Application of trehalose in preparation of medicine for treating spinal cord injury

Abstract

The invention relates to the technical field of nervous system injury repair, in particular to application of trehalose in preparation of a medicine for treating spinal cord injury. The invention proves that trehalose enhances the phagocytosis of macrophages on myelin fragments in vitro, and the optimal concentration is 20mM; it is proved that trehalose promotes treatment of myelin fragments in foam-like macrophages in vitro, and the optimal concentration is 20 mM; it is proved that trehalose promotes lipid phagocytosis and lipid treatment of foam-like macrophages after spinal cord injury; the trehalose treatment is proved to reduce inflammatory response and fiber scar formation after spinal cord injury; the trehalose treatment is proved to promote neuronal survival, axon regeneration and motor function recovery after spinal cord injury; the trehalose treatment is proved to enhance the autolysosome function of macrophages and the expression of TFEB after spinal cord injury; it is proved that trehalose treatment promotes removal of myelin fragments by enhancing autophagy.

Description

Technical Field

[0001] The invention relates to the technical field of nervous system injury repair, and in particular to an application of trehalose in the preparation of a medicine for treating spinal cord injury. Background Art

[0002] Spinal cord injury (SCI) is a severe central nervous system (CNS) injury characterized by extensive neuronal cell death and demyelination1,2. Accumulation of myelin debris at the injury site impedes axonal remyelination and growth cone formation3,4,5. Early infiltration of the lesion by blood-borne macrophages is essential for phagocytosis of this debris6,7. However, excessive myelin uptake leads to the formation of foamy macrophages8,9, which exhibit diminished migratory and phagocytic abilities, reduced lipid efflux, and increased pro-inflammatory cytokine production10,11. These foamy macrophages contribute to chronic inflammation, fibrotic scar formation, and impaired axonal regeneration, thereby impeding motor function recovery12,13. Therefore, enhancing myelin debris clearance and reducing foamy macrophage formation are critical for improving recovery outcomes after SCI14.

[0003] Autophagy is an important cellular process for clearing intracellular waste and metabolically accumulated lipids, etc.15,16 and plays an important role in cell health. In atherosclerosis, disrupted macrophage autophagy impairs lipophagy and lysosome-mediated cholesterol efflux, leading to inflammasome overactivation and accelerated plaque formation. Conversely, enhancing autophagy can counteract these adverse effects17-19. Trehalose is a naturally occurring non-reducing disaccharide that is known to induce autophagy20-22 and has been shown to improve neurodegenerative outcomes in mouse models by clearing abnormally aggregated proteins23,24. Transcription factor EB (TFEB) is a key player in the autophagy-lysosome system and regulates this process by increasing the expression of autophagy- and lysosome-related genes25-27. In models of atherosclerosis and motor neuron degeneration, trehalose has been shown to promote autophagy by inducing TFEB overexpression28,29. However, the effects of trehalose on macrophage TFEB expression, the autophagy-lysosome system, and recovery from spinal cord injury are still unclear. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention can be achieved through the following technical solutions:

[0005] Application of trehalose in the preparation of drugs for treating spinal cord injury.

[0006] Preferably, the drug for treating spinal cord injury is an injection.

[0007] Preferably, the concentration of trehalose in the injection is 20 mmol / L.

[0008] A medicine for treating spinal cord injury, comprising trehalose.

[0009] Application of trehalose as an autophagy activator in the treatment of spinal cord injury.

[0010] Beneficial effects of the present invention:

[0011] 1. The present invention proves that trehalose enhances the phagocytosis of myelin fragments by macrophages in vitro, and the optimal concentration is 20 mM.

[0012] 2. The present invention demonstrates that trehalose promotes the processing of myelin fragments in foamy macrophages in vitro, with the optimal concentration being 20 mM.

[0013] 3. The present invention demonstrates that trehalose promotes lipid phagocytosis and lipid processing of foamy macrophages after spinal cord injury.

[0014] 4. The present invention demonstrates that trehalose treatment reduces inflammatory response and fibrous scar formation after spinal cord injury.

[0015] 5. The present invention demonstrates that trehalose treatment promotes neuronal survival, axonal regeneration and motor function recovery after spinal cord injury.

[0016] 6. The present invention demonstrates that trehalose treatment enhances the autophagy-lysosome function of macrophages and the expression of TFEB after spinal cord injury.

[0017] 7. The present invention demonstrates that trehalose treatment promotes the clearance of myelin fragments by enhancing autophagy.

[0018] Trehalose can enhance the clearance of myelin fragments after spinal cord injury and reduce the formation of foam macrophages by inducing the expression of TFEB in macrophages and regulating the autophagy-lysosome system. Trehalose can promote axonal regeneration and motor function recovery by alleviating secondary inflammatory response, reducing fibrotic scars and injury area; therefore, trehalose has a positive effect in treating spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1Trehalose of the present invention promotes macrophages to phagocytize myelin fragments and foam macrophages in vitro to excrete myelin fragments; wherein: (a) immunofluorescence staining marks macrophages (CD68+, red); (b) myelin fragments are marked with ORO staining (red), and nucleus pulposus is marked with hematoxylin. Yellow, blue and purple arrows represent typical macrophages, and myelin fragments account for 1 / 3, 1 / 3 to 2 / 3 and more than 2 / 3 of the cytoplasm, respectively. The scales of a and b are both 40 μm; (c) and (d) quantitative analysis of the percentage of myelin-carrying macrophages in macrophages cultured with different concentrations of trehalose. The content of Dio-myelin debris in macrophages was divided into three levels: +, ++ and ++. Dio-myelin debris accounted for 1 / 3, 1 / 3 to 2 / 3 and more than 2 / 3 of the cytoplasm, respectively. Statistical analysis was performed to compare the percentage of cells with more than 1 / 3 of dio-myelin debris in the cytoplasm of macrophages in different trehalose treatment groups (i.e., ++ and +++ groups). Compared with the 0 mM group, *p<0.05, ***p<0.001. (e) Immunofluorescence staining of macrophages (CD68+, red); scale, 100 μm; myelin debris was marked with ORO staining (red), and nucleus pulposus was marked with hematoxylin; scale, 40 μm. (g and h) Quantitative analysis of the percentage of foamy macrophages containing myelin in different trehalose treatment groups. Statistical analysis was performed to compare the percentage of cells with more than 1 / 3 of dio-myelin debris in the cytoplasm of foamy macrophages in different trehalose treatment groups (i.e., ++ and +++ groups). *p<0.05, ****p<0.0001, ***p<0.001 compared with 0 mM group. The results are expressed as mean ± SEM. **p<0.01, one-way ANOVA showed no statistically significant difference. Co-24h in (a) and (b) indicates that RAW 264.7 cells were incubated with 0.5 mg / ml myelin fragments and different concentrations of trehalose for 24 hours. (e) and (f) indicate that cells were incubated in medium containing 0.5 mg / ml myelin fragments (green) for 24 hours and then treated with different concentrations of trehalose for 24 hours.

[0021] Figure 2Trehalose in the present invention promotes lipid phagocytosis and clearance of myelin debris after spinal cord injury; wherein: (a) intraperitoneal injection time and behavioral assessment; (b) representative images of dMBP (red) and F4 / 80 (green) immunofluorescence staining at 7, 14 and 28 dpi in the PBS and trehalose treatment groups. Scale bar: 40 μm (low magnification), 20 μm (high magnification); (c) Quantification of the proportion of dMBP+ macrophages in the PBS and trehalose treatment groups at 7, 14 and 28 dpi. Two-way ANOVA ****p<0.0001; (d) representative images of dMBP (red) and GFAP (green) immunofluorescence staining at 7, 14 and 28 dpi. Scale bar: 200 μm (low magnification), 20 μm (high magnification); (e) quantitative analysis of dMBP+ areas in the injured area of ​​PBS and trehalose treatment groups at various time points after spinal cord injury. ns, not significant. **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA. n=3 animals in (c) and (e). Asterisks indicate the lesion center. Regions of interest (ROI) indicate the boxed areas on the left.

[0022] Figure 3Trehalose in the present invention can inhibit the inflammatory response after spinal cord injury, reduce the area of ​​injury, and inhibit the formation of fibrotic scars; wherein: (a) Representative images of ORO staining of the lesion area in the PBS and trehalose treatment groups at 14 and 28 dpi; (b) Quantitative analysis of ORO+ area in the PBS and trehalose treatment groups at 14 and 28 dpi. *p<0.05, ****p<0.0001; (c) Representative images of GFAP (red) and CD68 (green) at 14 and 28 dpi; (d) Quantitative analysis of CD68+ area in the PBS and trehalose treatment groups at 14 and 28 dpi. *p<0.05, ****p<0.0001; (e) Immunofluorescence staining of PDGFRβ (red) and GFAP (green) in the sagittal plane of the PBS and trehalose treatment groups at 28 dpi. ROI represents the boxed area on the left, showing the fibrotic / astrocytic scar boundary; (f) and (g) Quantitative analysis of PDGFRβ+ and GFAAParea in PBS and trehalose-treated groups at 28 dpi. **p<0.01 by student t test. (h) Representative images of fibronectin (green) and GFAP (red) at 28 dpi. Representative images of laminin (red) and PDGFRβ (green) at 28 dpi. (i) and (j) Quantitative analysis of fibronectin+ and laminin+ areas in PBS and trehalose-treated groups at 28 dpi. ***p<0.001 by student t test. n=3 animals in (b), (d), (f), (g), (i), and (j). Asterisks indicate the center of the lesion. Scale bars: 200 μm (low power images), 20 μm (high power images).

[0023] Figure 4Trehalose in the present invention promotes axon regeneration and motor function recovery after spinal cord injury in mice. Wherein: (a) Representative images of NFH (red) and GFAP (green) immunofluorescence staining at 28dpi. ROI represents the left framed area, showing detailed immunostaining of NFH+ neurofilaments and GFAP. The arrow points to the typical NFH+ neurofilaments in the center of the lesion; (b) Quantitative analysis of the proportion of NFH+ neurofilament area to the dotted line enclosed area, and the area of ​​150μm on both sides of the epicenter of the lesion is separated by dotted lines. ****p<0.0001 by Student's t test; (c) Representative images of 5-HT (red) and GFAP (green) immunofluorescence staining at 28dpi. The square area on the left represents the rostral side and the injury center, showing detailed 5-HT immunostaining. The arrow points to a typical 5HT+ axon. R and C represent the rostral and caudal sides of the spinal cord, respectively; (d) Quantitative analysis of the area of ​​5-HT+ axons in the rostral side or the injury center. ns, not statistically significant. ****p<0.0001 by Student's t-test; (e) Representative images of immunofluorescence staining of NeuN (red) and PDGFRβ (green) at 28 dpi; (f) Quantification of the average density of NeuN+ cells in the dotted enclosed areas (Z1-Z3) of the two groups. ****p<0.0001 by Student's t-test; (g) Motor function was assessed by BMS at 0, 3, 7, 14, 21 and 28 dpi. **p<0.01, **p<0.001, ****p<0.0001 (PBS and trehalose groups at 14, 21, 28 dpi) by two-way ANOVA; (h) Representative images of foot print analysis at 28 dpi in the uninjured, PBS and trehalose treated groups. The hind paw is represented by red dye and the forepaw is represented by green dye; (i)-(k) Quantification of stride length, stride width and paw rotation at 28 dpi. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; n=3 animals in (b), (d) and (f), n=5 animals in (g), (i), (j) and (k). Asterisks indicate the injury center of the lesion. Scale bars: 200 μm (low power images), 40 μm (high power images).

[0024] Figure 5Trehalose in the present invention enhances the autophagy lysosome function of macrophages after spinal cord injury in mice and promotes TFEB expression; wherein (a) representative images of immunofluorescence staining of LC3B (red) and F4 / 80 (green) in the lesion area at 7 and 14 dpi. Mice were intraperitoneally injected with PBS and trehalose; (b) representative images of immunofluorescence staining of Lamp1 (red) and F4 / 80 (green) in the lesion area at 7 and 14 dpi; (c) representative images of immunofluorescence staining of P62 (red) and F4 / 80 (green) in the lesion area at 7 and 14 dpi; (d) representative images of immunofluorescence staining of TFEB (red) and F4 / 80 (green) in the lesion area at 7 and 14 dpi. The white arrow indicates that TFEB is located in the nucleus; (e) Quantification of the proportion of LC3B+ macrophages in a macrophage. After two-way ANOVA, *p<0.05, ***p<0.001; (f) Quantification of the proportion of Lamp1+ macrophages in macrophages. *p<0.05, **p<0.01 by two-way ANOVA; (g) Quantification of the proportion of P62+ macrophages in macrophages. *p<0.05, **p<0.01 by two-way ANOVA; (h) Quantification of the proportion of TFEB+ macrophages in macrophages. ***p<0.001, ****p<0.0001 by two-way ANOVA; (i) Quantification of the proportion of TFEB nuclear localization. *p<0.05, ****p<0.0001; n=3 animals in (e)-(i). All results are expressed as mean ± SEM. Scale bar: 20 μm.

[0025] Figure 6Trehalose in the present invention enhances the autophagy lysosome function of macrophages, promotes the expression of TFEB and the clearance of myelin fragments. Wherein (a) RAW 264.7 cells were incubated with myelin and 20mM trehalose for 24 hours. The control group used the same volume of PBS. Immunofluorescence was used to detect the intracellular LC3B and P62 levels (red), and RAW 264.7 cells were labeled with CD68 (green); (b) and (c) are the quantification of the average intensity of LC3B and P62 per cell in (a). ****p<0.0001 by student t test. ≥60 cells per group; (d) Immunofluorescence was used to detect the intracellular TFEB level (red), and RAW264.7 cells labeled with CD68 (green). The cell nucleus was labeled with DAPI (blue); (e) is the quantification of the localization of TFEB with nucleus in each cell in (d). ****p<0.0001 by student t test. ≥60 cells per group; (f) is the quantification of the average TFEB intensity per cell in (d). ***p<0.001 by Student's t-test. ≥60 cells per group; (g), (k), (i), (m) RAW 264.7 cells treated with 0.5 mg / mL Dio-myelin fragments and co-cultured with 20 mM trehalose or chloroquine for 24 h (g, k) or 24-24 h (im). Myelin fragments were labeled with ORO staining (red). (k) and (m) Immunofluorescence staining of RAW 264.7 cells (CD68+ red). Yellow, blue, and purple arrows indicate typical macrophages with myelin fragments occupying less than 1 / 3, between 1 / 3 and 2 / 3, and more than 2 / 3 of the cytoplasm. (h), (l), (j), (n) Statistical analysis of the proportion of cells with more than 1 / 3 of Dio-positive myelin fragments in the cytoplasm of macrophages in different groups (i.e., ++ and +++ groups). Compared with the PBS group, **p<0.01, ***p<0.001. One-way ANOVA, **p<0.01, **p<0.001, ns, no statistically significant difference. All results are expressed as mean ± SEM. Scale bar: 40 μm. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example

[0028] Animals and cells

[0029] All procedures involving animals were approved by the Ethics Committee of Anhui Medical University (approval number: LLSC20211113) and issued on October 12, 2021. 8-week-old C57BL / 6 female mice (weight 20-25 g) were purchased from the Animal Experiment Center of Anhui Medical University and randomly placed in standard cages. The ambient temperature and humidity were controlled, and food and water were supplied ad libitum in a 12-h light / 12-h dark cycle. All experiments in this study were performed and reported in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

[0030] Mouse mononuclear macrophage leukemia cells (RAW264.7) were provided by the Stem Cell Bank of the Chinese Academy of Sciences and cultured in Dulbecco's modified Eagle's medium containing 1% glutamine, 1% sodium pyruvate and 10% fetal bovine serum. The cells were cultured at 5% CO2 and 37°C.

[0031] Establishment of spinal cord injury model in mice

[0032] Fasting (food and water) for 24 h before surgery. Mice were intraperitoneally injected with sodium pentobarbital (P-010; Sigma, St. Louis, MO, USA) at a dose of 50 mg / kg. The mice were placed in a supine position, their back hair was shaved, and the skin was disinfected. The T10 segment of the spinal cord was exposed by excising the lamina. The T10 spinal cord was completely clamped from both sides with a calibrated Dumont No. 5 forceps for 5 seconds, resulting in a moderate compressive spinal cord injury30,31. The incision was disinfected and sutured layer by layer. Routine anti-infection treatment and assisted urination care were given after spinal cord injury twice a day.

[0033] Trehalose treatment

[0034] In vivo, trehalose (T0176, SIGMA) was intraperitoneally injected 4 hours after surgery, dissolved in phosphate-buffered saline, at a dose of 3 g / kg / day. The treatment lasted for 14 consecutive days, and the control group was injected with an equal amount of PBS.

[0035] Four different concentrations of trehalose were prepared in vitro: 0mM, 10mM, 20mM and 30mM, and added to macrophages or foam macrophages containing 0.5mg / ml myelin. After determining the optimal concentration, 20mM was used for the next experiment. The control group was given an equal amount of PBS, the experimental group was given trehalose 20mM, and the inhibition group was given an equal amount of chloroquine at a concentration of 30mg / ml.

[0036] Tissue preparation

[0037] After the mice were anesthetized, the chest was opened to expose the heart and perfused with 0.1 M PBS plus 4% paraformaldehyde. The spinal cord tissue was taken 0.5 mm from the center of the lesion, immersed in 4% PFA for 4 hours, and then placed in 30% sucrose solution and dehydrated at 4°C until the tissue settled to the bottom. Subsequently, the tissue was cut into 16 μm thick serial sagittal sections using a cryostat at -20°C.

[0038] Immunofluorescence staining

[0039] In vivo, sagittal sections containing the lesion area were used. Sections were dried at 50°C for 1 hour, washed three times with PBS, and blocked for 1 hour at room temperature with blocking solution containing 5% donkey serum, 0.3% Triton X-100 in PBS. Next, sections were incubated overnight at 4°C with the following primary antibodies: goat anti-5-HT, rat anti-F4 / 80, rabbit anti-F4 / 80, rat anti-GFAP, rabbit anti-degraded phospholipid basic protein (dMBP), rabbit anti-NeuN, rat anti-LAMP1, rat anti-CD68, rabbit anti-fibronectin, rabbit anti-laminin, rabbit anti-neurofilament heavy polypeptide (NFH), goat anti-PDGFRβ, rabbit anti-GFAP, rabbit anti-LC3B, mouse anti-p62, rabbit anti-TFEB. The sections were washed four times with PBS and incubated with the following secondary antibodies for 1 hour at room temperature in the dark: donkey anti-rabbit Alexa Fluor 555, donkey anti-rabbit Alexa Fluor 488, donkey anti-mouse Alexa Fluor 488, donkey anti-goat Alexa Fluor 488, donkey anti-mouse Alexa Fluor 488, donkey anti-goat Alexa Fluor 555, and donkey anti-mouse Alexa Fluor 555. Finally, the sections were stained with 4′6-diamidino-2-phenylindole. Negative control sections were incubated with secondary antibodies only.

[0040] In vitro, cell culture slides were fixed with 4% PFA for 15 minutes, washed three times with PBS, and then blocked with 5% donkey serum albumin for 30 minutes. The primary antibody was incubated overnight at 4°C. Then it was washed three times with PBS and incubated with the secondary antibody for 1 hour in the dark at room temperature. Finally, the slides were mounted with DAPI, and representative images were obtained by fluorescence microscopy.

[0041] Preparation of Myelin and Dio-Myelin

[0042] Myelin fragments were extracted from 8-12 week old mice. After euthanasia, brain tissue was obtained and homogenized with 0.32M crystal sugar. The myelin fragment solution was purified by sucrose density gradient centrifugation using an ultra-high speed centrifuge. The prepared myelin fragment solution was then labeled with 3,3'-Dioctadecyloxcarbocyanine perchlorates and incubated at 37°C for 20 minutes. The final myelin was successfully prepared32.

[0043] Oil Red O (ORO) staining

[0044] ORO staining was performed using the Oil Red O staining kit. ORO staining detects myelin debris accumulated in the lesion area and RAW264.7 cells after spinal cord injury. Tissue experiments: Spinal cord sections were dried, ORO stained for 10 min, soaked in 60% isopropanol for 3 min, and washed with distilled water. Representative images were obtained under a fluorescence microscope. Cell experiments: Cell coverslips were washed with PBS, fixed with ORO fixative for 15 min, and dehydrated with 60% isopropanol for 4 min. The 60% isopropanol was removed and freshly prepared ORO staining solution was added for 10 min. The stain was removed and coverslips were washed 6 times with PBS until there was no excess. Mayer's hematoxylin staining solution was added for 1 min, the dye was removed, and coverslips were washed 6 times with PBS. Cell coverslips were soaked in ORO buffer for 1 min and then observed under a microscope33,34.

[0045] Image acquisition and quantitative analysis

[0046] Representative images were obtained by fluorescence microscopy comparison and analysis, ensuring consistent light intensity in all images. All quantitative analyses were performed in a blinded manner. Quantitative image analysis was performed using image J version 2.0. To assess the accumulation of myelin debris in foamy macrophages, these cells were divided into three levels: +, ++, and +++. These levels represent the proportion of lipid droplets occupying the cytoplasm <1 / 3, 1 / 3-2 / 3, and >2 / 3, respectively. The number of cells in each group was counted using a Zeiss microscope and ZEN imaging software (n ≥ 60 / group). The proportion of lipid droplets occupying the cytoplasm and cells 33-35 were used for statistical analysis. To quantify the accumulation of myelin debris in the lesion area after spinal cord injury, we calculated the ratio of dMBP-positive area to GFAP-negative area. The ratio of ORO-positive area to the observed field of view was used to quantify the formation of foamy macrophages after spinal cord injury.

[0047] Tissue recovery was assessed by calculating the ratio of the quantified GFAP-negative area to the observed field of view. The area of ​​inflammation was assessed by quantifying the immunoreactive area of ​​CD68, and the area of ​​fibrotic scar was assessed by quantifying the immunoreactive area of ​​PDGFRβ, laminin, and fibronectin. These measurements were normalized to 4 × sections across the lesion area. Axon regeneration was assessed by quantifying the area of ​​NFH+ neurofilaments in a 300 μm-long box-shaped area centered on the lesion center and calculating its ratio to the total area. Neuronal survival was assessed by quantifying the density of NeuN+ neurons in three 250 μm-long areas (Z1-Z3) in the center of the spinal cord lesion. To assess axon regeneration, the area of ​​5-HT+ axons in the epicenter and rostral to the epicenter was quantified. At least three animals were examined in each group of the above experiments, and the cross-section of the lesion area and two adjacent sections separated by 180 μm were selected for staining, and the average value was taken as 1 for quantification.

[0048] On the 40x images of the lesion area, the number of dMBP+ macrophages was counted on days 7, 14, and 28 to assess their myelin phagocytosis. LC3B+ and P62+ macrophages were counted to assess macrophage autophagy, and Lamp1+ macrophages were counted to assess lysosomal function. The final result for each sample was the average of 3 random 40x images, with at least 3 animals in each group.

[0049] Behavioral assessment

[0050] All behavioral assessments were performed in a double-blind manner to ensure the fairness of the results. The Basso Mouse Scale (BMS) is commonly used to assess the recovery of motor function in mice after spinal cord injury. Scores range from 0 (no ankle joint activity) to 9 (full recovery of function), and movement is scored based on hindlimb joint movement, toe clearance, trunk position and stability, paw position, step coordination, and tail position. BMS in this study was performed in an open field according to the protocol developed by Basso and colleagues. All mice underwent BMS assessment before spinal cord injury to confirm normal motor function and after surgery to verify successful spinal cord injury modeling. All mice were observed and evaluated by 3 independent researchers on days 0, 3, 7, 14, 21, and 28 after injury (5 mice per group), and the final results were averaged 37.

[0051] Footprint analysis was used to further evaluate gait recovery and motor coordination in mice at 28 dpi. Dyes of different colors were applied to the forepaws and hindpaws. Stride length was the distance from the starting point to the end point of the hindpaw in one gait cycle. Stride length was determined by the distance from the lateralmost toe of the left paw to the lateralmost toe of the right paw. Paw rotation was assessed by the angle between the axis of the body centerline and the axis of the hindpaw. All assessments included more than three consecutive gait cycles on each side, with an average of five animals per group. Uninjured mice were divided into the uninjured group38,39 for comparison. Footprints were digitized and representative images were used to evaluate coordination.

[0052] Statistical analysis

[0053] Each experiment was repeated independently in at least 3 animals, 3 sections were stained in each sample, and the data are expressed as mean ± standard deviation (SEM). One-way or two-way analysis of variance (ANOVA) was performed; a post hoc Tukey-Kramer test was performed to compare the differences between multiple groups. The Student's t test was used for comparison between two groups. GraphPadPrism 8.3.2 software was used for data analysis and graphics. Significance is reported as ns, no significant difference, *p<0.05, **p<0.01, ***p<0.001 or ****p<0.0001.

[0054] in conclusion:

[0055] Trehalose enhances the phagocytosis of myelin fragments by macrophages in vitro, with the optimal concentration being 20 mM

[0056] We first studied the effect of trehalose on the phagocytosis of myelin fragments by macrophages in vitro. It is known that the phagocytic ability of macrophages reaches saturation at 0.5 mg / ml of myelin fragments35. In order to study whether trehalose would further affect the phagocytosis of myelin fragments by macrophages that have already experienced phagocytic saturation, we incubated macrophages with different concentrations of trehalose and 0.5 mg / ml of dio-dye-labeled myelin fragments. Immunofluorescence staining results showed that the proportion of myelin fragments in macrophages increased with the increase of trehalose concentration, reaching a peak at 20 mM. Beyond this concentration, there was no significant change in phagocytic ability ( Figure 1 a, c). ORO staining confirmed these findings, showing that the proportion of neutral lipids (myelin degradation products) in macrophages increased up to 20 mM trehalose, with no further changes observed ( Figure 1 b, d). Trehalose enhances the phagocytosis of myelin fragments by macrophages, and the effect is optimal at 20 mm.

[0057] Trehalose promotes the processing of myelin fragments in foam macrophages in vitro, with an optimal concentration of 20 mM

[0058] Next, we investigated the effect of trehalose on foamy macrophages in vitro. Initially, the foamy macrophage model was established by incubating macrophages with 0.5 mg / ml dio-dye-labeled myelin fragments for 24 h. Subsequently, the effects of different concentrations of trehalose on these foamy macrophages were evaluated. Immunofluorescence analysis showed that myelin fragments in foamy macrophages gradually decreased with increasing trehalose levels and stabilized at 20 mM ( Figure 1 e, g). ORO staining confirmed these results. At a trehalose concentration of 20 mM, macrophages were able to effectively process myelin debris accumulated in vitro ( Figure 1 f, h).

[0059] Trehalose treatment enhances lipid phagocytosis by foamy macrophages after spinal cord injury and promotes lipid processing

[0060] Demyelination occurs after spinal cord injury, forming a large amount of myelin fragments and triggering a secondary inflammatory response. Macrophages, as primary phagocytes, play a key role in the phagocytosis of myelin fragments32,40. The transformation of macrophages that phagocytize myelin fragments into foamy macrophages can lead to tissue necrosis and chronic inflammation. Reducing the formation of foamy macrophages helps in the recovery of spinal cord injury41,42. Therefore, we studied the effect of trehalose on the clearance of myelin fragments by macrophages after spinal cord injury. Trehalose (3 g / kg / day) was injected intraperitoneally starting from 4 hours after spinal cord injury20 until the 14th day. The recovery status of each group after injury was analyzed ( Figure 2 a). Myelin basic protein was specifically labeled with dMBP, and GFAP-negative areas represent the outer cortex of the lesion 43. Macrophages were labeled with F4 / 80 44. At 7, 14, and 28 dpi, staining showed that F4 / 80+ macrophages in the trehalose-treated group showed greater colocalization with dMBP+ myelin basic protein compared to the control group ( Figure 2 b, c). The fluorescence results showed that the level of dMBP+ myelin basic protein in the lesion area of ​​the control group mice was significantly higher than that of the trehalose treatment group ( Figure 2 d, e). This suggests that trehalose enhances debris clearance or reduces the formation of foamy macrophages. Therefore, we explored the effect of trehalose treatment on foamy macrophage formation. The accumulation of foamy macrophages in the lesion area was observed by ORO staining33,34, where positive ORO staining indicated the presence of foamy macrophages42. The results showed that the ORO+ area in the trehalose-treated group at 14 and 28 dpi was significantly smaller than that in the control group ( Figure 3 a, b). This indicates that foamy macrophages are abundant and exist for a long time after spinal cord injury, and trehalose treatment can reduce the formation of foamy macrophages after spinal cord injury.

[0061] Trehalose treatment reduces inflammation and fibrotic scarring after spinal cord injury

[0062] Due to the presence of foamy macrophages, which cause tissue necrosis and chronic inflammation42, we focused on the inflammatory response during the chronic phase of SCI. Our results showed that at 14 and 28 dpi, the area of ​​CD68+ inflammatory cells in the trehalose-treated group was significantly reduced compared with that in the control group ( Figure 3 c, d). This reduction suggests that reducing the formation of foamy macrophages alleviates chronic inflammation after SCI, which is consistent with previous studies36. After SCI, perivascular fibroblasts proliferate, migrate, and deposit a large amount of extracellular matrix (ECM), including fibronectin and laminin. They eventually form a fibrous scar that envelops macrophages in the center of the lesion and is closely associated with astrocytes at the edge of the lesion, hindering axonal regeneration and functional recovery8,45. The inflammatory response promotes the formation of fibrotic scars after SCI and aggravates the injury36. Therefore, we used immunofluorescence to detect PDGFRβ, fibronectin, and laminin to evaluate changes in fibroblasts and fibrotic ECM, detected GFAP to evaluate changes in astrocytic scars46, and used the GFAP-negative area to represent lesion size1. At 28dpi, the trehalose-treated group showed an interweaving of fibrosis and astrocytic scars with unclear boundaries, while the control group showed dense, continuous fibrotic scars ( Figure 3 In addition, the fibrotic scar area, including PDGFRβ+ fibroblasts and fibrotic EMCs positively shown by fibronectin or laminin, was significantly reduced in the trehalose-treated group compared with the control group ( Figure 3 hj). It is widely accepted that the dense continuous border formed by excessive deposition of fibroblasts after spinal cord injury is one of the main reasons that hinder axon regeneration and functional recovery44,46. Therefore, our study suggests that trehalose treatment can alleviate chronic inflammation after spinal cord injury, effectively inhibit the formation of fibrotic scars, and promote the formation of astrocyte scars, thereby providing a good internal environment for the recovery of spinal cord injury.

[0063] Trehalose treatment enhances neuronal survival, axonal regeneration and motor function recovery after spinal cord injury

[0064] To verify the effect of trehalose on axon regeneration after spinal cord injury, we used immunofluorescence staining to evaluate NFH fibers and serotonergic 5HT axons. The results showed that the density of NFH fibers in the trehalose-treated group was significantly increased ( Figure 4 a, b). In addition, the 5-HT axon area in the trehalose-treated group increased significantly, and the 5-HT axons extended from the rostral side to the caudal side across the lesion area, while the 5-HT axons in the control group did not cross the lesion area ( Figure 4c, d)47. Immunofluorescence staining showed that the density of NeuN+ neurons in the lesion center and surrounding areas (Z1-Z3) in the trehalose-treated group was significantly higher than that in the control group (Figures e, f)48, indicating that trehalose enhanced the survival of NeuN+ neurons in the lesion center and promoted the regeneration of NFH fibers. These findings collectively indicate that trehalose treatment promotes axon regeneration and neuronal survival after spinal cord injury, probably due to the reduction of foamy macrophage formation and the inhibition of inflammatory and fibrotic scar formation. Enhanced axon regeneration and neuronal survival are generally associated with better recovery of motor function. Therefore, we used BMS scores and footprint analysis to further evaluate the recovery of motor function in mice. There was no statistically significant difference in BMS scores between the two groups before 7dpi. However, compared with the control group, the trehalose-treated group showed better recovery of hindlimb motor function and higher BMS scores at 14, 21, and 28dpi ( Figure 4 g). Footprint analysis at 28 dpi showed that the trehalose-treated group had better recovery of motor function, including narrower paw rotation, longer stride length and increased step frequency ( Figure 4 hk). These results indicate that trehalose significantly improves neuronal survival, axonal regeneration, and motor function recovery after spinal cord injury.

[0065] Trehalose treatment enhances autophagic lysosomal function and TFEB expression in macrophages after spinal cord injury

[0066] Previous studies have shown that trehalose inhibits the formation of foam macrophages, reduces inflammation, promotes axonal growth, and contributes to the recovery of motor function after injury. However, its role in promoting the degradation of phagocytic substances by macrophages is still unclear. In recent years, trehalose has been shown to induce autophagy in Alzheimer's disease and Huntington's disease23,49. As an important pathway for the degradation and metabolism of intracellular substances, autophagy contributes to the transfer of endogenous or exogenous cellular substances for degradation in lysosomes50. Therefore, we focused on the autophagy and lysosomal function of macrophages after trehalose treatment after spinal cord injury. In the control group, immunofluorescence results showed that in the first two weeks after spinal cord injury, P62+ autophagic substances20, 51 gradually accumulated, while LC3B+ autophagosomes20, 51 and Lamp1+ lysosomal-associated membrane proteins52 decreased, indicating that the autophagy-lysosomal function of macrophages in the lesion area gradually decreased over time ( Figure 5 ac, eg). Trehalose treatment increased the levels of autophagy-related protein LC3B and lysosome-related protein Lamp1, and decreased the expression of P62, indicating that trehalose enhanced the autophagy-lysosome function of macrophages after spinal cord injury, thereby promoting the degradation of intracellular substances ( Figure 5ac, eg). TFEB plays a key role in activating autophagy by regulating the expression of autophagy- and lysosome-related genes52. The results showed that compared with the control group, the number of TFEB+ cells in macrophages in the trehalose-treated group increased significantly, and the nuclear localization of TFEB was enhanced ( Figure 5 d,h,i). Consistent with this, further in vitro experiments showed that the autophagy and lysosome function of RAW 264.7 cells was enhanced after trehalose treatment ( Figure 6 bd,h), TFEB expression increased ( Figure 6 Therefore, trehalose can enhance the expression of TFEB, thereby activating the autophagy-lysosome function of macrophages after spinal cord injury.

[0067] Trehalose treatment may promote the clearance of myelin debris by enhancing autophagy

[0068] We have demonstrated that the presence of trehalose not only promotes macrophage phagocytosis but also enhances the degradation of engulfed materials by macrophages, resulting in a decrease in the formation of foamy macrophages. We hypothesized that this process may be attributed to the enhancement of cellular autophagy by trehalose, thereby promoting the engulfment of myelin fragments by macrophages and the production of neutral lipid droplets from foamy macrophages. To test this hypothesis, we used a lysosomal inhibitor, chloroquine (CQ), which inhibits the fusion of autophagosomes with lysosomes, thereby inhibiting autophagy53-55. Macrophages were cultured in vitro in a medium containing myelin fragments (0.5 mg / ml), trehalose (20 mM), and chloroquine (30 mg / ml) for 24 hours, and ORO staining was performed. The results showed that after autophagy was inhibited by chloroquine, the phagocytic ability of macrophages to myelin fragments was significantly reduced, similar to that of the PBS group ( Figure 6 i, m). Staining and quantification of myelin fragments and CD68+ macrophages labeled with Dio dye showed similar results ( Figure 6 j, n). It can be seen that trehalose may promote macrophages to phagocytize myelin fragments by enhancing autophagy. We further studied whether trehalose improves the ability of foam macrophages to excrete intracellular lipids by enhancing cell autophagy. Macrophages were incubated in a medium containing myelin fragments (0.5 mg / ml) for 24 hours, and then incubated in a medium containing trehalose (20 mM) with or without chloroquine (30 mg / ml) for 24 hours. ORO staining results showed that chloroquine eliminated the ability of trehalose to enhance the ability of foam macrophages to process intracellular lipids ( Figure 6 Immunofluorescence and subsequent quantitative analysis showed similar trends ( Figure 6 l, p). Therefore, we concluded that trehalose may promote the phagocytosis of myelin debris by macrophages and the expulsion of lipid droplets from foamy macrophages by enhancing autophagy.

[0069] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0070] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. Application of trehalose in the preparation of drugs for the treatment of spinal cord injury.

2. The use according to claim 1, characterized in that: The medicine for treating spinal cord injury is an injection.

3. The use according to claim 2, characterized in that: In the injection, the concentration of trehalose is 20 mmol / L.

4. A drug for treating spinal cord injury, characterized in that: The medicine contains trehalose.

5. Application of trehalose as an autophagy activator in the treatment of spinal cord injury.

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