MSC derived EV loaded with miRNA‐22 inhibits the inflammatory response and nerve function recovery after spinal cord injury in rats

Abstract Our previous research has found that miRNA‐22 can inhibit the occurrence of pyroptosis by targeting GSDMD and decrease the production and release of inflammatory factors. In consideration of the therapeutic effects of mesenchymal stem cells (MSCs), MSCs‐EV were loaded with miRNA‐22 (EV‐miRNA‐22) to investigate the inhibitory effect of EV‐miRNA‐22 on the inflammatory response in SCI in rats in this study. LPS/Nigericin (LPS/NG) was used to induce pyroptosis in rat microglia in vitro. Propidium iodide (PI) staining was performed to observe cell permeability, lactate dehydrogenase (LDH) release assay was adopted to detect cytotoxicity, flow cytometry was conducted to detect pyroptosis level, immunofluorescence (IF) staining was utilized to observe the expression level of GSDMD (a key protein of pyroptosis), Western blot was performed to detect the expression of key proteins. For animal experiments, the T10 spinal cord of rats was clamped by aneurysm clip to construct the SCI model. BBB score, somatosensory evoked potential (SEP) and motor evoked potential (MEP) were performed to detect nerve function. HE staining and Nissl staining were used to detect spinal cord histopathology and nerve cell damage. EV‐miRNA‐22 could inhibit the occurrence of pyroptosis in microglia, suppress the cell membrane pore opening, and inhibit the release of inflammatory factors and the expression of GSDMD. In addition, EV‐miRNA‐22 showed higher pyroptosis‐inhibiting ability than EV. Consequently, EV‐miRNA‐22 could inhibit the nerve function injury after SCI in rats, inhibit the level of inflammatory factors in the tissue and the activation of microglia. In this study, we found that miRNA‐22‐loaded MSCs‐EV (EV‐miRNA‐22) could cooperate with EV to inhibit inflammatory response and nerve function repair after SCI.


| BACKG ROU N D
Spinal cord injury (SCI) can cause severe dyskinesia and sensory disturbance. 1 Inflammation caused by inflammatory factors can lead to secondary damage. The mechanism of inflammatory response of SCI is relatively complicated, involving both nerve cell injury and the participation of the immune system. 2 6,7 Theoretically, the inhibition of GSDMD can suppress pyroptosis, and simultaneously inhibit the activation of microglia and inflammatory response. Our team has previously found that miRNA-22 is a non-coding RNA regulated by GSDMD mRNA, which can inhibit neuroinflammatory response and is simultaneously validated in the Alzheimer's disease model. 8 In recent years, stem cell transplantation has been fully investigated in the treatment of SCI. 9 Present studies have demonstrated that mesenchymal stem cells (MSCs) can repair the nerve function injury of SCI to a certain extent, promote the regeneration of nerve axons and enhance the repair of motor nerves as well as functional repair. 10,11 However, the transplantation of MSCs may cause side effects such as tissue immune rejection, teratogenesis, and tumorigenesis, which is simultaneously accompanied with such problems as stem cell survival rate after transplantation. 12 Therefore, it is urgently needed to exploit a new approach to replace stem cell therapy. EV are vesicle-like structures secreted by cells. Studies have found that the EV derived from MSCs also exert good effects on the nerve repair of SCI. 13,14 In addition, MSC-derived EV greatly attenuate the various side effects caused by MSCs. Therefore, in this study, miRNA-22 was loaded into the MSC-derived EV, which was speculated to promote the regeneration of nerve cells and inhibit neuroinflammation, thereby playing a synergistic role.

| Detection of dynamic expression of miRNA-22 after SCI in rats
Animal experiments have been reviewed and approved by the Jiaxing University ethics committee. The entire animal experiment conforms to the relevant regulations of animal ethics and welfare, and the whole process conforms to ethical norms.
A total of 36 adult male rats (raised by the Animal Experiment Center of Jiaxing University) were divided into Control, SCI-12h, SCI-24h, SCI-48h, SCI-72h and SCI-7D groups (six rats in each group). In brief, the spinal cord was clamped by aneurysm clips to construct the SCI model. The rats were sacrificed after 12 h, 24 h, 48 h, 72 h and 7D, followed by resection of the spinal cord tissue of the T10 segment for examination. To construct the SCI model, rats were fasted for 12 h before the operation, subjected to anaesthesia with 4 mg/kg chloral hydrate, fixed on the plate, and routinely disinfected, followed by 3-cm longitudinal incision of the connection of the 13th rib and the 13th thoracic vertebrae to expose the skin, fascia and muscles.
The T9-T11 were positioned upwards from the 13 thoracic spinous process, and the T11 spinous process was exposed laterally. After cutting off the supraspinous ligament and interspinous ligament, the Mosquito Clamp was used to remove the T9 and T11, the spinal canal at the T10 spinous process was excised to expose the spinal cord and the aneurysm clamp (clamping force −0.88N) was utilized to clamp the T10 spinal cord for 10 s. The posterior limb of the rats showed no muscle tension, and the spinal cord was congested and swollen, which was sutured after compression to stop bleeding. Rats were maintained in the clean environment and intraperitoneally injected with penicillin once daily for anti-infection. When rats were sacrificed, the spinal cord was exposed along the original surgical incision, and the T9-T11 segments were excised and stored at −80°C.

| Extraction of bone marrow MSCs and their EV and miRNA-22 loading
Six-week-old SD rats were sacrificed by cervical dislocation, followed by isolation of the femur and tibia. After exposing the bone marrow cavity, DMEM (containing FGF2) (Gibco) was used to wash the bone marrow cavity to disperse the cells into single cell suspension. Cells were transferred to the petri dish and the culture medium was changed every three days to discard non-adherent cells Afterwards, 10 μl of 2% uranyl acetate was aspirated and dropped on the copper mesh for 1 min, followed by absorption of the floating liquid by the filter paper and drying for several minutes at room temperature. Finally, the samples were observed and photographed at 80 kv-120 kv.
2. The number and size of the EV were directly tracked by the rate of Brownian motion of EV using the NanoSight NS 300 system (NanoSight Technology, Malvern), configured with a high-sensitivity sCMOS camera, fast video capture and particle-tracking software (NanoSight). The samples were diluted 150-3000 times with Dulbecco's PBS (DPBS) without any nanoparticles to attain a concentration of 1-20 × 10 8 particles per millilitre for analysis. Each sample was measured in triplicate at the camera, which recorded and tracked each visible particle.
EVsome numbers and size distribution were explored using the Stokes-Einstein equation.
When cells reached to 70% confluency, cells were divided into Control group, LPS/NG group, EV group and EV-miRNA-22 group.
Cells of LPS/NG group were pre-treated with 1 μg/ml lipopolysaccharide (LPS) (Sigma, USA) for 5 h, followed by addition of 10 μM After the reagents were added and incubated according to the manufacturer's instructions, the absorbance value was detected at 450 nm by a microplate reader (BioTek) and the results were shown as pg/ml. 1. The nerve function of rats was evaluated by BBB method, which was assessed every 1-3-9-15 days after drug administration.

| The effects of EV-miRNA-22 on SCI in rats
2. Somatosensory evoked potential (SEP) assay: Rats were anaesthetized with 2% pentobarbital with disappeared corneal reflex and even breath. The anaesthetized rats were placed on the operating floor. After drilling rat's skull to expose the right cerebral sensory cortex, the electrode was inserted under the frontal skin to expose the sciatic nerve, followed by electrical stimulation and subsequent analysis for 100 ms. The latency of SEP N1 wave was recorded and the electrophysiological recovery was cautiously observed.
3. Motor evoked potential (MEP) assay: After drug administration, the nerve conduction waveform and the latency and amplitude changes of N1 wave were evaluated every 1-3-9-15 days (the operation method was consistent with SEP assay).

| Statistical analysis
All measurement data were expressed as ±s, and SPSS 17.0 was used for analysis and data processing. After the homogeneity of variance test, two independent sample t-test was used for comparison between the two groups. One-way ANOVA was used for comparison among three groups, followed by LSD method for subsequent pairwise comparisons between the groups. Two-sided p < 0.05 indicated statistical significance.

| The expression of miRNA-22 in SCI model and the identification of EVsome
The expression of miRNA-22 was detected on the 7D after SCI in rats. As a result, the expression of miRNA-22 in the spinal cord tissue of rats were increased during the acute phase (12 h) and later down-regulated. The expression of miRNA-22 was significantly lower than Control group after 48 h ( Figure 1A). EV and EV-miRNA-22 were characterized and found that miRNA-22-loaded MSC-derived EV had a pie-like shape similar to EV, which was consistent with the appearance of EV. In addition, the particle size analysis showed that EV and EV-miRNA-22 were both at 100 nm, indicating that miRNA-22 did not affect the stability of EV ( Figure 1C). When detecting EVsome markers, we found that the expression of CD63, CD81 and ALIX was significantly higher in EV and EV-miRNA-22 than MSCs ( Figure 1B).
After miRNA-22 overexpression in MSCs, the level of miRNA-22 was significantly increased ( Figure 1D). And the level of miRNA-22 in EV-miRNA-22 was significantly higher than that of EV ( Figure 1E).

| The effects of EV-miRNA-22 on pyroptosis in microglia
The relative PI uptake assay could detect the opening degree of cell membrane pores. As a result, the relative uptake rate of PI was low in Control group, without significant increase over time, indicating the low opening level of cell membrane pores. In LPS + NG group, the opening level of cell membrane was significantly enhanced and the PI uptake rate was increased; while the PI uptake rate was decreased in EV and EV-miRNA-22 groups, which was significantly lower than that in LPS + NG group (Figure 2A). When detecting LDH release rate, we found that the LDH release rate was significantly higher in LPS + NG group than that of Control group, and the inhibition level of LDH release rate was significantly higher in EV-miRNA-22 group than that of EV group ( Figure 2B). EtBr is a macromolecular dye, while EthD2 is a micromolecular dye. Both EtBr and EthD2 could enter into cells in LPS + NG group, which had a high opening level of membrane pores.
While EV-miRNA-22 could inhibit the opening of cell membrane

| The effects of EV-miRNA-22 on the nerve function of SCI rats
The BBB score of SCI, EV group and EV-miRNA-22 group was 0.
Rats were unable to move with posterior limbs and could only crawl with anterior limbs. After post-operative intervention of EV and EV-miRNA-22, the neurological function of rats was recovered to different degrees, which was significantly different from SCI model (p < 0.05). The neurological recovery level of rats in F I G U R E 2 The effects of EV-miRNA-22 on pyroptosis in microglia. (A) The PI uptake rate showed that PI was not significantly uptaken in cells of Control group, the PI uptake rate was significantly increased in LPS+NG group and the PI uptake rate was decreased in EV and EV-miRNA-22 groups. Moreover, the PI uptake rate was significantly lower in EV-miRNA-22 group than that of EV group. Comparison with Control group, *p < 0.05; comparison with LPS + NG group, # p < 0.05. (B) LDH release rate showed that LPS/NG could increase LDH release, indicating obvious cell injury. While EV and EV-miRNA-22 could inhibit the release of LDH. And the release rate of EV-miRNA-22 was significantly lower than that of EV. Comparison with Control group, *p < 0.05; comparison with LPS + NG group, # p < 0.05. (C) EtBr/ EthD2 staining. EtBr/EthD2 and EtBr/EthD2 could enter into cells of LPS/NG groups. While EV-miRNA-22 could inhibit EtBr/EthD2 entry and EV could inhibit EtBr/EthD2 entry to a certain extent. (D and E) Detection of pyroptosis. EV-miRNA-22 could inhibit the occurrence of pyroptosis and decrease the proportion of pyroptosis. Compared with Control group, *p < 0.05; comparison with LPS + NG group, # p < 0.05 EV-miRNA-22 group was significantly higher than that of EV group, suggesting that EV-miRNA-22 could better promote the nerve function recovery in rats. Results were shown in Table 1.
Somatosensory evoked potential assay revealed that the evoked potential waveform of rats in Sham group was normal, and the evoked potential waveform disappeared after SCI. After EV and EV-miRNA-22 intervention, the SEP value of rats was significantly changed from SCI (p < 0.05). The conduction function of posterior limbs of rats were significantly recovered. And the improvement was more obvious in EV-miRNA-22 group, which was significantly different from EV group. The results were shown in Table 2. was significantly up-regulated in pyroptosis, which was higher than that of Control group. EV and EV-miRNA-22 could inhibit the release of inflammatory factors. In particular, the level of inflammatory factors was lower in EV-miRNA-22 group than that in EV group. Comparison with Control group, *p < 0.05; comparison with LPS + NG group, # p < 0.05. (E and F) Protein detection revealed that EV-miRNA-22 could inhibit the expression of GSDMD and p30-GSDMD. Comparison with Control group, *p < 0.05; comparison with LPS + NG group, # p < 0.05

TA B L E 1 BBB score(mean ± SD, Score)
After constructing SCI model, the N1 latency was significantly higher than that in Sham group. After EV and EV-miRNA-22 intervention, the N1 latency was significantly shortened than SCI model (p < 0.05). The conduction function of posterior limbs of rats was significantly recovered. And the improvement in EV-miRNA-22 group was more obvious, which was significantly different from EV group.
The results were shown in Table 3.

| DISCUSS ION
Spinal cord injury is one of the common causes of disability in young people. 13 The pathological process of SCI includes two processes: namely, primary injury and secondary injury. Primary injury is defined as the direct damage of SCI by external force, causing the rupture of nerve cell membrane, thereby leading to irreversible nerve cell necrosis. 14,15 For secondary damage, at this stage, cell contents are rapidly released after cell necrosis, 16 such as glutamate, reactive oxygen species (ROS), potassium ions and cathepsin B (CTSB). [17][18][19] The leakage of these substances can cause inflammation of the nervous tissue and even lead to cell death. Cell death includes multiple types, such as autophagic death, programmed necrosis and py- preventing pyroptosis in nerve cells. 28 The main source of inflammatory damage in SCI is the activation of microglia. More studies have also found that microglia, as a special type of macrophages, will express a large amount of inflammatory factors in the case of pyroptosis, which is the essence of the inflammatory response.
Inhibition of scorch death in SCI has also become a key means to inhibit inflammation.
Our team has previously found that miRNA-22 is a regulatory non-coding RNA of GSDMD, and miRNA-22 has been confirmed to inhibit the activation and pyroptosis of microglia by inhibiting GSDMD. 8  superior to that of EV group. The BBB score and electrophysiological test also showed that EV and EV-miRNA-22 could repair the nerve function of rats and partially restore nerve function.

| CON CLUS ION
In this study, we have found that miRNA -

E TH I C A L A PPROVA L A N D CO N S ENT TO PA RTI CI PATE
The study was approved by Ethics Committee.

CO N S E NT FO R PU B LI C ATI O N
This article was published with the approval of all the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.