate dehydrogenase (LDH) levels. These changes were found to occur in M2 microglia triggered by ucMSC-exos, suggesting that ucMSC-exo-DFO treatment may suppress ferroptosis by enhancing histone lactylation in M2 microglia. Finally, a 3 mm mouse spinal hemisection model was used to validate the in vivo regenerative effects of the NSC microfibers with ucMSC-exos and DFO. The scaffold successfully inhibited ferroptosis, while additionally suppressed inflammation, promoted neurogenesis and angiogenesis, and ultimately restored motor and sensory functions. In summary, this study elucidated a potential mechanism for ferroptosis inhibition in SCI and provided a promising therapeutic strategy for SCI repair. spinal cord injury ferroptosis histone lactylation exosomes deferoxamine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Spinal cord injury (SCI) repair still remains a medical challenge at current stage. SCI could cause substantial vessel damage, axon disruption and neuronal death during its progression, leaving glial scars and lesion cavities that impede axonal regrowth [ 1 ][ 2 ] . Since the emergence of tissue engineering, scaffold transplantation has been developed as a main strategy for SCI repair due to its functionalities of bridging the lesion gap, guiding axon regrowth, inhibiting glial scar formation and other effects of regulating local microenvironments [ 3 ]−[ 6 ] . Meanwhile, scaffold transplantation could replenish neural cells for the damaged spinal cord by carrying stem cells and providing well-designed stem cell niches to facilitate their survival, migration and differentiation [ 7 ]−[ 9 ] . The implanted stem cells could further promote neural regeneration by their paracrine effect or their participation in the formation of local neural circuits [ 9 ]−[ 11 ] . Moreover, the microglia is another critical factor in SCI repair which could regulate inflammation, tissue regeneration and scar formation [ 12 ] . Therapeutic strategies to reestablish microglia homeostasis after SCI include the use of metal ions, proteins, small molecule drugs or exosomes [ 13 ]−[ 16 ] . Among these strategies, exosome therapy is a promising way for central system repair since they could cross blood-brain barrier and precisely deliver therapeutic proteins or RNAs to target cells [ 16 ][ 17 ] . Apart from regulating microglia homeostasis, the transplantation of exosome-loaded scaffolds could also promote angiogenesis, protect neurons and enhance neuronal differentiation [ 18 ][ 19 ] . However, as an emerging approach, the potential effects of exosomes in SCI treatment still require further investigation. Ferroptosis is an iron-dependent programmed cell death induced by the disruption of iron balance and the peroxidation of membrane lipid [ 20 ][ 21 ] . Ferroptosis could occur in SCI due to the hemorrhage-induced iron overload and subsequently induced reactive oxygen species (ROS) accumulation by Fenton reaction [ 22 ] . In SCI, ferroptosis could affect various types of neural cells, including but not limited to neurons, microglia, endothelial cells and astrocytes, thereby inhibiting neural regeneration [ 23 ] . Therefore, ferroptosis inhibition is considered beneficial to alleviate secondary injury, facilitate axon regrowth and regain motor function [ 21 ][ 22 ] . There have been several strategies to eliminate iron or ROS to prevent cells from ferroptosis. Deferoxamine (DFO) is an iron chelator approved by Food and Drug Administration which could reduce iron accumulation [ 22 ][ 24 ] . Functional hydrogels are also proved effectual to scavenge ROS and thus inhibit membrane lipid peroxidation [ 25 ][ 26 ] . In addition, exosome therapy has been extensively investigated and several exosome-mediated pathways that inhibit ferroptosis after SCI have been identified [ 27 ][ 28 ] . More importantly, exosomes have also been demonstrated to modulate the polarization of M2 microglia, an anti-inflammatory phenotype which plays a pivotal role in regulating the local inflammatory microenvironment after SCI [ 28 ][ 29 ] . Since M2 microglia have been reported to be more sensitive to ferroptosis [ 30 ][ 31 ] , investigating the protective effects of exosomes on M2 microglia is still of significant importance. Lactate is an energy source and metabolism byproduct inside human bodies. In recent years, it has been discovered that lactate could participate in the modification of histone lysine residues and thus directly stimulate gene transcription, which is called histone lactylation [ 32 ] . Hu et al have demonstrated that higher lactate level is beneficial to microglia proliferation, axon regrowth and motor function recovery after SCI due to the lactylation of histone H4 lysine 12 (H4K12) and triggering lactate/H4K12la/PD-1 signaling [ 33 ] . Meanwhile, PD-1, fully known as programmed death-1, has also been found to facilitate the polarization of M1 microglia toward the M2 phenotype [ 34 ] . However, as reported, the lactate level after SCI peaks at day 7 and subsequently declines, resulting in decreased lactylation level [ 35 ] . Lactate administration could have advantageous effects on inhibiting ferroptosis after SCI [ 33 ] . However, excessively high concentrations of lactate might induce mitophagy through alternative lactylation pathways, consequently leading to neuronal apoptosis [ 36 ] . It is still necessary to explore therapeutic approaches with minimum side effects. In our previous research, an combinatorial therapy with coaxial bioprinted neural stem cell (NSC) –laden hydrogel scaffolds and umbilical cord mesenchymal stem cell-derived exosomes (ucMSC-exos) was proposed for SCI defect repair [ 19 ] . Based on recent evidence demonstrating the anti-ferroptotic effects of exosomes on M2 microglia [ 30 ] , we hypothesize that the combinatorial therapy may promote M2 polarization of microglia while concurrently inhibit M2 microglia ferroptosis, thereby enhancing the regenerative capacity of the NSC scaffolds. In this study, we combined NSC-loaded hydrogel microfibers with DFO and ucMSC-exos to propose a multifactorial SCI therapy focusing on inhibiting ferroptosis. The beneficial effects of ucMSC-exos and DFO such as iron elimination and ROS scavenging were demonstrated in vitro . Moreover, this combinatorial approach was further confirmed to increase lactate level and lactate dehydrogenase (LDH) expression while finally enhanced histone lactylation. Based on previous findings that ucMSC-exos could facilitate M2 polarization of microglia [ 19 ] , we further demonstrated elevated histone lactylation level (H4K12la) and decreased ferroptosis in M2 microglia after treated with ucMSC-exos and DFO. This ferroptosis inhibition effect could be attributed to H4K12 lactylation according to a recent report [ 37 ] . Finally, this finding was also verified in in vivo experiments. 2. Materials and methods 2.1. Cell isolation and cell culture Primary NSCs were isolated from the cortex of fetal ICR mice as previously reported [ 38 ] . Pregnant mice were sacrificed by cervical dislocation at day 13 or 15 to obtain the fetuses. The obtained fetal cortex tissue was immersed in 5 mL DPBS with 2.5 mL 0.25% Trypsin-EDTA and 100µL DNase (Solarbio) at 37℃ for 10 min. The supernatant was removed afterwards and Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12, Gibco) with 10% FBS was added to neutralize the solution. The dissociated cell suspension was further processed with 40µm filters and centrifuged at 1000r/min to finally get the NSC pellets. NSC pellets were resuspended and maintained in DMEM/F12 with 2% B27, 1% N2, 1% Glutamine, 20ng/mL bFGF, 20ng/mL EGF and 1% penicillin-streptomycin at 37 ℃, 5% CO 2 . NSCs were passaged every 3 days. Furthermore, the BV-2 microglia (Pricella, CL-0493) were cultured in DMEM with 10% serum and 1% penicillin-streptomycin at 37℃. 2.2. ucMSC-exo acquisition and identification ucMSC-exos were purchased from Huamei BioTech Co. Ltd ( www.hmcell.com , ZA0010). The average size and zeta potential were analyzed on nanoparticle tracking analyzer (Zetaview) with laser scattering microscopy. Briefly, before analyzation, ucMSC-exos were suspended in PBS solution (pH = 7.0) at a concentration of 3.9 × 10 7 /mL. The analyzation was conducted at room temperature. 2.3. Biocompatibility evaluation of DFO and ucMSC-exos on primary NSCs and BV-2 For evaluation of NSC viability with ucMSC-exo addition, 1×10 8 primary NSCs were incubated for 24h with 1×10 8 ucMSC-exos. Cell Counting Kit-8 (CCK-8, Life-iLab, AC11L053/AC11L054) was used to detect the proliferative activity. Furthermore, for evaluation of NSC viability with DFO addition, primary NSCs were incubated with DFO (TargetMol, T124358) at 6 different concentrations: 0, 10, 20, 50, 100, 200 µg/mL. At the DFO concentration of 100µg/mL, a separate group with 1×10 8 /mL ucMSC-exo addition was established to evaluate the combined effects of ucMSC-exos and DFO on the viability of primary NSCs. CCK-8 assay was performed to detect the viability of each group. Finally, for evaluation of ucMSC-exo and DFO addition on BV-2, 4 experiment groups were established: BV-2, BV-2 with 100µg/mL DFO, BV-2 with 1×10 8 ucMSC-exos, BV-2 with 100ug/mL DFO and 1×10 8 ucMSC-exos. Each group was incubated for 24h. BeyoClick™ EdU-555 (C0075S) was used to detect the proliferation activity of BV-2 cells. 2.4. Ammonium iron citrate (AIC) treatment of BV-2 and intracellular iron content detection BV-2 microglia were treated with AIC (Sigma, F5879) to induce intracellular iron accumulation. Briefly, AIC treatment were conducted at 9 different concentrations: 0, 100, 200, 300, 400, 500, 600, 700, 800µM. 1×10 4 BV-2 microglia were incubated with AIC at corresponding concentrations for 3h. Moreover, ucMSC-exos and DFO were added to investigate their capacity for iron content clearance. 5 experiment groups were established: untreated BV-2 (control), AIC-treated BV-2 (AIC), AIC-treated BV-2 with DFO addition (DFO), AIC-treated BV-2 with ucMSC-exo addition (exo), AIC-treated BV-2 with DFO and ucMSC-exo addition (DFO + exo). Meanwhile, for the DFO + exo group, DFO and ucMSC-exos were pre-incubated at 37℃ for 2 h to facilitate DFO loading into the ucMSC-exos. DFO and ucMSC-exo treatments were administered for 1h following AIC treatment. FerroOrange (Dojindo, F374) was used to detect intracellular iron content for each group according to the manufacturer’s instruction. 2.5. Erastin and LPS treatment of BV-2 and in vitro BV-2 ferroptosis evaluation 1×10 4 BV-2 microglia were treated with 10µM erastin and 1µg/mL lipopolysaccharide (LPS, Sigma, L4391) to induce ferroptosis and BV-2 polarization. For investigation of the effects of DFO and ucMSC-exos on ferroptosis regulation and glial polarization in BV-2, 5 experiment groups were established: untreated BV-2 (control), Erastin-LPS treated BV-2 (E-LPS), Erastin-LPS treated BV-2 with DFO addition (DFO), Erastin-LPS treated BV-2 with ucMSC-exo addition (exo), Erastin-LPS treated BV-2 with ucMSC-exo and DFO addition (DFO + exo). Each group were incubated for 1 day before evaluation. JC-1 (TargetMol, T15609) fluorescence staining was used to detect the mitochondrial function. Red/green fluorescence indicates normal/impaired mitochondrial function, respectively. ROS assay kit (Solarbio, CA1410) was used to detect ROS accumulation. All experiments were performed according to the manufacturer’s instructions. 2.6. OHA synthesis and hydrogel preparation OHA was synthesized by oxidation of hyaluronic acid (HA, Freda) with sodium periodate as previously reported [ 39 ] . Briefly, the reaction product was dialysed and lyophilized to obtain the final solid OHA. CMC and OHA were separately dissolved in PBS at 2%. Rheologic characterization of OHA-CMC blend was reported in our previous report. For core-shell microfiber fabrication, CMC and OHA solutions were mixed in equal volume to form the hydrogel precursor. Digested NSCs were gently resuspended in the hydrogel precursor at 5 × 10 7 /mL to generate the final bioink for the core portion. 2.7. Fabrication of core-shell NSC microfibers with ucMSC-exo and DFO loading Core-shell microfibers were fabricated as previously reported [ 38 ] . Briefly, 5 × 10 7 primary NSCs were harvested and gently resuspended in the 2% OHA-CMC hydrogel precursor containing 1×10 8 ucMSC-exo and 100µg/mL DFO as bioink for the core portion. 3% sodium alginate solution was used as bioink for the shell portion. Both bioinks were loaded in 2 separate 10 mL syringes, which were subsequently fixed on 2 seperate channels of a microsyringe pump and connected to the core and shell inlets of a coaxial nozzle. The outlet of the nozzle was immersed in 3% calcium chloride bath. 2.8. Western blot Western blot (WB) assay was performed to quantify the protein expression level in every relevant section. All WB assays in this research were performed as follows. For in vitro WB, cells were harvested, grinded and lysed with cell lysis buffer (Life-iLab, AP01L013) on ice. The supernatant was collected after centrifuging at 10000 r/min for 20 min. Total protein concentration was quantified with BCA Protein Quantification Kit (Vazyme) following the manufacturer’s instructions. The proteins were transferred to PVDF membranes by electrophoresis and blocked with 5% skim milk for 1 h. Membranes were incubated with primary antibodies at 4℃ overnight. Primary antibodies used in this experiment include Tuj-1 (Proteintech, 66375), GFAP (Proteintech, 16825), Olig2 (Proteintech, 13999), Bax (Proteintech, 50599), Bcl2 (Affinty, AF6139), CD206 (Proteintech, 18704), Arg1 (Proteintech, 66129), iNOS (Invitrogen, AB2572890), IL-6 (Affinity, DF6087), TNF-α (Affinity, AF7014), IL-10 (ABclonal, A12255), LDH (Affinity, DF6280), Hif (Proteintech, 20960), Kla (PTMBIO, PTM-1401RM), H4K12la (PTMBIO, PTM-1411), 4-Hydroxynonenal (Invitrogen, MA5-27570), GPX4 (Proteintech, 67763), FTH (Affinity, DF6278), DMT1 (Proteintech, 20507), xCT (Affinity, DF12509), ACSL4 (abcam, AB155282), Tom20 (Proteintech, 11802), HRP-GAPDH (Beyotime, AF2823), HRP-β-tubulin (Beyotime, AF2839). On the next day, membranes were washed with TBST and incubated with corresponding secondary antibodies (Proteintech, SA00001, SA00002, SA00001-5) for 1 h. Immune bands were visualized with BeyoECL Star Kit (Beyotime, P0018AM) on imaging instrument. 2.9. Animal model All experimental methods performed in this experiment were performed in accordance with the approved guidelines and regulations by the Medical Ethical Committee and the Inspection of the Sichuan Provincial People’s Hospital (No.2021179). 24 8-week-old ICR Kunming mice were used to establish spinal cord hemisection models. Briefly, animals were anesthetized by isoflurane inhalation and immobilized on the operating table. T9-T11 laminectomy was performed after vertebrae exposure. A 3mm –long unilateral hemisection of spinal cord was completely removed. 3 separate experimental groups were established: NSC microfiber implantation with DFO and ucMSC-exos, NSC microfiber implantation alone and an untreated group (n = 8). All transplantations were performed immediately following spinal cord resection. After implantation, the musculature and skin were carefully sutured and the animals were kept in clean cages. Bladder evacuation was performed during the initial 2 weeks. 2.10. Locomotor and sensory function assessment Open field tests were performed at weeks 1, 2, 3, and 4 post surgery to evaluate locomotor function recovery in animal models. Briefly, each animal was placed in an open field for 3 min and the behavior was recorded by a video camera. The locomotion was scored by 2 independent observers according to Basso-Beattie-Bresnahan (BBB) scale. The swimming performances of experimental animals were also evaluated with Louisville swimming score. Meanwhile, on week 4, a 30 min open-field behavior was recorded and analyzed by VisuTrack software to calculate the total movement distance and the average speed. Furthermore, footprint analysis was performed on week 4 to evaluate the locomotor function of the injured hindlimb. Briefly, the left and right paws for animal hindlimbs were stained with blue and red inks, respectively. The animals were allowed to walk through a rectangular narrow box lined with white paper. The footprints were collected and analyzed. Meanwhile, Von Frey test was performed at week 2 and 5 post surgery. Briefly, the animals were habituated to mesh platform and stimulated with Von Frey filaments. The withdrawal thresholds were recorded and analyzed. 2.11. Histological analysis On week 4 post surgery, the spinal cord samples were isolated by heart perfusion. Briefly, the animals were administrated with overdose anesthesia and perfused with PBS through the left ventricle. Subsequently, 4% paraformaldehyde was perfused to fix the spinal cord tissues. The isolated samples were kept in 4% paraformaldehyde for 24h at 4℃ and immersed in 30% sucrose solution for another 3 days. Hematoxylin-eosin (HE) staining and Nissl staining were performed for the visualization of peripheral tissues, neurons, and neurites. For HE staining, briefly, the paraffin sections were dewaxed with xylene solution and a series of ethanol solutions. The sections were first stained with hematoxylin solution, followed by treatment with acid alcohol solution. Afterwards, the sections were stained with eosin solution, dehydrated, transparentized and mounted. For Nissl staining, the sections were stained with 1% Toluidine blue solution at 56℃ for 20 min, treated with acid alcohol solution, dehydrated, transparentized and mounted. 2.12. Immunostaining Immunostaining was performed to further visualize the cell phenotypes at lesion sites. Briefly, the sections were dewaxed, dehydrated, treated with 0.5% Triton and blocking solution successively. The sections were then incubated with primary antibodies at 4 ℃ overnight. On the next day, the sections were washed with DPBS and incubated with secondary antibodies for 2 h. The antibodies used in this experiment were from the same commercial sources as specified in section 2.8 except for MAP2 (Invitrogen, PA5-85755), MBP (Proteintech, 66003) and CD31 (Proteintech, 28083). Additionally, after staining with relevant antibodies, antifade mounting medium containing DAPI was added on the surface before microscopic observation. 2.13. Transmission electron microscopy (TEM) TEM was performed to assess mitochondrial activity and myelination in spinal cord tissues. Briefly, before observation, the obtained spinal cord samples were fixed, dehydrated, infiltrated, embedded in Epon-812. The embedded samples were then sliced into ultra-thin sections of 60-90nm thickness with a slicer. Sections with abundant myelin sheaths were selected under an optical microscope and then transferred to a copper mesh for staining. TEM observation was conducted on a transmission electron microscope (JEM-1400FLASH, Japan Electronics Corporation, JEOL). 2.14 Statistical analysis All data was presented as mean ± standard deviation (Mean ± SD). One-way analysis of variance (ANOVA) with Bonferroni post-hoc test was used to compare results across multiple groups. Student’s t-test was used to compare results between separate groups. Statistical significance was considered as *p < 0.05, **p < 0.01 and ***p < 0.001. 3. Results and discussion 3.1. ucMSC-exos and DFO affected NSC proliferation and differentiation in vitro The promotive effects of ucMSC-exos on NSC proliferation and differentiation have already been reported [ 19 ] . On the other hand, it has long been reported that DFO could inhibit neural cell proliferation depending on its concentration [ 40 ] . It is necessary to investigate the combinatorial effects of ucMSC-exos and DFO on NSCs. The experimental design is shown in Fig. 2 A. ucMSC-exos used in this experiment exhibited typical cup-shaped morphology and the particle sizes were 118.7-132.8nm (Fig. 2 B and 2 C). Initially, the primary NSCs exhibited an 1.5-fold increase in proliferative activity after 24h ucMSC-exo treatment (Fig. 2 D). Meanwhile, if treated with varying concentrations of DFO (0-200µg/mL), NSCs would exhibit significant reduction in proliferative activity at 100µg/mL and above (Fig. 2 E). While treated with 100µg/mL DFO and ucMSC-exos simultaneously, the NSC proliferative activity increased again, significantly higher compared with the group treated with DFO alone (Fig. 2 F). These results suggested ucMSC-exos could counteract the inhibitory effect of DFO on NSC proliferative activity. Next, the effects of ucMSC-exos and DFO on NSC differentiation were examined. The NSCs were cultured in differential medium for 7 days following treatment of ucMSC-exos, DFO, or their combination. The WB results indicate that all three treatments brought about significant promotion in early neuron marker Tuj-1 expression and no statistical significance was observed among the three groups (Fig. 2 G). The promotive effects of DFO on neuron differentiation has been reported [ 41 ] . However, there was no further improvement of neuronal differentiation observed with combined treatment. In addition, immunostaining provided further evidence including NSC identification (Oct4+/Sox2+/Nestin+), neuron location in neurospheres, and scattered NSC differentiation into oligodendrocytes (Olig2+) and astrocytes (GFAP+) (Fig. 2 H). In summary, ucMSC-exos could mitigate the negative effects of high-concentration DFO on NSC proliferative activity, while both components could promote neuronal differentiation. Therefore, the combined application of ucMSC-exos and DFO, along with NSC scaffold transplantation, is a feasible strategy for SCI treatment. 3.2 ucMSC-exos and DFO reduced iron overload and ROS accumulation in microglia in vitro The regulation effects of exosomes on microglia polarization to M2 phenotype have been extensively reported [ 16 ],[ 42 ][ 43 ] . However, as previously mentioned, M2 microglia are highly sensitive to ferroptosis while M1 microglia being resistant [ 30 ] . Since the anti-inflammatory function of M2 microglia plays an important role in SCI repair, in vitro experiments were preformed to validate the synergistic effect of ucMSC-exos and DFO in inhibiting microglia ferroptosis (Fig. 3 A). First, BV-2 microglia also exhibited a reduction in proliferative activity while treated with 100µg/mL DFO as examined with Edu assay. The addition of ucMSC-exos could partially counteracted the inhibitory effects (Fig. 3 B and 3 E). Next, since iron overload is the initial cause of ferroptosis by triggering ROS accumulation [ 44 ] , the BV-2 microglia were treated with AIC to induce iron accumulation. Consistent with previously reported [ 22 ] , DFO significantly alleviated iron overload in microglia. Moreover, we further found that ucMSC-exos failed to lower iron level, whereas DFO + exo significantly enhanced iron clearance compared with DFO alone (Fig. 3 C and 3 F). This finding suggested ucMSC-exos might facilitate the cellular uptake of DFO. It had been demonstrated in our previous report that ucMSC-exos could shift BV-2 from M1 to M2 phenotype under LPS treatment [ 19 ] . In this research, BV-2 microglia were treated with LPS and ferroptosis inducer Erastin to simultaneously induce BV-2 polarization and ferroptosis. Following treatment, ROS assay was performed to evaluate the ROS removal effects of ucMSC-exos and DFO. The result indicated that both DFO and ucMSC-exos alone could significantly reduce ROS. DFO + exo group exhibited superior ROS scavenging capacity, while failed to show statistical significance compared with DFO group (Fig. 3 D and 3 G). JC-1 assay was performed to further evaluate mitochondrial function, a factor reflecting ferroptosis degree. The DFO + exo group also showed statistical significance compared with E-LPS group (Fig. 3 D and 3 G). In summary, ucMSC-exos and DFO could synergistically reduced ferroptosis of microglia. ucMSC-exos could enhance microglia proliferative activity. DFO, as an iron chelator, could eliminate iron overload while ucMSC-exos help DFO cellular uptake. Both components could facilitate ROS scavenging and finally reduce microglia ferroptosis. 3.3 ucMSC-exos and DFO alleviated ferroptosis by facilitating histone lactylation of M2 microglia in vitro As presented in Fig. 4 A and 4 C, LPS successfully induced M1 polarization of BV-2 microglia which is marked by iNOS. Meanwhile, ucMSC-exos decreased the proportion of iNOS + cells and increased the proportion of CD206 + or Arg1 + cells, demonstrating the shift from M1 to M2 microglia. The increasing of M2 microglia further resulted in lowered inflammation level marked by the decreased expression of pro-inflammatory cytokine TNF-α and increased expression of anti-inflammatory cytokine IL-10. On the other hand, DFO has also been reported to reduce neuroinflammation by lowering the expression of pro-inflammatory cytokines [ 45 ] . Similar results were also observed in this experiment. DFO treatment alone induced upregulated expression of IL-10 and downregulated expression TNF-α. DFO + exo treatment further improved the beneficial effects compared with ucMSC-exo treatment alone. The expressions of ferroptosis-related factors were then evaluated. As presented in Fig. 4 B and 4 C, the ferritin marker FTH expression was decreased after Erastin treatment, indicating impaired iron-binding capacity of BV-2 microglia. DFO + exo treatment could restore FTH expression. DMT1 is a divalent metal transporter. DMT1 knockdown has been demonstrated to inhibit ferroptosis [ 46 ] . Erastin treatment promoted DMT1 expression. Compared with DFO or ucMSC-exo treatment alone, DFO + exo more effectively reduced DMT1 expression level. ACSL4 is a key enzyme in lipid metabolism facilitating ferroptosis. TOM20 is a mitochondrial membrane protein which is positive correlation with ferroptosis susceptibility. Following Erastin treatment, ACSL4 expression was upregulated while TOM20 expression was downregulated, while DFO + exo treatment effectively restored both proteins to baseline levels. Exogenous lactate treatment has been confirmed to stimulate histone lactylation level in microglia and thus facilitating SCI repair through lactate/H4K12la/PD-1 signaling pathway [ 33 ] . In this research, it was first demonstrated that DFO + exo treatment elevated the level of lactate dehydrogenase (LDH), which could catalyze pyruvate to lactate‌ conversion (Fig. 5 A and 5 C). Meanwhile, the addition of DFO initiated the expression of HIF-1α which had been reported to promote lactate production [ 47 ] (Fig. 5 A). Therefore, DFO + exo treatment successfully lowered the level of 4HNE, a product of lipid peroxidation, and elevated the level of GPX4, which plays a central role in ferroptosis inhibition to directly remove lipid ROS [ 22 ] (Fig. 5 A and 5 C). On the other hand, ucMSC-exo treatment promoted the polarization of M2 microglia (CD206+) while DFO + exo treatment elevated the level of lactylation modification in M2 microglia (CD206 + Kla+) (Fig. 5 B and 5 C). Finally, DFO + exo treatment promoted the histone lactylation in M2 microglia (CD206 + H4K12la+) and led to ferroptosis inhibition (4HNE + H4K12la+) (Fig. 5 B and 5 C). In summary, we first demonstrated that ucMSC-exo treatment facilitated BV-2 polarization to M2 phenotype and alleviated inflammation. Next, DFO + exo treatment could reduce ferroptosis with related markers variation. After that, we found that DFO + exo treatment elevated LDH level to facilitate pyruvate to lactate conversion. Meanwhile, DFO could induce HIF-1α upregulation to further promote lactate production. With abundant lactate, histone lactylation occurred in M2 microglia to inhibit their ferroptosis with upregulation of GPX4. Therefore, DFO + exo treatment could effectively inhibit ferroptosis in M2 microglia in vitro by histone lactylation. 3.4 NSC microfiber transplantation in SCI model with ucMSC-exos and DFO to promote neural regeneration and inflammation alleviation in vivo DFO + exo scaffolds had been reported to treat SCI by diminish inflammation and neuronal ferroptosis [ 28 ] . In this research, we combined ucMSC-exos and DFO with NSC-loaded hydrogel microfibers for SCI treatment. NSCs play a key role in neural regeneration after SCI to reestablish local neural circuits [ 10 ]−[ 11 ] . Moreover, we have previously demonstrated the neurotrophic and neuroprotective effects of ucMSC-exos on NSCs [ 19 ] . Based on these beneficial effects, we further added DFO, aiming at suppressing ferroptosis in both neurons and M2 microglia after SCI for better therapeutic effects. The NSC microfibers were fabricated in a previously reported manner [ 48 ] . Abundant NSCs were embedded in dynamic crosslinking OHA-CMC hydrogel to facilitate their self-organization. The hydrogel network generated via Schiff base reaction had been reported to successfully encapsulate DFO [ 49 ] . During in vitro culture, NSCs were more inclined to form neurospheres with DFO + exo addition compared with DFO addition alone (Fig. 6 A). NSCs also exhibited significantly higher viability in DFO + exo microfibers (98.89 ± 0.26% vs 70.46 ± 9.51%, Fig. 6 B). Moreover, ucMSC-exos strengthened NSC proliferative activity as evaluated by Edu + cells (35.55 ± 5.36% vs 11.20 ± 4.37%, Fig. 6 C). These results indicated that ucMSC-exos could mitigate the negative effects of DFO in 3D hydrogel networks as well. In in vivo experiments, microfibers were transplanted into 3mm hemisection defects in mice (Fig. 7 A). At week 4 post surgery, DFO + exo group exhibited better spinal tissue integration with fewer hydrogel residual compared with NSC group (Fig. 7 B). Meanwhile, at lesion site, DFO + exo and NSC groups both exhibited matured neuron and myelin sheath formation (MAP2+/MBP+). All groups exhibited oligodendrocyte formation (Olig2+) (Fig. 7 C). These regeneration effects of NSCs and exosomes have been extensively reported [ 7 ],[ 10 ][ 11 ],[ 18 ] and further confirmed by WB results here (Fig. 7 E). On the other hand, the spinal tissues were extracted at day 7 (week 1) post injury to evaluate the inflammation level and formation of M2 microglia. Except for DFO + exo group, both NSC and blank group exhibited intensive expression of inflammation cytokine TNF-α at lesion site. While at day 28, inflammation level decreased in NSC and blank group, which is consistent with our previous report [ 19 ] . In addition, at day 28, both DFO + exo and NSC groups exhibited vascular formation at lesion site (CD31+). As for blank group, while CD31 expression was detectable, no distinct ring-like structures were observed (Fig. 7 D). The WB results further confirmed the downregulation of pro-inflammatory cytokine IL-6 and upregulation of anti-inflammatory cytokine IL-10 in DFO + exo group (Fig. 7 E). Therefore, these results collectively indicated the neuroregenerative and angiogenic effects, particularly the anti-inflammatory effect of the NSC microfibers with ucMSC-exos and DFO. 3.5 NSC microfibers with ucMSC-exos and DFO alleviated ferroptosis by facilitating histone lactylation of M2 microglia in vivo DMT1 expression could be induced by TNF-α as previously reported [ 50 ] . In this research, lowered expression of DMT1 was observed in DFO + exo group at day 7 post injury, which was highly correlated with the expression of TNF-α as previously mentioned. At day 28, DMT1 expressed scatteredly at lesion site for the three groups. Meanwhile, FTH expression increased at day 28 in DFO + exo group (Fig. 8 A). Furthermore, 4HNE + regions were observed in NSC and blank groups at day 7 while not in DFO + exo group. However, at day 28, scattered 4HNE + regions were observed in all three groups while 4HNE expression level was still lower in DFO + exo group. There were also regions where GPX4 strongly expressed in DFO + exo group at day 28 (Fig. 8 B and 8 D). Since 4HNE is pro-ferroptotic and GPX4 is anti-ferroptotic, it is considered that ferroptosis was alleviated through DFO + exo treatment, especially during the initial days. This finding is consistent with previous findings that variations of ferroptosis markers predominantly occurred within the initial 7 days [ 22 ] . On the other hand, higher level of lactylation modification (CD206 + Kla+) and histone lactylation (CD206 + H4K12la+) was observed in M2 microglia in DFO + exo group. The histone lactylation level in NSC group was higher than blank group, yet lower than DFO + exo group (Fig. 8 C and 8 D). Considering that M2 microglia is more sensitive to ferroptosis and the inhibitory effect of histone lactylation on this process, integrated with in vitro and in vivo evidence from this study, it is reasonable to conclude that this therapeutic approach suppresses M2 microglial ferroptosis by promoting histone lactylation. 3.6 NSC microfibers with ucMSC-exos and DFO promoted motor and sensory function recovery of SCI mice Figure 9 A showed the BBB scores for the three groups on their lesion side and DFO + exo scaffolds significantly enhanced the locomotor function. In addition, we performed swimming test at week 4 to evaluate the swimming performance of the lesion side, including the evaluation of hindlimb movement, forelimb dependency and body position [ 51 ] . DFO + exo group exhibited significantly improved functional recovery (Fig. 9 B). Furthermore, DFO + exo scaffolds significantly enhanced the sensory function of the lesion hindlimbs as measured by Von Frey test (Fig. 9 C). Figure 9 D presented 30min motion trails for the three groups. The moving distances and active time were calculated. The total distance and active time of DFO + exo group was slightly higher than the other two groups (Fig. 9 E). Although these differences did not reach statistical significance, we propose that this might be attributed to motor capacity of the unlesion limbs. Accordingly, footprint analysis indicated that DFO + exo group had better locomotor function recovery than the other two groups (Fig. 9 F). Finally, according to the TEM images, DFO + exo group had less disintegrated myelin sheathes (white arrow) and damaged mitochondria (black arrow) (Fig. 9 G), indicating the DFO + exo scaffolds’ effects on myelin preservation and ferroptosis inhibition. Ultimately, the restoration of motor and sensory functions can be attributed to the DFO + exo scaffolds’ inhibition of ferroptosis, which subsequently reduced neural apoptosis and preserved neural fibers. In the previous section, we demonstrated the ferroptosis-inhibition effects of the NSC microfibers with ucMSC-exos and DFO. Compared with reported DFO- or exosome-loaded SCI scaffolds, the DFO + exo + NSC scaffolds have more comprehensive effects to replenish, protect and nourish neural cells while reduce ferroptosis and regulate inflammation. However, since histone lactylation mediated by ucMSC-exos and DFO might serve as a principal mechanism here, further investigation is required to independently delineate the complete signaling pathways for ucMSC-exos and DFO that driving histone lactylation. 4. Conclusion In this study, we proposed a therapeutic strategy for transactional SCI that integrated NSC-loaded hydrogel microfibers with ucMSC-exos and DFO. ucMSC-exo and DFO treatment facilitated NSC proliferation and neuronal differentiation in vitro , while enhanced M2 polarization of microglia. Most importantly, ucMSC-exos and DFO reduced ferroptosis in M2 microglia by promoting histone lactylation. In this procedure, DFO eliminated accumulated iron and induced HIF-1α expression to generate more lactate. Meanwhile, DFO + exo treatment upregulated LDH expression. The ferroptosis inhibition effect was further confirmed in in vivo hemisection SCI models. As a result, lesion-site inflammation was alleviated by increased M2 microglia. Moreover, the implantation of DFO + exo + NSC microfibers could improve neural regeneration and tissue vascularization while ultimately improve better restoration of motor and sensory function. In summary, this study provided validation of the significance of inhibiting ferroptosis during SCI and proposed a feasible multifactorial approach for SCI treatment. Declarations Competing Interests The authors declare no competing interests. Funding This research is supported by National Key Research and Development Program of China (Grant No. 2023YFF1204200), National Natural Science Foundation of China (Grant No. 82501661), National Natural Science Foundation of China (Grant No. 82571686). Author Contribution J.Z, X.L, T.X, R.X., W.D.: Conceptualization; J.Z, C.X., H.X, L.G, X.L.: Methodology; J.Z, W.Q, X.L.: Formal analysis; C.X, Y.W., W.L., X.L.: Data curation; L.G, W.Q, Y.Y., C.X.: Validation; J.Z., H.X.,L.G.,W.Q., J.Z., X.L.: Investigation; Y.Z., B.L: Resources; X.L., R.X.: Funding acquisition; X.L.: Writing-original draft; J.Z, C.X, W.D, Q.L, T.X.: Writing-review & editing. All authors reviewed the manuscript. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References McDonald JW, Sadowsky C. Spinal-cord injury. Lancet. 2002;359(9304):417–25. 10.1016/S0140-6736(02)07603-1 . Thuret S, Moon LD, Gage FH. 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School of Medicine, University of Electronic Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Qidan","middleName":"","lastName":"Liu","suffix":""},{"id":582915959,"identity":"090db4e5-b8de-414c-90d1-1cd1b4a73652","order_by":12,"name":"Yi Zhang","email":"","orcid":"","institution":"Department of Research and Development, Huaqing Zhimei (Shenzhen) Biotechnology Co. 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Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Boxun","middleName":"","lastName":"Liu","suffix":""},{"id":582915961,"identity":"b8b9f16f-93e8-4de4-b390-0dcd9096b9e0","order_by":14,"name":"Weibei Dou","email":"","orcid":"","institution":"Department of Electronic Engineering, Beijing National Research Center for Information Science and Technology, Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Weibei","middleName":"","lastName":"Dou","suffix":""},{"id":582915962,"identity":"76d7e93c-b73b-4fa8-8e15-ca5488b1cdbe","order_by":15,"name":"Tao Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYDCCAxBKDojZiNXCDKaMYVokiNaS2EC0Fr7b5w9++Nh2OH3D8eZnDxh32NQR1CJ5LplZcsaZw7kbzhwzN2A8k0bYFoMzzAzSPBVALTcSzCQY2w4TpYX5N4/B4XSD+8+/AbX8J0oLG8iWBIMbPCBbDhDWInmG2cxyxpl0w5lncsokEtuSJRsIaeE7w/j4xsc2a3m+48e3SXxss+MnaAscKBwAEgnEqwcCeYIOGgWjYBSMghELAHSaPOLe0C2MAAAAAElFTkSuQmCC","orcid":"","institution":"Center for Bio-intelligent Manufacturing and Living Matter Bioprinting, Research Institute of Tsinghua University in Shenzhen, Tsinghua University","correspondingAuthor":true,"prefix":"","firstName":"Tao","middleName":"","lastName":"Xu","suffix":""},{"id":582915963,"identity":"3e0ef9c5-5749-4cbc-aef0-7211816976cf","order_by":16,"name":"Xinda Li","email":"","orcid":"","institution":"Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Xinda","middleName":"","lastName":"Li","suffix":""},{"id":582915964,"identity":"1ceac993-7746-497d-baa4-cf9cbd49db98","order_by":17,"name":"Ruxiang Xu","email":"","orcid":"","institution":"Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Ruxiang","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2026-01-05 12:23:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8521282/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8521282/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101703199,"identity":"cd024ecd-1138-42a9-88e0-c8718e13c1c0","added_by":"auto","created_at":"2026-02-02 18:40:11","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":206245,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic view of ferroptosis inhibition through histone lactylation by ucMSC-exos and DFO.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8521282/v1/53a6ebc1681e6ec9777011e9.jpeg"},{"id":101703215,"identity":"cbe44cb2-7186-4370-8c09-1c8d76710bf3","added_by":"auto","created_at":"2026-02-02 18:40:15","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":560634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ein vitro\u003c/em\u003e evaluation of NSC proliferation and differentiation. (A) schematic illustration of experiment design. (B) SEM image of ucMSC-exo (Scale bar: 100nm). (C) distribution of ucMSC-exo particle size. (D) CCK-8 profile of NSCs with ucMSC-exo addition. (E) CCK-8 profile of NSCs with DFO of different concentrations. (F) CCK-8 profile of NSCs with DFO and DFO+exo. (G) WB results and analysis of Tuj-1 and GFAP after NSC differentiation culture. (H) Immunostaining results of Oct4, Sox2 and Nestin before NSC differentiation culture and MAP2, Olig2, GFAP after differentiation culture with DFO, exo or DFO+exo (Scale bar: 100μm).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8521282/v1/5266fdd9aa44356bd6001220.jpeg"},{"id":101703232,"identity":"8c19bc52-c265-45d2-bb81-e101520ae5b4","added_by":"auto","created_at":"2026-02-02 18:40:18","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":443118,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ein vitro\u003c/em\u003e evaluation of iron and ROS in BV-2 microglia. (A) schematic illustration of experiment design. (B) Edu staining results of BV-2 cultured with DFO, exo or DFO+exo (Scale bar: 100μm). (C) FerroOrange staining results of BV-2 after AIC treatment (Scale bar: 100μm). (D) ROS and JC-1 staining results of BV-2 after LPS and Erastin treatment (Scale bar: 100μm). (E-G) Analysis of staining results from B, C and D.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8521282/v1/98ec6fa08096c83c0e1ffec0.jpeg"},{"id":101703214,"identity":"8e6415b4-6ce7-4a1e-9db0-11b6068637df","added_by":"auto","created_at":"2026-02-02 18:40:15","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":431289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ein vitro\u003c/em\u003e evaluation of microglial and ferroptosis markers of BV-2 microglia. (A) Immunostaining results of microglia and inflammatory markers iNOS, CD206, TNF-α and IL-10 (Scale bar: 100μm). (B) Immunostaining results of ferroptosis markers FTH, DMT1, ASCL4 and TOM20 (Scale bar: 100μm). (C) WB results of iNOS, Arg1, IL-10, FTH, DMT1, ACSL4 and TOM20.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8521282/v1/9a421c08fb2569e04bf0612d.jpeg"},{"id":101703226,"identity":"68bb51a8-145d-4b90-b211-8544738af5a3","added_by":"auto","created_at":"2026-02-02 18:40:17","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":492570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ein vitro\u003c/em\u003e evaluation of ferroptosis and histone lactylation markers of BV-2 microglia. (A) Immunostaining results of lactylation and ferroptosis markers LDH, HIF-1α, 4HNE and GPX4 (Scale bar: LDH and HIF-1α: 100μm, 4HNE and GPX4: 50μm). (B) Immunostaining results of M2 microglia and histone lactylation markers CD206, Kla and H4K12la (Scale bar: Kla/CD206 and H4K12la: 250μm, H4K12la/CD206: 100μm, 4HNE/H4K12la: 50μm). (C) WB results of 4HNE, LDH, GPX4, Kla and H4K12la..\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8521282/v1/1da3585aa1a53652df3427d7.jpeg"},{"id":101703198,"identity":"a83db8c7-4308-43b9-917b-85a17ee5500f","added_by":"auto","created_at":"2026-02-02 18:40:10","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":339444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ein vitro\u003c/em\u003e culture of NSC microfibers with ucMSC-exos and DFO. (A) schematic illustration of experiment design. (B) Microscope images of NSC microfibers on day 3. (C) Calcein-AM/PI results and analysis of NSCs (Scale bar: 100μm). (D) Edu results and analysis of NSCs (Scale bar: 100μm).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8521282/v1/7c1bef11668ab15de36722e0.jpeg"},{"id":101703225,"identity":"5d82db44-5327-4b9a-a5b9-1a32bd857f75","added_by":"auto","created_at":"2026-02-02 18:40:17","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":899374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ein vivo\u003c/em\u003e evaluation of neuro-regeneration and anti-inflammation effects of NSC microfibers with ucMSC-exos and DFO. (A) schematic illustration of experiment design. (B) HE and Nissl staining results of spinal tissue at week 4 post surgery (Scale bar: 1mm). (C) Immustaining results of neuro-regeneration markers Tuj-1, GFAP, MAP2, Olig2 and MBP (Scale bar: gross views: 500μm, detailed views: 20μm). (D) Immustaining results of inflammation and angiogenesis markers TNF-α and CD31 (Scale bar: gross views: 500μm, detailed views: 20μm). (E) WB results and analysis of Tuj-1, GFAP, MBP, IL-6 and IL-10.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8521282/v1/ec63dbcaa939795cd63e64d1.jpeg"},{"id":101703230,"identity":"eaa98169-57e1-457c-a92c-01b7753f00db","added_by":"auto","created_at":"2026-02-02 18:40:18","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1237274,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ein vivo \u003c/em\u003eevaluation of anti-ferroptosis effects of NSC microfibers with ucMSC-exos and DFO. (A) Immustaining results of ferroptosis markers DMT1 and FTH (Scale bar: gross views: 500μm, detailed views: 20μm). (B) Immustaining results of ferroptosis markers 4HNE and GPX4 (Scale bar: gross views: 500μm, detailed views: 20μm). (C) Immustaining results of microglial and lactylation markers CD206, H4K12la, Kla and LDH (Scale bar: gross views: 500μm, detailed views: 20μm) (E) WB results and analysis of DMT1, 4HNE, GPX4 and H4K12la.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8521282/v1/bd07028fedf7234bf1f61b01.jpeg"},{"id":101703234,"identity":"74745eae-22be-46da-8cb4-35cbcc31fc0d","added_by":"auto","created_at":"2026-02-02 18:40:18","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":729886,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of motor and sensory function recovery. (A) BBB score on week 4. (B) Swimming evaluation score. (C) Von Frey test result. (D-E) Open field locomotor trails and analysis. (F) Footprint analysis. (G) TEM images of the cross sections of spinal tissues on week 4 (Scale bar: 2μm).\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8521282/v1/7a407e87c465f74b73b8a548.jpeg"},{"id":102159769,"identity":"38d33373-d1e6-4d61-9eda-aca859326014","added_by":"auto","created_at":"2026-02-08 21:24:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6426483,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8521282/v1/3e6bb07a-69f1-482d-ba35-3a14e982d18f.pdf"},{"id":101703235,"identity":"161c9138-e3ae-4ec7-b3f1-0ee7b4704131","added_by":"auto","created_at":"2026-02-02 18:40:19","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19277405,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.zip","url":"https://assets-eu.researchsquare.com/files/rs-8521282/v1/4bd5ec6a176714fed4074e37.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exosome-deferoxamine loaded bioengineered neural stem cell microfibers inhibited ferroptosis after severe spinal cord injury via H4K12 lactylation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSpinal cord injury (SCI) repair still remains a medical challenge at current stage. SCI could cause substantial vessel damage, axon disruption and neuronal death during its progression, leaving glial scars and lesion cavities that impede axonal regrowth\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e][\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Since the emergence of tissue engineering, scaffold transplantation has been developed as a main strategy for SCI repair due to its functionalities of bridging the lesion gap, guiding axon regrowth, inhibiting glial scar formation and other effects of regulating local microenvironments\u003csup\u003e[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u0026minus;[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, scaffold transplantation could replenish neural cells for the damaged spinal cord by carrying stem cells and providing well-designed stem cell niches to facilitate their survival, migration and differentiation\u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u0026minus;[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The implanted stem cells could further promote neural regeneration by their paracrine effect or their participation in the formation of local neural circuits\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u0026minus;[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Moreover, the microglia is another critical factor in SCI repair which could regulate inflammation, tissue regeneration and scar formation\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Therapeutic strategies to reestablish microglia homeostasis after SCI include the use of metal ions, proteins, small molecule drugs or exosomes\u003csup\u003e[\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u0026minus;[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Among these strategies, exosome therapy is a promising way for central system repair since they could cross blood-brain barrier and precisely deliver therapeutic proteins or RNAs to target cells\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e][\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Apart from regulating microglia homeostasis, the transplantation of exosome-loaded scaffolds could also promote angiogenesis, protect neurons and enhance neuronal differentiation\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e][\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. However, as an emerging approach, the potential effects of exosomes in SCI treatment still require further investigation.\u003c/p\u003e \u003cp\u003eFerroptosis is an iron-dependent programmed cell death induced by the disruption of iron balance and the peroxidation of membrane lipid\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e][\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Ferroptosis could occur in SCI due to the hemorrhage-induced iron overload and subsequently induced reactive oxygen species (ROS) accumulation by Fenton reaction\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In SCI, ferroptosis could affect various types of neural cells, including but not limited to neurons, microglia, endothelial cells and astrocytes, thereby inhibiting neural regeneration\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Therefore, ferroptosis inhibition is considered beneficial to alleviate secondary injury, facilitate axon regrowth and regain motor function\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e][\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. There have been several strategies to eliminate iron or ROS to prevent cells from ferroptosis. Deferoxamine (DFO) is an iron chelator approved by Food and Drug Administration which could reduce iron accumulation\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e][\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Functional hydrogels are also proved effectual to scavenge ROS and thus inhibit membrane lipid peroxidation\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e][\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. In addition, exosome therapy has been extensively investigated and several exosome-mediated pathways that inhibit ferroptosis after SCI have been identified\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e][\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. More importantly, exosomes have also been demonstrated to modulate the polarization of M2 microglia, an anti-inflammatory phenotype which plays a pivotal role in regulating the local inflammatory microenvironment after SCI\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e][\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Since M2 microglia have been reported to be more sensitive to ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e][\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, investigating the protective effects of exosomes on M2 microglia is still of significant importance.\u003c/p\u003e \u003cp\u003eLactate is an energy source and metabolism byproduct inside human bodies. In recent years, it has been discovered that lactate could participate in the modification of histone lysine residues and thus directly stimulate gene transcription, which is called histone lactylation\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Hu et al have demonstrated that higher lactate level is beneficial to microglia proliferation, axon regrowth and motor function recovery after SCI due to the lactylation of histone H4 lysine 12 (H4K12) and triggering lactate/H4K12la/PD-1 signaling\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, PD-1, fully known as programmed death-1, has also been found to facilitate the polarization of M1 microglia toward the M2 phenotype\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. However, as reported, the lactate level after SCI peaks at day 7 and subsequently declines, resulting in decreased lactylation level\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Lactate administration could have advantageous effects on inhibiting ferroptosis after SCI\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. However, excessively high concentrations of lactate might induce mitophagy through alternative lactylation pathways, consequently leading to neuronal apoptosis\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. It is still necessary to explore therapeutic approaches with minimum side effects. In our previous research, an combinatorial therapy with coaxial bioprinted neural stem cell (NSC) \u0026ndash;laden hydrogel scaffolds and umbilical cord mesenchymal stem cell-derived exosomes (ucMSC-exos) was proposed for SCI defect repair\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Based on recent evidence demonstrating the anti-ferroptotic effects of exosomes on M2 microglia\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, we hypothesize that the combinatorial therapy may promote M2 polarization of microglia while concurrently inhibit M2 microglia ferroptosis, thereby enhancing the regenerative capacity of the NSC scaffolds.\u003c/p\u003e \u003cp\u003eIn this study, we combined NSC-loaded hydrogel microfibers with DFO and ucMSC-exos to propose a multifactorial SCI therapy focusing on inhibiting ferroptosis. The beneficial effects of ucMSC-exos and DFO such as iron elimination and ROS scavenging were demonstrated \u003cem\u003ein vitro\u003c/em\u003e. Moreover, this combinatorial approach was further confirmed to increase lactate level and lactate dehydrogenase (LDH) expression while finally enhanced histone lactylation. Based on previous findings that ucMSC-exos could facilitate M2 polarization of microglia\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, we further demonstrated elevated histone lactylation level (H4K12la) and decreased ferroptosis in M2 microglia after treated with ucMSC-exos and DFO. This ferroptosis inhibition effect could be attributed to H4K12 lactylation according to a recent report\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Finally, this finding was also verified in \u003cem\u003ein vivo\u003c/em\u003e experiments.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell isolation and cell culture\u003c/h2\u003e \u003cp\u003ePrimary NSCs were isolated from the cortex of fetal ICR mice as previously reported\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Pregnant mice were sacrificed by cervical dislocation at day 13 or 15 to obtain the fetuses. The obtained fetal cortex tissue was immersed in 5 mL DPBS with 2.5 mL 0.25% Trypsin-EDTA and 100\u0026micro;L DNase (Solarbio) at 37℃ for 10 min. The supernatant was removed afterwards and Dulbecco\u0026rsquo;s Modified Eagle Medium/F12 (DMEM/F12, Gibco) with 10% FBS was added to neutralize the solution. The dissociated cell suspension was further processed with 40\u0026micro;m filters and centrifuged at 1000r/min to finally get the NSC pellets. NSC pellets were resuspended and maintained in DMEM/F12 with 2% B27, 1% N2, 1% Glutamine, 20ng/mL bFGF, 20ng/mL EGF and 1% penicillin-streptomycin at 37 ℃, 5% CO\u003csub\u003e2\u003c/sub\u003e. NSCs were passaged every 3 days. Furthermore, the BV-2 microglia (Pricella, CL-0493) were cultured in DMEM with 10% serum and 1% penicillin-streptomycin at 37℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. ucMSC-exo acquisition and identification\u003c/h2\u003e \u003cp\u003eucMSC-exos were purchased from Huamei BioTech Co. Ltd (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.hmcell.com\" target=\"_blank\"\u003ewww.hmcell.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.hmcell.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, ZA0010). The average size and zeta potential were analyzed on nanoparticle tracking analyzer (Zetaview) with laser scattering microscopy. Briefly, before analyzation, ucMSC-exos were suspended in PBS solution (pH\u0026thinsp;=\u0026thinsp;7.0) at a concentration of 3.9 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e/mL. The analyzation was conducted at room temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Biocompatibility evaluation of DFO and ucMSC-exos on primary NSCs and BV-2\u003c/h2\u003e \u003cp\u003eFor evaluation of NSC viability with ucMSC-exo addition, 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e primary NSCs were incubated for 24h with 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e ucMSC-exos. Cell Counting Kit-8 (CCK-8, Life-iLab, AC11L053/AC11L054) was used to detect the proliferative activity. Furthermore, for evaluation of NSC viability with DFO addition, primary NSCs were incubated with DFO (TargetMol, T124358) at 6 different concentrations: 0, 10, 20, 50, 100, 200 \u0026micro;g/mL. At the DFO concentration of 100\u0026micro;g/mL, a separate group with 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e/mL ucMSC-exo addition was established to evaluate the combined effects of ucMSC-exos and DFO on the viability of primary NSCs. CCK-8 assay was performed to detect the viability of each group. Finally, for evaluation of ucMSC-exo and DFO addition on BV-2, 4 experiment groups were established: BV-2, BV-2 with 100\u0026micro;g/mL DFO, BV-2 with 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e ucMSC-exos, BV-2 with 100ug/mL DFO and 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e ucMSC-exos. Each group was incubated for 24h. BeyoClick\u0026trade; EdU-555 (C0075S) was used to detect the proliferation activity of BV-2 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Ammonium iron citrate (AIC) treatment of BV-2 and intracellular iron content detection\u003c/h2\u003e \u003cp\u003eBV-2 microglia were treated with AIC (Sigma, F5879) to induce intracellular iron accumulation. Briefly, AIC treatment were conducted at 9 different concentrations: 0, 100, 200, 300, 400, 500, 600, 700, 800\u0026micro;M. 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e BV-2 microglia were incubated with AIC at corresponding concentrations for 3h. Moreover, ucMSC-exos and DFO were added to investigate their capacity for iron content clearance. 5 experiment groups were established: untreated BV-2 (control), AIC-treated BV-2 (AIC), AIC-treated BV-2 with DFO addition (DFO), AIC-treated BV-2 with ucMSC-exo addition (exo), AIC-treated BV-2 with DFO and ucMSC-exo addition (DFO\u0026thinsp;+\u0026thinsp;exo). Meanwhile, for the DFO\u0026thinsp;+\u0026thinsp;exo group, DFO and ucMSC-exos were pre-incubated at 37℃ for 2 h to facilitate DFO loading into the ucMSC-exos. DFO and ucMSC-exo treatments were administered for 1h following AIC treatment. FerroOrange (Dojindo, F374) was used to detect intracellular iron content for each group according to the manufacturer\u0026rsquo;s instruction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Erastin and LPS treatment of BV-2 and \u003cem\u003ein vitro\u003c/em\u003e BV-2 ferroptosis evaluation\u003c/h2\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e4\u003c/sup\u003e BV-2 microglia were treated with 10\u0026micro;M erastin and 1\u0026micro;g/mL lipopolysaccharide (LPS, Sigma, L4391) to induce ferroptosis and BV-2 polarization. For investigation of the effects of DFO and ucMSC-exos on ferroptosis regulation and glial polarization in BV-2, 5 experiment groups were established: untreated BV-2 (control), Erastin-LPS treated BV-2 (E-LPS), Erastin-LPS treated BV-2 with DFO addition (DFO), Erastin-LPS treated BV-2 with ucMSC-exo addition (exo), Erastin-LPS treated BV-2 with ucMSC-exo and DFO addition (DFO\u0026thinsp;+\u0026thinsp;exo). Each group were incubated for 1 day before evaluation. JC-1 (TargetMol, T15609) fluorescence staining was used to detect the mitochondrial function. Red/green fluorescence indicates normal/impaired mitochondrial function, respectively. ROS assay kit (Solarbio, CA1410) was used to detect ROS accumulation. All experiments were performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. OHA synthesis and hydrogel preparation\u003c/h2\u003e \u003cp\u003eOHA was synthesized by oxidation of hyaluronic acid (HA, Freda) with sodium periodate as previously reported\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Briefly, the reaction product was dialysed and lyophilized to obtain the final solid OHA. CMC and OHA were separately dissolved in PBS at 2%. Rheologic characterization of OHA-CMC blend was reported in our previous report. For core-shell microfiber fabrication, CMC and OHA solutions were mixed in equal volume to form the hydrogel precursor. Digested NSCs were gently resuspended in the hydrogel precursor at 5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e/mL to generate the final bioink for the core portion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Fabrication of core-shell NSC microfibers with ucMSC-exo and DFO loading\u003c/h2\u003e \u003cp\u003eCore-shell microfibers were fabricated as previously reported\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Briefly, 5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e primary NSCs were harvested and gently resuspended in the 2% OHA-CMC hydrogel precursor containing 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e ucMSC-exo and 100\u0026micro;g/mL DFO as bioink for the core portion. 3% sodium alginate solution was used as bioink for the shell portion. Both bioinks were loaded in 2 separate 10 mL syringes, which were subsequently fixed on 2 seperate channels of a microsyringe pump and connected to the core and shell inlets of a coaxial nozzle. The outlet of the nozzle was immersed in 3% calcium chloride bath.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Western blot\u003c/h2\u003e \u003cp\u003eWestern blot (WB) assay was performed to quantify the protein expression level in every relevant section. All WB assays in this research were performed as follows. For \u003cem\u003ein vitro\u003c/em\u003e WB, cells were harvested, grinded and lysed with cell lysis buffer (Life-iLab, AP01L013) on ice. The supernatant was collected after centrifuging at 10000 r/min for 20 min. Total protein concentration was quantified with BCA Protein Quantification Kit (Vazyme) following the manufacturer\u0026rsquo;s instructions. The proteins were transferred to PVDF membranes by electrophoresis and blocked with 5% skim milk for 1 h. Membranes were incubated with primary antibodies at 4℃ overnight. Primary antibodies used in this experiment include Tuj-1 (Proteintech, 66375), GFAP (Proteintech, 16825), Olig2 (Proteintech, 13999), Bax (Proteintech, 50599), Bcl2 (Affinty, AF6139), CD206 (Proteintech, 18704), Arg1 (Proteintech, 66129), iNOS (Invitrogen, AB2572890), IL-6 (Affinity, DF6087), TNF-α (Affinity, AF7014), IL-10 (ABclonal, A12255), LDH (Affinity, DF6280), Hif (Proteintech, 20960), Kla (PTMBIO, PTM-1401RM), H4K12la (PTMBIO, PTM-1411), 4-Hydroxynonenal (Invitrogen, MA5-27570), GPX4 (Proteintech, 67763), FTH (Affinity, DF6278), DMT1 (Proteintech, 20507), xCT (Affinity, DF12509), ACSL4 (abcam, AB155282), Tom20 (Proteintech, 11802), HRP-GAPDH (Beyotime, AF2823), HRP-β-tubulin (Beyotime, AF2839). On the next day, membranes were washed with TBST and incubated with corresponding secondary antibodies (Proteintech, SA00001, SA00002, SA00001-5) for 1 h. Immune bands were visualized with BeyoECL Star Kit (Beyotime, P0018AM) on imaging instrument.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Animal model\u003c/h2\u003e \u003cp\u003eAll experimental methods performed in this experiment were performed in accordance with the approved guidelines and regulations by the Medical Ethical Committee and the Inspection of the Sichuan Provincial People\u0026rsquo;s Hospital (No.2021179). 24 8-week-old ICR Kunming mice were used to establish spinal cord hemisection models. Briefly, animals were anesthetized by isoflurane inhalation and immobilized on the operating table. T9-T11 laminectomy was performed after vertebrae exposure. A 3mm \u0026ndash;long unilateral hemisection of spinal cord was completely removed. 3 separate experimental groups were established: NSC microfiber implantation with DFO and ucMSC-exos, NSC microfiber implantation alone and an untreated group (n\u0026thinsp;=\u0026thinsp;8). All transplantations were performed immediately following spinal cord resection. After implantation, the musculature and skin were carefully sutured and the animals were kept in clean cages. Bladder evacuation was performed during the initial 2 weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Locomotor and sensory function assessment\u003c/h2\u003e \u003cp\u003eOpen field tests were performed at weeks 1, 2, 3, and 4 post surgery to evaluate locomotor function recovery in animal models. Briefly, each animal was placed in an open field for 3 min and the behavior was recorded by a video camera. The locomotion was scored by 2 independent observers according to Basso-Beattie-Bresnahan (BBB) scale. The swimming performances of experimental animals were also evaluated with Louisville swimming score. Meanwhile, on week 4, a 30 min open-field behavior was recorded and analyzed by VisuTrack software to calculate the total movement distance and the average speed. Furthermore, footprint analysis was performed on week 4 to evaluate the locomotor function of the injured hindlimb. Briefly, the left and right paws for animal hindlimbs were stained with blue and red inks, respectively. The animals were allowed to walk through a rectangular narrow box lined with white paper. The footprints were collected and analyzed. Meanwhile, Von Frey test was performed at week 2 and 5 post surgery. Briefly, the animals were habituated to mesh platform and stimulated with Von Frey filaments. The withdrawal thresholds were recorded and analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Histological analysis\u003c/h2\u003e \u003cp\u003eOn week 4 post surgery, the spinal cord samples were isolated by heart perfusion. Briefly, the animals were administrated with overdose anesthesia and perfused with PBS through the left ventricle. Subsequently, 4% paraformaldehyde was perfused to fix the spinal cord tissues. The isolated samples were kept in 4% paraformaldehyde for 24h at 4℃ and immersed in 30% sucrose solution for another 3 days. Hematoxylin-eosin (HE) staining and Nissl staining were performed for the visualization of peripheral tissues, neurons, and neurites. For HE staining, briefly, the paraffin sections were dewaxed with xylene solution and a series of ethanol solutions. The sections were first stained with hematoxylin solution, followed by treatment with acid alcohol solution. Afterwards, the sections were stained with eosin solution, dehydrated, transparentized and mounted. For Nissl staining, the sections were stained with 1% Toluidine blue solution at 56℃ for 20 min, treated with acid alcohol solution, dehydrated, transparentized and mounted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Immunostaining\u003c/h2\u003e \u003cp\u003eImmunostaining was performed to further visualize the cell phenotypes at lesion sites. Briefly, the sections were dewaxed, dehydrated, treated with 0.5% Triton and blocking solution successively. The sections were then incubated with primary antibodies at 4 ℃ overnight. On the next day, the sections were washed with DPBS and incubated with secondary antibodies for 2 h. The antibodies used in this experiment were from the same commercial sources as specified in section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e2.8\u003c/span\u003e except for MAP2 (Invitrogen, PA5-85755), MBP (Proteintech, 66003) and CD31 (Proteintech, 28083). Additionally, after staining with relevant antibodies, antifade mounting medium containing DAPI was added on the surface before microscopic observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Transmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eTEM was performed to assess mitochondrial activity and myelination in spinal cord tissues. Briefly, before observation, the obtained spinal cord samples were fixed, dehydrated, infiltrated, embedded in Epon-812. The embedded samples were then sliced into ultra-thin sections of 60-90nm thickness with a slicer. Sections with abundant myelin sheaths were selected under an optical microscope and then transferred to a copper mesh for staining. TEM observation was conducted on a transmission electron microscope (JEM-1400FLASH, Japan Electronics Corporation, JEOL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data was presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). One-way analysis of variance (ANOVA) with Bonferroni post-hoc test was used to compare results across multiple groups. Student\u0026rsquo;s t-test was used to compare results between separate groups. Statistical significance was considered as *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1. ucMSC-exos and DFO affected NSC proliferation and differentiation \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe promotive effects of ucMSC-exos on NSC proliferation and differentiation have already been reported\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. On the other hand, it has long been reported that DFO could inhibit neural cell proliferation depending on its concentration\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. It is necessary to investigate the combinatorial effects of ucMSC-exos and DFO on NSCs. The experimental design is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. ucMSC-exos used in this experiment exhibited typical cup-shaped morphology and the particle sizes were 118.7-132.8nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Initially, the primary NSCs exhibited an 1.5-fold increase in proliferative activity after 24h ucMSC-exo treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Meanwhile, if treated with varying concentrations of DFO (0-200\u0026micro;g/mL), NSCs would exhibit significant reduction in proliferative activity at 100\u0026micro;g/mL and above (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). While treated with 100\u0026micro;g/mL DFO and ucMSC-exos simultaneously, the NSC proliferative activity increased again, significantly higher compared with the group treated with DFO alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results suggested ucMSC-exos could counteract the inhibitory effect of DFO on NSC proliferative activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, the effects of ucMSC-exos and DFO on NSC differentiation were examined. The NSCs were cultured in differential medium for 7 days following treatment of ucMSC-exos, DFO, or their combination. The WB results indicate that all three treatments brought about significant promotion in early neuron marker Tuj-1 expression and no statistical significance was observed among the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). The promotive effects of DFO on neuron differentiation has been reported\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. However, there was no further improvement of neuronal differentiation observed with combined treatment. In addition, immunostaining provided further evidence including NSC identification (Oct4+/Sox2+/Nestin+), neuron location in neurospheres, and scattered NSC differentiation into oligodendrocytes (Olig2+) and astrocytes (GFAP+) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). In summary, ucMSC-exos could mitigate the negative effects of high-concentration DFO on NSC proliferative activity, while both components could promote neuronal differentiation. Therefore, the combined application of ucMSC-exos and DFO, along with NSC scaffold transplantation, is a feasible strategy for SCI treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 ucMSC-exos and DFO reduced iron overload and ROS accumulation in microglia \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe regulation effects of exosomes on microglia polarization to M2 phenotype have been extensively reported\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e],[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e][\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. However, as previously mentioned, M2 microglia are highly sensitive to ferroptosis while M1 microglia being resistant\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Since the anti-inflammatory function of M2 microglia plays an important role in SCI repair, \u003cem\u003ein vitro\u003c/em\u003e experiments were preformed to validate the synergistic effect of ucMSC-exos and DFO in inhibiting microglia ferroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). First, BV-2 microglia also exhibited a reduction in proliferative activity while treated with 100\u0026micro;g/mL DFO as examined with Edu assay. The addition of ucMSC-exos could partially counteracted the inhibitory effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Next, since iron overload is the initial cause of ferroptosis by triggering ROS accumulation\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e, the BV-2 microglia were treated with AIC to induce iron accumulation. Consistent with previously reported\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, DFO significantly alleviated iron overload in microglia. Moreover, we further found that ucMSC-exos failed to lower iron level, whereas DFO\u0026thinsp;+\u0026thinsp;exo significantly enhanced iron clearance compared with DFO alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). This finding suggested ucMSC-exos might facilitate the cellular uptake of DFO.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt had been demonstrated in our previous report that ucMSC-exos could shift BV-2 from M1 to M2 phenotype under LPS treatment\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. In this research, BV-2 microglia were treated with LPS and ferroptosis inducer Erastin to simultaneously induce BV-2 polarization and ferroptosis. Following treatment, ROS assay was performed to evaluate the ROS removal effects of ucMSC-exos and DFO. The result indicated that both DFO and ucMSC-exos alone could significantly reduce ROS. DFO\u0026thinsp;+\u0026thinsp;exo group exhibited superior ROS scavenging capacity, while failed to show statistical significance compared with DFO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). JC-1 assay was performed to further evaluate mitochondrial function, a factor reflecting ferroptosis degree. The DFO\u0026thinsp;+\u0026thinsp;exo group also showed statistical significance compared with E-LPS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). In summary, ucMSC-exos and DFO could synergistically reduced ferroptosis of microglia. ucMSC-exos could enhance microglia proliferative activity. DFO, as an iron chelator, could eliminate iron overload while ucMSC-exos help DFO cellular uptake. Both components could facilitate ROS scavenging and finally reduce microglia ferroptosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 ucMSC-exos and DFO alleviated ferroptosis by facilitating histone lactylation of M2 microglia \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eAs presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, LPS successfully induced M1 polarization of BV-2 microglia which is marked by iNOS. Meanwhile, ucMSC-exos decreased the proportion of iNOS\u0026thinsp;+\u0026thinsp;cells and increased the proportion of CD206\u0026thinsp;+\u0026thinsp;or Arg1\u0026thinsp;+\u0026thinsp;cells, demonstrating the shift from M1 to M2 microglia. The increasing of M2 microglia further resulted in lowered inflammation level marked by the decreased expression of pro-inflammatory cytokine TNF-α and increased expression of anti-inflammatory cytokine IL-10. On the other hand, DFO has also been reported to reduce neuroinflammation by lowering the expression of pro-inflammatory cytokines\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Similar results were also observed in this experiment. DFO treatment alone induced upregulated expression of IL-10 and downregulated expression TNF-α. DFO\u0026thinsp;+\u0026thinsp;exo treatment further improved the beneficial effects compared with ucMSC-exo treatment alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expressions of ferroptosis-related factors were then evaluated. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, the ferritin marker FTH expression was decreased after Erastin treatment, indicating impaired iron-binding capacity of BV-2 microglia. DFO\u0026thinsp;+\u0026thinsp;exo treatment could restore FTH expression. DMT1 is a divalent metal transporter. DMT1 knockdown has been demonstrated to inhibit ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Erastin treatment promoted DMT1 expression. Compared with DFO or ucMSC-exo treatment alone, DFO\u0026thinsp;+\u0026thinsp;exo more effectively reduced DMT1 expression level. ACSL4 is a key enzyme in lipid metabolism facilitating ferroptosis. TOM20 is a mitochondrial membrane protein which is positive correlation with ferroptosis susceptibility. Following Erastin treatment, ACSL4 expression was upregulated while TOM20 expression was downregulated, while DFO\u0026thinsp;+\u0026thinsp;exo treatment effectively restored both proteins to baseline levels.\u003c/p\u003e \u003cp\u003eExogenous lactate treatment has been confirmed to stimulate histone lactylation level in microglia and thus facilitating SCI repair through lactate/H4K12la/PD-1 signaling pathway\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. In this research, it was first demonstrated that DFO\u0026thinsp;+\u0026thinsp;exo treatment elevated the level of lactate dehydrogenase (LDH), which could catalyze pyruvate to lactate\u0026zwnj; conversion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Meanwhile, the addition of DFO initiated the expression of HIF-1α which had been reported to promote lactate production\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Therefore, DFO\u0026thinsp;+\u0026thinsp;exo treatment successfully lowered the level of 4HNE, a product of lipid peroxidation, and elevated the level of GPX4, which plays a central role in ferroptosis inhibition to directly remove lipid ROS\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). On the other hand, ucMSC-exo treatment promoted the polarization of M2 microglia (CD206+) while DFO\u0026thinsp;+\u0026thinsp;exo treatment elevated the level of lactylation modification in M2 microglia (CD206\u0026thinsp;+\u0026thinsp;Kla+) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Finally, DFO\u0026thinsp;+\u0026thinsp;exo treatment promoted the histone lactylation in M2 microglia (CD206\u0026thinsp;+\u0026thinsp;H4K12la+) and led to ferroptosis inhibition (4HNE\u0026thinsp;+\u0026thinsp;H4K12la+) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, we first demonstrated that ucMSC-exo treatment facilitated BV-2 polarization to M2 phenotype and alleviated inflammation. Next, DFO\u0026thinsp;+\u0026thinsp;exo treatment could reduce ferroptosis with related markers variation. After that, we found that DFO\u0026thinsp;+\u0026thinsp;exo treatment elevated LDH level to facilitate pyruvate to lactate conversion. Meanwhile, DFO could induce HIF-1α upregulation to further promote lactate production. With abundant lactate, histone lactylation occurred in M2 microglia to inhibit their ferroptosis with upregulation of GPX4. Therefore, DFO\u0026thinsp;+\u0026thinsp;exo treatment could effectively inhibit ferroptosis in M2 microglia \u003cem\u003ein vitro\u003c/em\u003e by histone lactylation.\u003c/p\u003e \u003cp\u003e3.4 NSC microfiber transplantation in SCI model with ucMSC-exos and DFO to promote neural regeneration and inflammation alleviation \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e \u003cp\u003eDFO\u0026thinsp;+\u0026thinsp;exo scaffolds had been reported to treat SCI by diminish inflammation and neuronal ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In this research, we combined ucMSC-exos and DFO with NSC-loaded hydrogel microfibers for SCI treatment. NSCs play a key role in neural regeneration after SCI to reestablish local neural circuits\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u0026minus;[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Moreover, we have previously demonstrated the neurotrophic and neuroprotective effects of ucMSC-exos on NSCs\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Based on these beneficial effects, we further added DFO, aiming at suppressing ferroptosis in both neurons and M2 microglia after SCI for better therapeutic effects.\u003c/p\u003e \u003cp\u003eThe NSC microfibers were fabricated in a previously reported manner\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Abundant NSCs were embedded in dynamic crosslinking OHA-CMC hydrogel to facilitate their self-organization. The hydrogel network generated via Schiff base reaction had been reported to successfully encapsulate DFO\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. During \u003cem\u003ein vitro\u003c/em\u003e culture, NSCs were more inclined to form neurospheres with DFO\u0026thinsp;+\u0026thinsp;exo addition compared with DFO addition alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). NSCs also exhibited significantly higher viability in DFO\u0026thinsp;+\u0026thinsp;exo microfibers (98.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26% vs 70.46\u0026thinsp;\u0026plusmn;\u0026thinsp;9.51%, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Moreover, ucMSC-exos strengthened NSC proliferative activity as evaluated by Edu\u0026thinsp;+\u0026thinsp;cells (35.55\u0026thinsp;\u0026plusmn;\u0026thinsp;5.36% vs 11.20\u0026thinsp;\u0026plusmn;\u0026thinsp;4.37%, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). These results indicated that ucMSC-exos could mitigate the negative effects of DFO in 3D hydrogel networks as well.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn \u003cem\u003ein vivo\u003c/em\u003e experiments, microfibers were transplanted into 3mm hemisection defects in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). At week 4 post surgery, DFO\u0026thinsp;+\u0026thinsp;exo group exhibited better spinal tissue integration with fewer hydrogel residual compared with NSC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Meanwhile, at lesion site, DFO\u0026thinsp;+\u0026thinsp;exo and NSC groups both exhibited matured neuron and myelin sheath formation (MAP2+/MBP+). All groups exhibited oligodendrocyte formation (Olig2+) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These regeneration effects of NSCs and exosomes have been extensively reported\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e],[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e][\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e],[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e and further confirmed by WB results here (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). On the other hand, the spinal tissues were extracted at day 7 (week 1) post injury to evaluate the inflammation level and formation of M2 microglia. Except for DFO\u0026thinsp;+\u0026thinsp;exo group, both NSC and blank group exhibited intensive expression of inflammation cytokine TNF-α at lesion site. While at day 28, inflammation level decreased in NSC and blank group, which is consistent with our previous report\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. In addition, at day 28, both DFO\u0026thinsp;+\u0026thinsp;exo and NSC groups exhibited vascular formation at lesion site (CD31+). As for blank group, while CD31 expression was detectable, no distinct ring-like structures were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The WB results further confirmed the downregulation of pro-inflammatory cytokine IL-6 and upregulation of anti-inflammatory cytokine IL-10 in DFO\u0026thinsp;+\u0026thinsp;exo group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Therefore, these results collectively indicated the neuroregenerative and angiogenic effects, particularly the anti-inflammatory effect of the NSC microfibers with ucMSC-exos and DFO.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.5 NSC microfibers with ucMSC-exos and DFO alleviated ferroptosis by facilitating histone lactylation of M2 microglia \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e \u003cp\u003eDMT1 expression could be induced by TNF-α as previously reported\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. In this research, lowered expression of DMT1 was observed in DFO\u0026thinsp;+\u0026thinsp;exo group at day 7 post injury, which was highly correlated with the expression of TNF-α as previously mentioned. At day 28, DMT1 expressed scatteredly at lesion site for the three groups. Meanwhile, FTH expression increased at day 28 in DFO\u0026thinsp;+\u0026thinsp;exo group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Furthermore, 4HNE\u0026thinsp;+\u0026thinsp;regions were observed in NSC and blank groups at day 7 while not in DFO\u0026thinsp;+\u0026thinsp;exo group. However, at day 28, scattered 4HNE\u0026thinsp;+\u0026thinsp;regions were observed in all three groups while 4HNE expression level was still lower in DFO\u0026thinsp;+\u0026thinsp;exo group. There were also regions where GPX4 strongly expressed in DFO\u0026thinsp;+\u0026thinsp;exo group at day 28 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Since 4HNE is pro-ferroptotic and GPX4 is anti-ferroptotic, it is considered that ferroptosis was alleviated through DFO\u0026thinsp;+\u0026thinsp;exo treatment, especially during the initial days. This finding is consistent with previous findings that variations of ferroptosis markers predominantly occurred within the initial 7 days\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. On the other hand, higher level of lactylation modification (CD206\u0026thinsp;+\u0026thinsp;Kla+) and histone lactylation (CD206\u0026thinsp;+\u0026thinsp;H4K12la+) was observed in M2 microglia in DFO\u0026thinsp;+\u0026thinsp;exo group. The histone lactylation level in NSC group was higher than blank group, yet lower than DFO\u0026thinsp;+\u0026thinsp;exo group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Considering that M2 microglia is more sensitive to ferroptosis and the inhibitory effect of histone lactylation on this process, integrated with \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e evidence from this study, it is reasonable to conclude that this therapeutic approach suppresses M2 microglial ferroptosis by promoting histone lactylation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6 NSC microfibers with ucMSC-exos and DFO promoted motor and sensory function recovery of SCI mice\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA showed the BBB scores for the three groups on their lesion side and DFO\u0026thinsp;+\u0026thinsp;exo scaffolds significantly enhanced the locomotor function. In addition, we performed swimming test at week 4 to evaluate the swimming performance of the lesion side, including the evaluation of hindlimb movement, forelimb dependency and body position\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. DFO\u0026thinsp;+\u0026thinsp;exo group exhibited significantly improved functional recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Furthermore, DFO\u0026thinsp;+\u0026thinsp;exo scaffolds significantly enhanced the sensory function of the lesion hindlimbs as measured by Von Frey test (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD presented 30min motion trails for the three groups. The moving distances and active time were calculated. The total distance and active time of DFO\u0026thinsp;+\u0026thinsp;exo group was slightly higher than the other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). Although these differences did not reach statistical significance, we propose that this might be attributed to motor capacity of the unlesion limbs. Accordingly, footprint analysis indicated that DFO\u0026thinsp;+\u0026thinsp;exo group had better locomotor function recovery than the other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF). Finally, according to the TEM images, DFO\u0026thinsp;+\u0026thinsp;exo group had less disintegrated myelin sheathes (white arrow) and damaged mitochondria (black arrow) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG), indicating the DFO\u0026thinsp;+\u0026thinsp;exo scaffolds\u0026rsquo; effects on myelin preservation and ferroptosis inhibition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUltimately, the restoration of motor and sensory functions can be attributed to the DFO\u0026thinsp;+\u0026thinsp;exo scaffolds\u0026rsquo; inhibition of ferroptosis, which subsequently reduced neural apoptosis and preserved neural fibers. In the previous section, we demonstrated the ferroptosis-inhibition effects of the NSC microfibers with ucMSC-exos and DFO. Compared with reported DFO- or exosome-loaded SCI scaffolds, the DFO\u0026thinsp;+\u0026thinsp;exo\u0026thinsp;+\u0026thinsp;NSC scaffolds have more comprehensive effects to replenish, protect and nourish neural cells while reduce ferroptosis and regulate inflammation. However, since histone lactylation mediated by ucMSC-exos and DFO might serve as a principal mechanism here, further investigation is required to independently delineate the complete signaling pathways for ucMSC-exos and DFO that driving histone lactylation.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, we proposed a therapeutic strategy for transactional SCI that integrated NSC-loaded hydrogel microfibers with ucMSC-exos and DFO. ucMSC-exo and DFO treatment facilitated NSC proliferation and neuronal differentiation \u003cem\u003ein vitro\u003c/em\u003e, while enhanced M2 polarization of microglia. Most importantly, ucMSC-exos and DFO reduced ferroptosis in M2 microglia by promoting histone lactylation. In this procedure, DFO eliminated accumulated iron and induced HIF-1α expression to generate more lactate. Meanwhile, DFO\u0026thinsp;+\u0026thinsp;exo treatment upregulated LDH expression. The ferroptosis inhibition effect was further confirmed in \u003cem\u003ein vivo\u003c/em\u003e hemisection SCI models. As a result, lesion-site inflammation was alleviated by increased M2 microglia. Moreover, the implantation of DFO\u0026thinsp;+\u0026thinsp;exo\u0026thinsp;+\u0026thinsp;NSC microfibers could improve neural regeneration and tissue vascularization while ultimately improve better restoration of motor and sensory function. In summary, this study provided validation of the significance of inhibiting ferroptosis during SCI and proposed a feasible multifactorial approach for SCI treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research is supported by National Key Research and Development Program of China (Grant No. 2023YFF1204200), National Natural Science Foundation of China (Grant No. 82501661), National Natural Science Foundation of China (Grant No. 82571686).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.Z, X.L, T.X, R.X., W.D.: Conceptualization; J.Z, C.X., H.X, L.G, X.L.: Methodology; J.Z, W.Q, X.L.: Formal analysis; C.X, Y.W., W.L., X.L.: Data curation; L.G, W.Q, Y.Y., C.X.: Validation; J.Z., H.X.,L.G.,W.Q., J.Z., X.L.: Investigation; Y.Z., B.L: Resources; X.L., R.X.: Funding acquisition; X.L.: Writing-original draft; J.Z, C.X, W.D, Q.L, T.X.: Writing-review \u0026amp; editing. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcDonald JW, Sadowsky C. Spinal-cord injury. Lancet. 2002;359(9304):417\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0140-6736(02)07603-1\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(02)07603-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThuret S, Moon LD, Gage FH. Therapeutic interventions after spinal cord injury. 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Acta Neurochir (Wien). 2018;160(3):449\u0026ndash;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00701-017-3389-4\u003c/span\u003e\u003cspan address=\"10.1007/s00701-017-3389-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\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, ferroptosis, histone lactylation, exosomes, deferoxamine","lastPublishedDoi":"10.21203/rs.3.rs-8521282/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8521282/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFerroptosis occurs after spinal cord injury (SCI), leading to neuronal loss and impairment of neural regeneration. Therefore, combining the beneficial effects of existing neural stem cell (NSC) scaffolds with ferroptosis inhibition strategies might lead to better therapeutic outcomes. This study proposes a combinatorial approach integrating NSC microfibers, umbilical cord mesenchymal stem cell-derived exosomes (ucMSC-exos), and deferoxamine (DFO) for SCI repair. \u003cem\u003eIn vitro\u003c/em\u003e experiments demonstrated that the combined application of ucMSC-exos and DFO successfully inhibited ferroptosis by clearing iron ions and reactive oxygen species (ROS). Furthermore, ucMSC-exos and DFO promoted cellular lactylation by increasing lactate and lactate dehydrogenase (LDH) levels. These changes were found to occur in M2 microglia triggered by ucMSC-exos, suggesting that ucMSC-exo-DFO treatment may suppress ferroptosis by enhancing histone lactylation in M2 microglia. Finally, a 3 mm mouse spinal hemisection model was used to validate the \u003cem\u003ein vivo\u003c/em\u003e regenerative effects of the NSC microfibers with ucMSC-exos and DFO. The scaffold successfully inhibited ferroptosis, while additionally suppressed inflammation, promoted neurogenesis and angiogenesis, and ultimately restored motor and sensory functions. In summary, this study elucidated a potential mechanism for ferroptosis inhibition in SCI and provided a promising therapeutic strategy for SCI repair.\u003c/p\u003e","manuscriptTitle":"Exosome-deferoxamine loaded bioengineered neural stem cell microfibers inhibited ferroptosis after severe spinal cord injury via H4K12 lactylation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 18:40:00","doi":"10.21203/rs.3.rs-8521282/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":"afdc9fa3-6338-4ab5-a6c9-98681e0fe655","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-08T21:24:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-02 18:40:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8521282","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8521282","identity":"rs-8521282","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}