Engineered Exosomes Derived From Primary M2 Macrophages With Anti-inammatory And Neuroprotective Properties For The Treatment of Spinal Cord Injury.

Background: Uncontrollable inammation and nerve cell apoptosis are the most destructive pathological response after spinal cord injury (SCI). So, inammation suppression combined with neuroprotection is one of the most promising strategies to treat SCI. Engineered exosomes with anti-inammatory and neuroprotective properties are promising candidates for the implementation of this strategies for the treatment of SCI. Results: By combining nerve growth factor (NGF) and curcumin (Cur), we prepared stable engineered exosomes of approximately 120 nm from primary M2 macrophages with anti-inammatory and neuroprotective properties (Cur@EXs -cl-NGF ). Notably, NGF was coupled with EXs by matrix metalloproteinase 9 (MMP9)-cleavable linker to accurately release at the injured site. Through targeted experiments, we found that these exosomes could actively and effectively accumulate at the injured site of SCI mice, which greatly improved the bioavailability of the drugs. Subsequently, Cur@EXs -cl-NGF reached the injured site and could effectively inhibit the uncontrollable inammatory response to protect the spinal cord from secondary damage; in addition, Cur@EXs -cl-NGF could release NGF into the microenvironment in time to exert a neuroprotective effect against nerve cell damage. Conclusions: A series of in vivo and in vitro experiments showed that the engineered exosomes signicantly improved the microenvironment after injury and promoted the recovery of motor function after SCI. We provide a new method for inammation suppression combined with neuroprotective strategies to treat SCI.

exosomes derived from M2 macrophages (EXs) can inherit diverse anti-in ammatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor (TGF-β), as well as various chemokine receptors from the macrophage surface, such as chemokine receptor type 2 (CCR2) [13]. More signi cantly, EXs have played a central role in the treatment of in ammatory diseases, such as atherosclerosis, skin damage and stroke [13,14]. Thus, EXs with both in ammation suppression and targeting functions are ideal drug carriers. Previous research by our team has con rmed that EXs carrying berberine can effectively reduce the in ammatory response after SCI to promote the recovery of motor function [15].
Nerve growth factors (NGF) can reduce neuronal apoptosis, promote angiogenesis, and promote axon extension and functional recovery [16,17]. After SCI, hypoxia-ischemia leads to a signi cant decrease in endogenous expression of NGF at the injury site, highlighting the importance of delivering exogenous NGF [18,19]. However, previous research has indicated that the biological half-life of NGF after intravenous injection is approximately 2.3 h, so poor bioavailability greatly limits the accumulation of NGF in target cells [20]. To prolong the circulation time of NGF in the body, we coupled NGF to the surface of EXs by the matrix metalloproteinase 9 (MMP9) cleavable linker (cl), which had a sequence of RVGLP, to prepare EXs − cl−NGF [21,22]. Considering that MMP9 expression is signi cantly increased at the injury site after SCI, NGF in EXs − cl−NGF can dissociate from EXs and be released into the microenvironment over time [23,24]. The NGF is then taken up by nerve cells through NGF receptor-mediated endocytosis, avoiding consumption by in ammatory cells [25].
Curcumin (Cur) is a natural polyphenol with excellent anti-in ammatory properties that promotes the polarization of M1 macrophages to M2 macrophages [26,27]. Simultaneously, a low solubility, poor stability, low absorption rate and short biological half-life lead to its low bioavailability, which limits its anti-in ammatory application in vivo [28,29]. To overcome these shortcomings, Cur was loaded into EXs − cl−NGF , and Cur@EXs − cl−NGF was prepared for the treatment of SCI.
In this work, the designed Cur@EXs − cl−NGF had both anti-in ammatory and neuroprotective properties.
The design concept and mechanism of action are shown in the schematic diagram below ( Figure. 1).
These effects were veri ed in vitro using cell experiments and in vivo using animal experiments. We hope that this design can provide new ideas for the clinical treatment of SCI.

Results
Preparation and characterization of EXs − cl−NGF .
After IL-4 (20 ng/mL) stimulation for 48 h, ow cytometry showed that approximately 85.03% of primary macrophages obtained from the abdominal cavity of mice had differentiated into M2 macrophages with anti-in ammatory effects ( Figure.  (NTA) showed that after modi cation with NGF, the size of EXs − cl−NGF was slightly increased compared with that of EXs, while the zeta potential was signi cantly reduced to 33.56 mV from 42.17 mV ( Figure. 2H). Western blotting was used to detect the expression of exosome marker proteins (CD9 and TSG101), chemokine receptors (CCR2) mediating the chemotaxis of exosomes to in ammatory lesions, and in ammation inhibitory cytokines (IL-10) in EXs and EXs − cl−NGF . We found that expression of the marker protein and functional protein of the modi ed EXs did not change signi cantly ( Figure. 2I). Based on ELISA kits, we detected the release ability of NGF from EXs − cl−NGF . After adding MMP9 to the PBS solution of EXs − cl−NGF , the NGF linked on the surface of EXs was effectively dissociated. The solution with MMP9 and the MMP9 inhibitor GM6001 together did not dissociate NGF, which showed that the cleavable linker was speci c to MMP9 ( Figure. 2J). Subsequently, the PC12 cells growth rate was determined with an MTT assay. Comparing the NGF released from EXs − cl−NGF with the free form of NGF, we found no signi cant difference between free NGF and NGF in EXs − cl−NGF in promoting the proliferation of PC12 cells. This result indicated that the binding and release of NGF did not damage the activity of NGF ( Figure. 2K). Finally, we tested the stability of EXs − cl−NGF , and the results showed that the size (Figure. The ability of EXs − cl−NGF to target sites of in ammation and release NGF.
To evaluate whether EXs − cl−NGF , as a carrier of NGF, could effectively deliver NGF to the injured site, we used a living imaging system to detect the in ammation targeting ability of EXs − cl−NGF in SCI animal models. After SCI, the mice were randomly divided into 3 groups, NGF, RVs − cl−NGF , or EXs − cl−NGF (NGF in each group was labeled with CY7 uorescent dye), and different nanoparticles were injected through the tail vein. A living imaging system showed that the free form of NGF hardly accumulated at the injury site, and RVs − cl−NGF could exist at the injury site for a shorter time owing to the passive targeting ability of the red blood cell membrane nanoparticles. However, EXs − cl−NGF exhibited the strongest uorescence intensity and the longest accumulation time at the injury site, demonstrating the excellent ability of EXs − cl−NGF to target in ammation ( Figure. 3A). Quantitative statistics of the uorescence image demonstrated that NGF delivered by EXs − cl−NGF accumulated at the injured site within 2 h after injection, reached a peak at approximately 12 h after injection and persisted for more than 72 h ( Figure. 3B). Fluorescence images of the main organs (heart, liver, spleen, lung and kidney) and spinal cord from different groups were collected 12 h after tail vein injection, further indicating the satisfactory targeting ability of EXs − cl−NGF for the injured spinal cord ( Figure. 3C). Statistical analysis showed that the uorescence intensity of EXs − cl−NGF was approximately eight times that of free NGF in the spinal cord, while the kidneys of mice injected with free NGF had obvious uorescence accumulation ( Figure. 3D). An NGF ELISA kit was used to detect the biological half-life of NGF in the blood of each group. The results showed that the circulation time in the body of NGF in EXs − cl−NGF was signi cantly prolonged due to the long-term circulating ability of EXs ( Figure. 3E). Next, we constructed the Transwell™ coculture system to verify whether the cleavable linker contributed to the uptake of NGF by nerve cells. The upper layer of the Transwell™ coculture system consisted of PC12 cells, and the lower layer was primary M1 macrophages. Then, we added free NGF, EXs − NGF , or EXs − cl−NGF (NGF in each group was labeled with FITC) to the Transwell™ coculture system. The schematic diagram is shown ( Figure.

In vitro in ammation regulation and neuroprotective capabilities of EXs -cl-NGF
To test the anti-in ammatory and neuroprotective capabilities of EXs − cl−NGF , we conducted Transwell™ coculture tests. Brie y, primary macrophages were induced to differentiate into M1 macrophages with LPS for 24 h and then cultured in the lower layer of the system. After H 2 O 2 pretreatment for 12 h to simulate oxidative stress after SCI, PC12 cells were cultured in the upper layer of the system. After the addition of different nanoparticles to the system for 12 h, we evaluated the survival rate of PC12 cells and the degree of in ammation inhibition in each group. Flow cytometry (Figure. conditions. The ow cytometry results showed that under the action of EXs − cl−NGF , the apoptotic rate of PC12 cells decreased from 41.53-12.74%. The statistical results of live/dead cell staining further con rmed the neuroprotective effect of EXs − cl−NGF . Notably, compared with the PBS group, the survival rate of PC12 cells in the EXs group was also signi cantly improved, which suggested that inhibiting the in ammatory response in the system might be bene cial to the survival of injured nerve cells. Simultaneously, based on ELISA kits, we detected the expression of proin ammatory factors (TNF-α, IL-1β, and IL-6) and anti-in ammatory factors (TGF-β) in the supernatants ( Figure. 4C). As expected, in the EXs, EXs − NGF , and EXs − cl−NGF groups under the action of EXs, the expression of proin ammatory factors (TNF-α, IL-1β, and IL-6) was signi cantly reduced, and the expression of anti-in ammatory factors (TGFβ) was obviously increased. Flow cytometry detection of the macrophage phenotype showed that the proportion of M1 macrophage subpopulations (CD86+) dropped to 49.79% from 91.16% in the EXs − cl−NGF group, suggesting that LPS induction was signi cantly blunted ( Figure. 4D). Moreover, the proportion of M2 macrophage subpopulations (CD206+) rose to 10.27% from 1.59% ( Figure. 4E). It was rather remarkable that the EXs group and EXs − NGF group also had obvious in ammation inhibitory effects under the action of EXs.
Cur@EXs − cl−NGF showed better anti-in ammatory effects via Cur delivery.
As a small molecule drug, although Cur has excellent anti-in ammatory and antioxidant functions, its poor solubility and low bioavailability limit its effectiveness in vivo [27]. Therefore, we opted for Cur as a  Cur@EXs − cl−NGF promotes the functional recovery of mice with SCI.
To explore the therapeutic effect of Cur@EXs − cl−NGF , mice with SCI were randomly divided into 5 groups: (1) Sham, (2) Normal saline (NS), (3) Cur, (4) EXs − cl−NGF , and (5) Cur@EXs − cl−NGF . Since the in ammatory reaction occurs immediately and increases signi cantly within 3-6 h after the injury [30,31], we started administering treatment within 2-3 h after SCI. We selected 2 h, 2 d, 4 d, and 6 d after injury as the administration time, and the mice were sacri ced at 28 d after injury to evaluate the recovery status of each group. First, we assessed the ultrastructure of myelin sheaths of mice that received different treatments by TEM (Figure. 6A). The photo showed that the myelin sheaths in the sham group were arranged in a tight and order manner, the thickness was uniform, and the layered structure was clear. The ultrastructure of myelin sheaths in the mice treated with NS was the most severely damaged. However, after Cur treatment, the amelioration of myelin sheaths was very limited. After EXs − cl−NGF and Cur@EXs − cl−NGF treatments, the numbers, diameters and thickness of the myelin sheaths were signi cantly improved. Speci cally, the protective effect of Cur@EXs − cl−NGF on myelin sheaths was superior. The spinal cord from each group of mice was subjected to H&E staining to further investigate the lesion changes at the injury site ( Figure. 6B). The results showed that compared with the NS treatment group, the spinal cord in the Cur@EXs − cl−NGF treatment group was signi cantly more complete, its lesions were signi cantly reduced, and pathological changes were signi cantly improved. Moreover, both the number of surviving neuronal axon tubulin and the severity of scars caused by astrocytes were closely related to the recovery of motor function in injured mice. Therefore, we observed differences in the expression of GFAP and β3-Tubulin in each group of mice at 28 d after injury by immuno uorescence staining (Figure. 6C). The statistical analysis of β3-Tubulin uorescence showed that the number of surviving microtubule axon proteins in the EXs − cl−NGF and Cur@EXs − cl−NGF groups was signi cantly higher than that in the NS groups and Cur groups; in particular, the Cur@EXs − cl−NGF group had the strongest expression of β3-Tubulin ( Figure. 6D). The uorescence statistics of GFAP showed that scar formation in the EXs − cl−NGF and Cur@EXs − cl−NGF groups was signi cantly less than that in the NS and Cur groups ( Figure. 6D). These results further con rmed the excellent therapeutic effect of Cur@EXs − cl−NGF on mice with SCI. Finally, we evaluated the effects of different treatments on motor function recovery through behavioral analysis. Footprint behavioral assays showed that in the sham group with a steady stride, the hind foot prints were clearly on the ground, the forefoot stride length was the longest and the stride width was the narrowest (Figure. 6E). In contrast, the footprints of the NS group were the poorest, the hind feet showed a drag gait, the forefoot stride length was signi cantly shortened and the stride width was signi cantly increased. Moreover, the gait of the hind foot and the stride length and width of the forefoot showed the most obvious improvement in the Cur@EXs − cl−NGF group ( Figure. 6F). In addition, the BMS scores at multiple time points after injury in each group of mice further con rmed that the Cur@EXs − cl−NGF group had an excellent therapeutic effect on spinal cord injury ( Figure. 6G).
The role of Cur@EXs − cl−NGF in the early stages of injury.
We further explored the internal mechanism of Cur@EXs − cl−NGF treatment for SCI. Spinal cord tissues from each group were removed 7 d after injury to observe the pathological changes. The spinal cord 5 mm above and below the injury point was collected to make a single cell suspension. The ow cytometry results showed a changed polarization state of monocytes/macrophages in spinal cord tissue. The proportion of M1 monocytes/macrophages (F4/80+, CD86+) was signi cantly higher than that of M2 monocytes/macrophages (F4/80+, CD206+) in the NS group. Among them, the proportion of M1 monocytes/macrophages in total monocytes/macrophages increased to approximately 85.43%, while the proportion of M2 monocytes/macrophages was only 5%. With different treatments, the polarization state of monocytes/macrophages changed accordingly. Cur@EXs − cl−NGF displayed the most obvious regulatory effect on monocyte/macrophage polarization, balancing the ratio of proin ammatory M1 monocytes/macrophages and anti-in ammatory M2 monocytes/macrophages ( Figure. 7A, Figure.

Discussion
An anti-in ammatory combined neuroprotection strategy is one of the most promising options for the treatment of SCI [8,32]. Activation of microglia is one of the earliest in ammatory reactions after injury, and this activation is usually excessive [33]. Many proin ammatory factors are released into the damaged microenvironment, and many mononuclear macrophages are recruited to the in ammation site to form an uncontrollable in ammatory response [34]. We found that the microglia/macrophages that accumulated at the injury site in the early stage of injury were mainly the M1 type ( Figure. S1), which indicated that the polarization of microglia/macrophages was severely imbalanced under the action of proin ammatory factors [35]. Therefore, regulating the polarization direction of microglia/macrophages to achieve the balance between M1 microglia/macrophages and M2 microglia/macrophages is a potential treatment strategy for uncontrollable in ammatory responses in SCI [36]. Previous studies have shown that EXs can change the polarization state of M1-type macrophages after being taken up by M1-type macrophages [15,37]. We further found that the ratio of monocytes/macrophages in peripheral blood leukocytes increased signi cantly within 7 d after injury and reached a peak on day 3 ( Figure. S2). This nding implies that due to the recruitment of in ammation sites, EXs can actively target in ammation sites and exert in ammatory inhibitory effects [2,38].
In this study, the rst step was to design a stable drug delivery vehicle with anti-in ammatory, neuroprotective and targeting capabilities. The results of western blotting and cell proliferation experiments showed that the modi cation process did not affect the activity of NGF or the function of EXs ( Figure. 2I, Figure. (Figure. 3E). These advantages of EXs − cl−NGF greatly increased the amount of NGF that accumulated at the injured site and signi cantly improved the bioavailability of NGF. Considering that the target cells of EXs are in ammatory cells, the timely dissociation of NGF from EXs after reaching the injured site is also very meaningful. It has been reported that after SCI, the concentration of MMP9 at the injured site increases signi cantly, and we also discovered this phenomenon through ELISA. CLSM and ow cytometry showed that NGF coupled with EXs through a cleavable linker could be effectively taken up by PC12 cells (Figure. 3G, Figure. 3H). Researchers who use exosomes as carriers for NGF often overlook the consumption of NGF by in ammatory cells such as microglia/macrophages. Therefore, it is necessary to use cleavable linkers to ensure that NGF reaches the injured site and can be internalized by nerve cells as much as possible instead of macrophages.
In vitro cell experiments con rmed that EXs reduced the expression of proin ammatory factors (TNF-α, IL-1β, IL-6) in the injured microenvironment and increased the secretion of anti-in ammatory factors (TGF-β) ( Figure. 4C). Subsequently, we used ow cytometry to analyze the phenotype of the primary macrophages in the Transwell™ coculture system. The results showed that under the action of EXs, the number of M1 macrophages was reduced by approximately 40%, while the number of M2 macrophages was increased by approximately 10%. The results suggested that although EXs signi cantly inhibited the in ammatory response, the macrophages were still in a proin ammatory polarized state. Thus, using EXs might not be su cient to achieve a satisfactory therapeutic effect, so the drug carrier effect of EXs should also be fully utilized. The model drug Cur was loaded into EXs to improve the in ammationinhibiting effect of the system. As expected, compared with EXs − cl−NGF , Cur@EXs − cl−NGF had a more obvious inhibitory effect on proin ammatory M1 macrophages and signi cantly promoted the secretion of anti-in ammatory factors and the expression of the macrophage marker protein M2 (ARG-1) ( Figure. 5F, Figure. 5G). The phenotypic veri cation results of microglia/macrophages accumulating at the injury site showed that Cur@EXs − cl−NGF rebalanced microglia/macrophage polarization ( Figure. 6A). This nding is of great signi cance for reducing the secondary damage caused by uncontrollable in ammation.
Moreover, NGF coupled with EXs by cleavable linkers could effectively inhibit the apoptosis of PC12 cells after injury. Flow cytometry and CLSM images suggested that although EXs could indirectly inhibit apoptosis of PC12 cells by reducing the in ammatory response [39,40], the degree of inhibition was very limited (Figure. 4A, Figure.

Exosome isolation
Extraction and induction of peritoneal macrophages: The abdominal skin of the mice was disinfected with ethanol, and 1 mL of 5% starch broth solution was injected into the abdominal cavity of each mouse. After 48 h, the mice were sacri ced by cervical dislocation. The peritoneum of the mice was exposed under aseptic conditions, and the peritoneal cavity was injected with 5 mL precooled Dulbecco's Modi ed Eagle's Medium (DMEM). After gently massaging the mouse's abdomen for 5 min, a 5-mL syringe was used to repeatedly ush the peritoneal cavity lavage uid twice, and then the lavage uid was recovered. The lavage uid was centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the peritoneal cell concentration was adjusted to 1×10 6 /mL. One milliliter per well of the cell suspension was inoculated in a 12-well culture plate and placed in an incubator at 37°C with 5% carbon dioxide and saturated humidity to allow the macrophages to adhere to the wall. After incubating for 2 h to 4 h, the culture supernatant was discarded, and the cells were cultured in DMEM supplemented with exosome-free 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). After 24 h, interleukin-4 (IL-4) (20 µg/mL) was added to the medium to induce the differentiation of primary macrophages into M2 macrophages.
Exosome isolation: The cell supernatant of primary M2 macrophages was collected and placed in a centrifuge tube, which was sequentially centrifuged at 300 g for 10 min, 2,000 g for 10 min, 10,000 g for 30 min, and 100,000 g for 70 min at 4°C. The obtained precipitates were suspended in phosphatebuffered saline (PBS) and centrifuged at 100,000 g for 70 min to wash away impurities. The exosomes were resuspended in PBS for further characterization.
Modi cation of NGF with cleavable peptide substrate (NHS-Arg-Val-Gly-Leu-Pro-(6-Mal), RVGLP-(6-Mal)): The peptide (0.5 mg/mL) was added to the NGF solution (1 mg/mL) at a molar ratio of 50:1, keeping the linker in excess. The mixed solution was incubated at 4°C in PBS (pH 7.4) overnight. Then, the samples were ltered using a centrifugal lter device (Amicon Ultra-0.5, Millipore Co, Germany) at 7,000 g for 30 min to remove the excess linker. Next, the NGF was linked to EXs. The exosomes were pretreated with 1 mM TCEP at 37°C for 30 min to break the disul de bonds on the surface of the exosomes and expose the sulfhydryl groups. Then, "-cl-NGF" was dissolved in PBS (pH 7.4) and reacted with the EXs at 25°C for 1 h.
Finally, the EXS − cl−NGF was washed and collected by dialysis. Cur was dissolved in a 1:1 mixed solution of ethanol and acetonitrile. PBS was added to the mixed solution to a concentration of the organic solvent of 10%. EXs − cl−NGF was added at a concentration of 20% curcumin to the solution. Cur@EXS − cl−NGF was prepared by ultrasonic soaking using a 40-kHz and 100-W intermittent ultrasonic cleaner for 15 min and washing by centrifugation at 5000 rpm three times with a 100-kDa ultra ltration tube. The loading capacity or percent drug load was calculated using the following formula. Colocalization analysis of EXS − cl−NGF and Cur@EXS − cl−NGF : First, EXs were dyed with DID, and NGF was labeled with CY3. Subsequently, the double uorescently labeled nanomaterial EXS − cl−NGF was prepared using the above method. EXS − cl−NGF was imaged by confocal laser scanning microscopy (CLSM) (Nikon A1 Japan) and visualized with stimulated emission depletion (STED) (Leica SP8, Germany).
Release and biological activity of NGF from EXS − cl−NGF : MMP-9 was added to a PBS solution of EXS − cl−NGF with/without inhibitor, and the supernatant was removed at different time points. After ultra ltration, the content of NGF in the ltrate was detected with an NGF ELISA kit. The biological activity of NGF was detected by the MTT test in PC12 cells, in which different concentrations of NGF from EXS − cl−NGF /free NGF were added to the culture medium of PC12 cells. For the apoptosis rate of PC12 cells, the Transwell™ coculture model was removed after different stimulations, and the upper PC12 cells were resuspended. A total of 10 5 PC12 cells were mixed in 100 µL of binding buffer. Cells were stained with Annexin V-Alexa Fluor 488/PI for 15 min at room temperature to assess cell apoptosis.
Animals and the SCI model Adult male C56BL/6 mice (8-10 weeks old, 22-25 g) were used. All animal experiments were approved by the Animal Protection and Use Committee of Jinzhou Medical University. A contusion-induced SCI model was developed by an improved weightlessness method. In brief, after intraperitoneal anesthesia with 1% sodium pentobarbital (50 mg/kg), the mice were xed on a sterile operating table, fully exposing the T9 spinal cord. Using a 12.5-g impactor device (diameter: 2 mm), the uniform height was 5 cm down to the spinal cord, resulting in a moderate spinal cord contusion. After successful modeling, the surgical incision was sutured layer by layer, and antibiotics were given to prevent infection. From 2 d after surgery, the mouse bladder was massaged twice a day to help them urinate until the mice could urinate spontaneously. Sham-operated mice underwent the same procedures, except for contusion of the spinal cord. To investigate the treatment effect in vivo, 100 µL of 2 mg/mL Cur, EXS − cl−NGF or Cur@EXs − cl−NGF was injected through the tail vein at 2 h, 2 d, 4 d, and 6 d after injury.

In vivo imaging
For in vivo imaging of nanoparticles, 100 µL of NGF, red vesicles (RVs − cl−NGF ), or EXs − cl−NGF (2 mg/mL, Alexa Fluor® 647-labeled NGF) was injected via the tail vein. At different time points, mice were imaged using a Kodak Imaging System FX Pro to evaluate the uorescence signal distribution in vivo. Twelve hours after tail vein injection, the mice were sacri ced by cervical dislocation, and the mouse heart, liver, spleen, lung, kidney, and spinal cord were removed for uorescence imaging.

Immuno uorescence staining
For the cell uptake experiment, medium containing NGF, EXS − NGF , and EXS − cl−NGF (FITC-labeled NGF, 5 µg/mL) was added to the Transwell™ coculture model for 6 h. Cells were removed from the incubator, and the cells were xed with immune tissue xative. Finally, the cell membrane was labeled with phalloidin 561, and the cell nucleus was labeled with 4',6-diamidino-2-phenylindole (DAPI).

Behavioral analysis
The basso mouse scale (BMS) exercise rating scale was used to evaluate the functional recovery of injured animals on days 1, 3, 5, 7, 14, 21 and 28 according to scores ranging from 0 to 9 (9 for complete normality and 0 for complete paralysis). The animals were placed in the open eld and observed for 4 min. Three examiners who did not know the mouse grouping observed and scored the mouse's ankle joints, the touch degree of the sole and dorsum of the foot, trunk stability and tail position. The experiment was repeated three times.

Statistical analysis
All data are expressed as the average value of the data distribution evaluated by the Shapiro-Wilk test.
The Mann-Whitney U test was used for two-group comparisons. One-way ANOVA and Bonferroni post hoc tests were used to compare more than two groups. All data were graphed and statistically analyzed using GraphPad Prism 9. P < 0.05 was indicated with "*", P < 0.01 was indicated with "**", and P < 0.001 was indicated with "***".  indicates the difference analysis with the NS group. Data presented the mean ± SD (n = 6 per group) (*P < 0.05, **P < 0.01, ***P < 0.001 and ns: not signi cant)

Supplementary Files
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