Human umbilical cord mesenchymal stem cell‐derived exosome suppresses programmed cell death in traumatic brain injury via PINK1/Parkin‐mediated mitophagy

Abstract Aims Recently, human umbilical cord mesenchymal stem cell (HucMSC)‐derived exosome is a new focus of research in neurological diseases. The present study was aimed to investigate the protective effects of HucMSC‐derived exosome in both in vivo and in vitro TBI models. Methods We established both mouse and neuron TBI models in our study. After treatment with HucMSC‐derived exosome, the neuroprotection of exosome was investigated by the neurologic severity score (NSS), grip test score, neurological score, brain water content, and cortical lesion volume. Moreover, we determined the biochemical and morphological changes associated with apoptosis, pyroptosis, and ferroptosis after TBI. Results We revealed that treatment of exosome could improve neurological function, decrease cerebral edema, and attenuate brain lesion after TBI. Furthermore, administration of exosome suppressed TBI‐induced cell death, apoptosis, pyroptosis, and ferroptosis. In addition, exosome‐activated phosphatase and tensin homolog‐induced putative kinase protein 1/Parkinson protein 2 E3 ubiquitin–protein ligase (PINK1/Parkin) pathway‐mediated mitophagy after TBI. However, the neuroprotection of exosome was attenuated when mitophagy was inhibited, and PINK1 was knockdown. Importantly, exosome treatment also decreased neuron cell death, suppressed apoptosis, pyroptosis, and ferroptosis and activated the PINK1/Parkin pathway‐mediated mitophagy after TBI in vitro. Conclusion Our results provided the first evidence that exosome treatment played a key role in neuroprotection after TBI through the PINK1/Parkin pathway‐mediated mitophagy.


| INTRODUC TI ON
Traumatic brain injury (TBI) occurs due to direct impact or hit on the head caused by factors such as motor vehicles, crushes, and assaults. 1 Central nervous system (CNS) is highly sensitive to external mechanical damage, presenting a limited capacity for regeneration due to its inability to restore either damaged neurons or synaptic network. 2 Thus, TBI is identified as an important global health concern that represents a leading cause of death and disability. 3 The primary injury happens at the time of injury and is responsible for the initiation of secondary injury cascades such as inflammation, apoptosis, oxidative stress, and endoplasmic reticulum. 4 These cascades contribute to long-term brain damage including neurological deficits, brain edema, and blood brain barrier (BBB) disruption. 5 Despite the progress has been made in the prevention and treatment of TBI in the past, patients suffering from TBI usually end up with poor prognosis. 6 Thus, it is urgently needed to find optimal therapies and improve patients' long-term neurological functioning after TBI.
Recently, cell therapies, especially human umbilical cord mesenchymal stem cell (HucMSC) transplantation has been suggested to be a powerful method for the treatment of TBI. 7 HucMSC exhibits strong proliferative ability, low immunogenicity, and multi-potential differentiation. 8 Exogenous HucMSC can stimulate repair processes, suppress astrocyte activation, inhibit inflammatory factors, and decrease neuron loss after brain injury, resulting in improvement of behavioral outcomes and cerebral lesion volumes after brain damage. 9 It has been found that the therapeutic effects of HucMSC were depend on the release of exosome. Exosome is cup-like shapes with a diameter size range from 30 to 150 nm, it is produced by the membrane of multivesicular body (MVB). 10 When the endosome or MVB fuse with the plasma membrane, exosome is released extracellularly. 11 Exosome contains lipids, proteins, non-coding RNAs, and mRNAs, these contents are protected from degradation by the lipid bilayer of exosome. 12 Via releasing these components to neighboring cells, exosome is able to regulate cell-to-cell communications as well as multiple autocrine and paracrine cellular phenotypes. 13 In addition, exosome has extensive and unique advantages due to its favorable pharmacokinetic and ability to penetrate physiological barriers. 14 Exosome readily crosses the BBB and has the potential to specifically deliver molecules to CNS. 11 Recently, studies have revealed the effective role of exosome in the diagnosis and treatments of CNS diseases such as TBI. 15 It has been shown that HucMSC could provide neuroprotection after TBI. 16 However, the role of HucMSC-derived exosome in TBI has not been fully studied. In this study, we explored whether HucMSC-derived exosome could suppress cell death, apoptosis, pyroptosis, and ferroptosis after TBI and the underlying mechanisms.

| Animal preparation
All animal studies were carried out in accordance with the principles of the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were approved by the Institutional Animal Care and Use Committee of Nanjing University (Nanjing, China). Male ICR mice (28-32 g) were obtained from Animal Center of Jinling hospital.
Mice were housed on a 12 h light/dark cycle at 23 ± 1°C with free access to food and water.

| Primary culture of mouse cortical neurons
The culture of mouse cortical neurons was performed according to previous studies. 17,18 In brief, mouse cortical neurons were isolated from the embryos of time-mated pregnant mice and subsequently cultured in poly-D-lysine-coated 6-well dishes at a density of 1 × 10 6 cells per well. Then, the neurons were cultured in neurobasal medium

| TBI models
The in vivo (mouse) TBI model was performed using a Controlled Cortical Impact (CCI) model. Mice were anesthetized with 3% isoflurane and subsequently placed on the stereotaxic apparatus. With a 1.5 cm midline longitudinal incision of the scalp, the skull was exposed. The area to be impacted lies on the right frontal skull (2.5 mm lateral to the midline and 0.5 mm anterior to bregma). After confirming the correct impact location again, mice were subjected to cortical contusion injury by a 3.0 mm rounded impactor tip (piston velocity: 3.5 m/s; deformation depth: 1.5 mm, dwell time: 150 ms).
The mortality results from apnea were decreased by early respiratory support. Then the mice were returned to cages to recover for 24 h with free access to a standard diet. The sham-injured mice underwent the same procedures but did not undergo the CCI.
The in vitro (neurons) TBI model was conducted using a mechanical stretch injury model. Briefly, the 6-well plates were manually scratched with a sterile plastic needle followed by a 9 × 9 square grid (the space between every line was 4 mm). Then, neurons were cultured at 37°C in 5% CO 2 incubator for another 24 h without change of culture medium.

| Exosome isolation and characterization
Extraction of HucMSCs-derived exosome: after expanded culture, HucMSCs grew to about 85%, the culture medium was discarded, the cells were washed three times with sterile phosphate buffer saline (PBS), and serum-free culture medium was added. After 48 h of culture, the cell supernatant was collected and transferred to a 50 mL centrifuge tube. After centrifugation at 500 g, 4°C for 5 min, the supernatant was collected and then transferred to a new 50 mL centrifuge tube, centrifugated at 2000 g, 4°C for 30 min. Then, the supernatant was collected and centrifugated at 10,000 g for 60 min. Finally, the supernatant was collected, filtered with 0.22 μM sterile filter, and added to the ultra-high speed centrifugal tube. After centrifugation at 120,000 g, 4°C for 1 h, the supernatant was removed, and the precipitate was dissolved with 200 μL sterile PBS and resuspended. Measurement of the exosome particle size: exosomes were diluted 40 times with sterile PBS and then filtered with 0.22 μM sterile filter.
The particle size of exosomes was detected by nanoparticle tracking analysis (NTA) at Vivacell Biosciences with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) as previously described. 19 Detection of exosome by transmission electron microscopy (TEM): exosomes were washed with 1 mL of PBS for three times.
Then, 0.5 mL of 2% osmic acid solution was added and fixed at 4°C for 2 h. After washing with 1 mL of PBS for three times, 1 mL of 50%, 70%, 80%, and 90% ethanol were used to gradually dehydrate for 15 min each time and then 1 mL of 100% ethanol was used to dehydrate twice for 20 min each time. After dehydration, the sample was replaced twice with 1 mL acetone for 15 min each time. Then, the sample was immersed and put into the embedding plate, and the embedding plate was polymerized at 65°C for 48 h. Finally, it was stained with uranyl acetate and lead acetate for 10 min. After cleaning, exosomes were observed using TEM (JEM-1230, JEOL Ltd., Akishima, Japan) to identify the morphology.

| Neurological evaluation and brain water content
The neurological impairment was evaluated using the neurologic severity score (NSS) at 1 day, 3 days and 7 days after TBI. The investigators estimate the ability of mouse to perform 10 different tasks which demonstrate physiological behavior, alertness, and motor function. One point is given for failing to perform each task, thus 0 = minimum deficit and 10 = maximum deficit (Table 1). 20,21 The motor performance of mice was also evaluated at 1 day, 3 days, and 7 days after TBI using the grip test score 18 and neurological score. 22 Both tests were carried out by an investigator who was blinded to the experimental groups. For grip test score, mice were placed on a thin, horizontal, metal 45 cm long wire which was lay up between two vertical poles 45 cm above a foam pad. Zero point was given if the mice was unable to remain on the wire for <30 s; one point was given if the mice failed to hold on to the wire with both hind paws and forepaws together; two points were given if the mice held on to the wire with both hind paws and forepaws but not the tail; three points were given F I G U R E 1 Diagram showing the experimental design throughout the study.
if the mice used its tail along with both hind paws and forepaws; four points were given if the mice moved along the wire on all four paws plus tail; five points were given if mice that scored four points also ambulated down one of the posts used to support the wire. The grip test was performed in tree times and a total point was calculated for each mouse. For neurological score, an 18-point scoring system, ranging from 3 to 18, assessed the neurological deficits from six subtests. These tests comprise spontaneous activity (0-3 score), symmetry in all limbs' movements (0-3 score), forelimb extension (0-3 score), climbing (1-3 score), body proprioception (1-3 score), and response to vibrissae stimulation (1-3 score). Higher scores indicate better neurological function. The brain water content was conducted according to a previous study. 21 Briefly, mouse brain was taken out and placed onto a cooled brain matrix 1 day following TBI. The cerebellum and stem were taken away, and the ipsilateral tissue was weighed to get the wet weight (ww). Then, the hemisphere was dried for 72 h at 80°C and weighed again to get the dry weight (dw). The brain water content equals (ww−dw)/ww × 100%.

| Measurement of lesion volume
Lesion volume measurement was conducted based on previous studies. 18 Briefly, the brain sections were stained with cresyl violet. Then, the areas of lesion, injured, and non-injured brain tissues were estimated by an image analysis system. Area measurements from each section were obtained and summed, and corresponding volumes were calculated.

| Isolation of mitochondria
We used a tissue mitochondria isolation kit (Beyotime Biotechnology, Shanghai, China; catalog number: C3606) to separate cytoplasmic and mitochondria fractions. The mitochondria fractions separated from tissues were further lysed for extraction of mitochondrial proteins.

| Western blot analysis
Western blot analysis was performed according to previous studies. 24  Seeking behavior Physiological behavior as a sign of "interest" in the environment 0 1 Beam balancing Ability to balance on a beam 7 mm in width for at least 10 s 0 1 Round stick balancing Ability to balance on a round stick 5 mm in diameter for at least 10 s 0 1 Beam walk: 3 cm Ability to cross a beam (length × width, 30 × 3 cm) 0 1 Beam walk: 2 cm Same task but with increased difficulty (beam width = 2 cm) 0 1  . The ImageJ software quantified band intensities. All original blot images were available in Appendix S1.

| Immunofluorescence (IF) staining
Immunofluorescence staining was assessed according to a previ-

| Measurement of malondialdehyde (MDA) and glutathione peroxidase (GPx)
The injured cerebral cortex samples were homogenized in 2 mL of PBS. After centrifugation at 10,000 g for 25 min, the MDA and GPx in the supernatant were measured using an MDA assay kit (Beyotime

| Observation of mitochondria by TEM
Transmission electron microscopy was used to identify mitochondria as previously described. 25 Briefly, at 1 day after TBI, mice were killed and perfused with 2.5% buffered glutaraldehyde. The specimens were collected and fixed in glutaraldehyde with a 1% (w/v) solution of osmium tetroxide. The fixed specimens were then embedded, sectioned, double stained with lead citrate, and uranyl acetate, observed under a TEM JEM-1011 (JEOL, Japan).

| Cell viability analysis
To analyze the cell viability of primary cultured neuron cells, we first used the trypan blue (TB) staining assay. Cells were stained by

| HucMSC-derived exosome provided neuroprotection after TBI
To determine whether HucMSC-derived exosome (short for exosome) protected mice against TBI, exosome was isolated from HucMSC as described in Methods. The particle size of exosome was analyzed by NTA. We found that the average particle size of exosome was 146.8 nm, and the main peak of particle size was 153.2 nm ( Figure 2A). Moreover, the shape of exosome was observed by TEM (150,000×) ( Figure 2B). In addition, we detected the expression of the exosome surface marker CD9 and CD63 by Western blot, the results showed that the expression of CD9 and CD63 was significantly higher than that of HucMSC ( Figure 2C).
We then examined the protective effects of exosome in TBI.
We firstly used the NSS to evaluate the neurological impairment of mice after TBI. As showed in Figure 2D, exosome-treated mice showed better neurological function than vehicle-treated mice at 1 day. Moreover, a significant difference was still detectable at 3 days. However, there was no obvious difference between these two groups at 7 days (p > 0.05).
In addition, we used grip test score and neurological score to measure the motor performance of mice following TBI. Figure 2E , grip test score (E), and neurological score (F) were evaluated at 1, 3, and 7 days after TBI and exosome treatment. (G, H) Mice were subjected to TBI and received 100 μg/mL, 150 μg/mL, 200 μg/mL, 250 μg/mL, and 300 μg/mL of exosome or vehicle 30 min after TBI. Brain water content (G) and brain tissue loss (H) were examined at 1 day after TBI and exosome treatment. Data were presented as mean ± SD; ***p < 0.001 versus sham group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus TBI + vehicle group. β-Actin was used as a loading control.
obviously better than that of the vehicle-treated mice at 1 day and 3 days. However, there was no significant difference between these two groups at 7 days (p > 0.05).
Next, we used brain water content to validate the neuroprotective effects of exosome. As shown in Figure 2G, the brain edema was increased at 1 day after TBI. However, treatment of exosome decreased the brain edema. Significantly, all of NSS, grip test score, neurological score, and brain water content experiments suggested that 200 μg/mL of exosome showed the best neuroprotection.
Finally, we explored whether exosome could affect TBI-induced cortical lesion volume. Figure 2H showed that TBI induced significant brain tissue loss (red lines). While exosome treatment decreased the lesion volume ( Figure 2H). In conclusion, all experiments above indicated that exosome could provide neuroprotection after TBI and 200 μg/mL of exosome exhibited the best neuroprotection, which we would use in our subsequent in vivo experiments.

| Exosome reduced cell death and apoptosis after TBI
In the pathophysiology of TBI, cell death runs through the occurrence and development. Moreover, apoptosis plays an essential part in the progress of TBI, and its inhibition may help overcome TBI's negative consequences and improve functional recovery. 26 To study the occurrence of neural cell death, we used Nissl staining. In the sham group, the neurons were intact and clear, without edema around the cells ( Figure 3A). While in the TBI and TBI + vehicle groups, the damaged neuron cells were increased, exhibiting extensive degenerative changes including swollen cell bodies, shrunken cytoplasm, and oval or triangular nucleus ( Figure 3A). On the contrary, the severity of neuron damage was remarkably attenuated after exosome treatment ( Figure 3A).
To examine TBI-induced neural cell apoptosis, we applied TUNEL fluorescence staining. As shown in Figure 3B, few apoptotic-positive cells were detected in the sham group. However, apoptotic-positive cells were found in the TBI and TBI + vehicle groups ( Figure 3B).
While treatment of exosome significantly decreased the number of apoptotic-positive cells ( Figure 3B).
To further confirm the effects of exosome on apoptosis, we an-

| Exosome suppressed pyroptosis after TBI
It has been shown that pyroptosis was an important cause of neuronal damage after TBI. Pyroptosis is a type of inflammatory programmed cell death (PCD) that is characterized by the release of pro-inflammatory cytokines such as interleukin (IL), which triggers an inflammatory cascade response that results in cellular damage. 27 Therefore, we first measured the levels of pro-inflammation cytokines including IL-1β, IL-6, IL-18, and TNFα by ELISA. We found that the levels of IL-1β, IL-6, IL-18 and TNFα were increased at 1 day after TBI, which were significantly inhibited by exosome treatment compared to vehicle-treated group ( Figure 4A), indicating that exosome could inhibit TBI-induced inflammation.
We then examined some pyroptosis-related markers, including

| Exosome decreased ferroptosis after TBI
Current studies have indicated that ferroptosis was a crucial mechanism causing neurological deficit of TBI, and inhibition of ferroptosis may improve long-term outcomes of TBI. The characteristics of ferroptosis are the iron-dependent lipid peroxidation (LPO) accumulation and aggravated oxidative damage. To confirm that ferroptosis was initiated after TBI and exosome could alleviate TBI-induced ferroptosis, we first analyzed the LPO levels by measurement of MDA and GPx 1 day after TBI. We found that the levels of MDA were significantly up-regulated after TBI,  Next, we observed the cell morphology by TEM. Upon TBI, mitochondria became smaller with the mitochondrial ridges decreasing. In addition, the bilayer membrane density was also increased, which was considered as the symbol of ferroptosis ( Figure 7E). However, exosome treatment restored the morphological changes of mitochondria ( Figure 7E).

F I G U R E 3 Exosome suppressed TBI
The expression of ferroptosis-related protein was also analyzed after TBI. It has been shown that ACSL4 and GPX4 are the main tar-

| Exosome promoted mitophagy to inhibit brain injury after TBI
Previous studies have demonstrated that inhibition of mitophagy contributed to brain injury after TBI. Therefore, we wondered whether mitophagy was involved in the protective effects of exosome against TBI. We firstly used the IF staining of LC3, results of the fluorescence microscopy revealed that the TBI groups exhibited a significant increase in the number of neurons with LC3 compared with the sham groups, suggesting the formation of autophagosome ( Figure 9A). In addition, the LC3-positive neurons were further increased after exosome treatment ( Figure 9A).
The ability of exosome to induce autophagy was confirmed by TEM. In respond to TBI, the autophagic vacuoles containing cellular material or organelle were observed in the cytoplasm. Upon treatment of exosome, the number of autophagic vacuoles was up-regulated ( Figure 9B). To further verify that mitophagy was induced by exosome, we analyzed the expression of mitophagyrelated proteins, such as TOMM20, COX IV, Beclin-1 and LC3.
These data illustrated that exosome could promote TBI-induced mitophagy. To determine whether the neuroprotective effects of exosome in TBI were abated when mitophagy was blocked, we used a mitophagy inhibitor mitochondrial division inhibitor-1 (Mdivi-1) and studied the neurologic function, brain water content, oxidative damage, inflammation, pyroptosis, apoptosis, and ferroptosis.
As shown in Figure 11A,B, there was significant difference of the

| Exosome provided neuroprotection in TBI via PINK1/Parkin pathway-mediated mitophagy
PINK1/Parkin pathway has been demonstrated to play a key role in mitophagy and brain injury after TBI. Thus, we analyzed the involvement of PINK1/Parkin pathway in the neuroprotective effects of exosome. The results showed that the expression of PINK1 and Parkin was significantly increased after TBI, and treatment of exosome further increased PINK1 and Parkin expression ( Figure 13A,B).

| Exosome protected primary cultured neurons from TBI
The neuroprotective effects of exosome in TBI were also confirmed in primary-cultured neurons. TB staining and LDH release assay were firstly conducted in neurons treated with exosome. In TB staining, treatment of exosome significantly increased the percentage of viable cells ( Figure 14A). Similar results were found in LDH release assay ( Figure 14B). These results suggested that exosome provided neuroprotection after TBI in vitro and indicated that 100 μg/mL of exosome exhibited the best effect, which we used in the following in vitro experiments.
Then, to understand the effects of exosome on neuron lipid peroxides level, we used BODIPY staining. Figure 14C showed that compared with the control neurons, the level of lipid peroxides in the damaged neurons was significantly increased. When neurons were treated with exosome, the level of lipid peroxides was decreased ( Figure 14C).

| DISCUSS ION
To the best of our knowledge, this is the first study detailly examin- Ferroptosis is a recently discovered form of non-apoptotic PCD. 33 Biologically, the characteristics of ferroptosis are the irondependent LPO accumulation. 34 Ferroptosis plays a key role in TBI and is closely related to oxidative stress, immunity, and chronic injuries. The inhibitors against ferroptosis effectively improve iron homeostasis, lipid metabolism, redox stabilization, neuronal remodeling, and functional recovery after trauma. 35 48 We then wondered whether mitophagy was activated by exosome. In accordance with previous results, our data showed that TBI significantly up-regulated the expression of mitophagy-related proteins such as TOMM20, COX IV, Beclin-1, and LC3-II, exosome treatment further increased the expression of these proteins. In addition, both IF and TEM confirmed that mitophagy was activated after TBI and further promoted by exosome.
Recently, the interplay between mitophagy and different types of PCD, such as apoptosis, pyroptosis and ferroptosis has been proposed. In a rat subarachnoid hemorrhage (SAH) model, low serum triiodothyronine treatment reduced neuronal apoptosis and promoted mitophagy following SAH, inhibition of mitophagy by PINK1-siRNA reversed this preventative effect on apoptosis. 49 Moreover,

Han et al. indicated that Quercetin decreased pyroptosis-related
proteins, including NLRP3, cleaved caspase-1, GSDMD-N, and cleaved IL-1β through promoting mitophagy, thus preventing neuronal injury. 50 Consist with these results, the present study showed that inhibition of mitophagy by Mdivi-1 reversed the inhibitory effects of exosome on TBI-induced cell death, apoptosis, pyroptosis, and ferroptosis, indicating that exosome prevented TBI-induced PCD by enhancing mitophagy.
We further examined the potential activators of mitophagy in our system. The PINK1/Parkin-mediated ubiquitin pathway is the main pathway that drives mitophagy. 51 In Alzheimer's disease, Aβ1- In our study, we found that the expression of PINK1 and Parkin was increased after TBI, treatment of exosome further increased the expression of PINK1 and Parkin. Interestingly, we found that knockdown of PINK1 partly inhibited mitophagy and reversed the neuroprotection of exosome, suggesting that there might be other pathways involved in the activation of mitophagy by exosome. This was an interesting aspect for us to explore in the future.
There were some limitations in our study. Firstly, exosome owns a variety of properties such as release of non-coding RNAs. Thus, it should be clarified that whether the neuroprotection of exosome in TBI was contributed to other properties. Secondly, further studies were needed to investigate whether administration of exosome in different time courses might provide better neuroprotective effects against TBI.

| CON CLUS ION
Our study indicated that HucMSC-derived exosome could provide neuroprotection against TBI by combating cell death, apoptosis, pyroptosis, and ferroptosis through the PINK1/Parkin-mediated mitophagy. These results demonstrated that HucMSC-derived exosome may be a promising target for the treatment of TBI.

AUTH O R CO NTR I B UTI O N S
Li Zhang was responsible for the data collection, animal experiments, and manuscript writing. Yixing Lin was responsible for the design of the article. Wanshan Bai was responsible for the literature collection and manuscript review. Lean Sun was responsible for the data analysis. Mi Tian was responsible for the cell experiments and funding support. All authors read and approved the final manuscript.

FU N D I N G I N FO R M ATI O N
This work was supported by Grants from the National Natural Science Foundation of China (No. 82202392) from Mi Tian.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflict of interest.

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