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Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Attenuate Oxygen-Glucose Deprivation/Reperfusion-Induced Microglial Pyroptosis by Promoting FOXO3a-Dependent Mitophagy

Background Mesenchymal stem cell-derived exosomes (MSC-exos) have been recognized as a promising therapeutic strategy for neonatal hypoxic-ischemic brain damage (HIBD). Recently, microglial pyroptosis was shown to play a vital role in the progression of neonatal HIBD. However, whether MSC-exos improve HIBD by regulating microglial pyroptosis remains unknown. Methods Exosomes were isolated from human umbilical cord mesenchymal stem cells (huMSCs) and identified by transmission electron microscopy (TEM), western blot, and nanoparticle tracking analysis (NTA). BV-2 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to induce microglial ischemia/reperfusion (I/R) in vitro. CCK-8, ELISA, western blot, and Hoechst 33342/PI double staining were performed to detect the pyroptosis of BV-2 cells. Conditioned medium (CM) from BV-2 cells exposed to different treatments was used to investigate its effect on neuronal injury. Moreover, 3-methyladenine (3-MA) and mitochondrial division inhibitor-1 (mdi-1) were used to verify the involvement of mitophagy in the protection of MSC-exos against OGD/R-induced pyroptosis in BV-2 cells. Finally, FOXO3a siRNA was used to investigate the involvement of FOXO3a in MSC-exo-induced mitophagy and pyroptosis inhibition. Results Exosomes from huMSCs were successfully extracted. In OGD/R-exposed BV-2 cells, MSC-exos increased cell viability and decreased the expression of NLRP3, cleaved caspase-1, and GSDMD-N as well as the release of IL-1β and IL-18. Compared with CM from OGD/R-exposed BV-2 cells treated with PBS, CM from OGD/R-exposed BV-2 cells treated with MSC-exos significantly increased the viability of SH-SY5Y cells and decreased LDH release. MSC-exos also increased the expression of TOM20 and COX IV in OGD/R-exposed BV-2 cells. Additionally, 3-MA and mdi-1 attenuated the inhibition of pyroptosis with MSC-exo treatment. Furthermore, FOXO3a siRNA partially abolished the neuroprotective effect of MSC-exos and attenuated mitophagy and pyroptosis inhibition induced by MSC-exo treatment. Conclusions Our findings demonstrated that MSC-exos increased FOXO3a expression to enhance mitophagy, therefore protecting microglia from I/R-induced pyroptosis and alleviating subsequent neuronal injury.


Introduction
Neonatal hypoxic-ischemic brain damage (HIBD) is not only a serious threat to the lives of newborns but also the most important cause of long-term neurological dysfunction in infants [1,2]. Currently, only limited therapeutic inter-vention strategies are available to confer neuroprotective effects in this disease [1][2][3]. A preliminary clinical study by Kabataş et al. showed that Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) can effectively improve neurological function and quality of life in a patient with HIBD [4], suggesting that mesenchymal stem cell (MSC) transplantation may be a promising therapy for neonatal HIBD. However, direct application of MSCs is associated with potential risks and hazards, especially in babies who are considered a fragile population [5]. Recently, MSCderived exosomes (MSC-exos) have been identified as one of the key neuroprotective mechanisms of MSCs and can effectively ameliorate ischemia/reperfusion-(I/R-) induced brain injury by promoting angiogenesis, regulating immune responses, and inhibiting neuronal apoptosis [6][7][8][9][10]. MSCexos have emerged as an attractive therapeutic alternative that holds great regenerative potential and is cell-free [11]. Therefore, exploring the neuroprotective mechanism of MSC-exos is of great significance to their clinical application.
As the resident immune cells in the central nervous system, microglia play an important role in the occurrence and development of HIBD [12][13][14][15][16]. Previous studies have demonstrated that inhibition of microglial pyroptosis in neonatal HIBD mice could promote neuronal survival and improve brain injury [12,16]. The key signaling molecules in the classical pyroptosis pathway were found to be increased significantly in the serum of neonates with HIBD and positively correlated with the severity of the disease [16]. The above findings suggest that the reduction in microglial pyroptosis may be the key for curing neonatal HIBD. Recently, MSCexos were reported to inhibit pyroptosis in a variety of cells [17][18][19]. However, whether MSC-exos improve neonatal HIBD by inhibiting microglial pyroptosis remains unknown.
Mitophagy, a way to clear damaged mitochondria, plays a vital role in the activation and survival of microglia [20][21][22]. A recent study has showed that hypercapnia promotes microglial pyroptosis by inhibiting mitophagy in hypoxemic adult rats [23], suggesting that mitophagy acts as a negative regulator of microglial pyroptosis. To date, the effect of mitophagy on I/R-induced microglial pyroptosis remains unclear. Recently, MSC-exos have been shown to exert their protective role by promoting mitophagy in animal models of different diseases, such as nonalcoholic steatohepatitis [24] and cigarette smoke-(CS-) induced lung inflammation [25]. Therefore, it is worthwhile to explore whether mitophagy is involved in the protective effect of MSC-exos against I/R-induced microglial pyroptosis. In the present study, we investigated the effect and mechanism of MSC-exos against I/R-induced microglial pyroptosis in vitro using an oxygen-glucose deprivation/reperfusion (OGD/R) model in BV-2 cells.  Zhou). All cells were incubated in a humidified atmosphere of 5% CO 2 at 37°C.

Exosome Isolation and Characterization.
Exosomes were extracted from the cell culture supernatants of huMSCs using a VEX exosome isolation reagent in accordance with the manufacturer's instructions (cat. R601, Vazyme, Nanjing, China). In brief, an initial spin was performed at 3,000 g 4°C for 10 minutes to remove cells, and the supernatant was passed through a 0.22 μm filter (Millipore, Massachusetts, USA) to pelletize and exclude contaminating dead cells and debris. Then, 1/3 of the VEX Exosome Isolation Reagent was added proportionally to the starting sample volume, according to the manufacturer's instructions. Mixtures were vortexed and incubated at 4°C for up to 16 h and then centrifuged at 10,000 g 4°C for 30 minutes to precipitate exosome pellets. Pellets were resuspended in 1× PBS, and the resuspension volume for exosome pellets was 200 μl for 20 mL starting volumes according to the manufacturer's instructions. The bicinchoninic acid (BCA) protein assay kit (cat. BCA1-1KT, Sigma) was used to estimate exosome concentrations. The amount of MSC-exos obtained was 30 μg/mL medium. All exosomes were stored at -80°C immediately after isolation until further analysis. For transmission electron microscopy (JEM-1230, JEOL Ltd., Akishima, Japan), 10 μl of each sample was added to a copper mesh and precipitated for 3 min. The remaining liquid was carefully pipetted from the filter paper edge. Thereafter, the filter paper was rinsed with PBS, and phosphotungstic acid was used for negative staining prior to drying at room temperature for 2 min and imaging (operating voltage: 80-120 kV). The expression levels of exosome-specific biomarkers, CD9, CD63, and Alix, were analyzed by western blot. Nanoparticle tracking analysis (NTA) was performed as previously described [26]. Briefly, the exosome particle size and concentration were measured using NTA at Vivacell Biosciences with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and the corresponding software ZetaView 8.04.02. Isolated exosome samples were appropriately diluted using 1× PBS buffer to measure the particle size and concentration. NTA measurement was recorded and analyzed at 11 positions. The ZetaView system was calibrated using 110 nm polystyrene particles. Temperature was maintained at approximately 23°C and 30°C.
2.3. OGD/R Induction, Exosome Treatment, and Collection of Conditioned Medium. For OGD/R induction, BV-2 cells were cultured in glucose-free MEM (cat. PM150443, Procell, Wuhan, China) and then transferred to a sealed hypoxic box containing a mixture of 95% N 2 and 5% CO 2 at 37°C for 4 h. Thereafter, the cells were cultured in normal MEM with 10% FBS and maintained for 24 h in reoxygenation under normoxic conditions. BV-2 cells cultured in growth culture medium under normoxic conditions served as a control. After being cultured in oxygen-glucose deprivation (OGD) for 4 h, BV-2 cells were treated with fresh normal MEM with 10% FBS containing 40 μg/mL MSC-exos and placed back in a 5% CO 2 incubator for 24 h. PBS incubation was set as the control. Conditioned medium (CM) from BV-2 cells with  2.12. Lactate Dehydrogenase (LDH) Activity Detection. The LDH content in the supernatants of SH-SY5Y cells was measured by using an LDH kit (cat. no. A020-2-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's manual. The absorbance OD value at 450 nm was measured by using a plate reader (ELx800, BioTek Instruments, Inc.). The LDH level of the control group was expressed as 100%, and the LDH levels of the other groups were normalized to this value.
2.13. Statistical Analysis. The data represent the mean ± standard deviation (SD) from at least three separate experiments. Statistical analyses were carried out using SPSS software version 15.0 (SPSS Inc., Chicago, IL). Student's t-test was used to analyze differences between two groups. When comparisons between multiple groups were carried out, one-way ANOVA followed by SNK tests was employed. Statistical significance was considered when P < 0:05.

Isolation and Identification of MSC-exos.
To investigate the potential roles of MSC-exos in I/R-induced neuronal injury, MSC-exos were first isolated and verified with transmission electron microscopy (TEM), western blot, and nanoparticle tracking analysis (NTA). The TEM results revealed that MSC-exos exhibited round-shaped morphology (Figure 1(a)), which is consistent with the typical exosomal morphology. Western blot results indicated that the levels of specific exosome surface markers (CD9, CD63, and Alix) were significantly upregulated in MSCs-exos (Figure 1(b)). NTA data indicated that the diameters of the MSC-exos were mostly approximately 100 nm (Figure 1(c)). Collectively, these results confirmed that the MSC-exos (30 μg/mL medium) were successfully isolated and identified.

Discussion
Although Kabataş et al. clinically demonstrated the neural repair effect of MSCs in a patient with HIBD [4], the underlying mechanism remained largely unknown. MSC-exos were demonstrated to be one of the key mediators of MSC paracrine signaling and exerted a potential neuroprotective effect against hypoxic-ischemic-(H/I-) induced brain injury in vitro and in vivo [8,9,35,36]. This suggested that MSCexos may be more suitable for the treatment of HIBD than MSCs. However, the neuroprotective mechanism of MSCexos has not been thoroughly elucidated. The present study investigated the potential protective mechanism of MSCexos against I/R-induced neuronal injury. In the current study, TEM, NTA, and surface marker proteins confirmed that MSC-exos were successfully isolated and met international standards [37]. A further study showed that MSCexos at a dose of 40 μg/mL attenuated the injury of SH-SY5Y cells induced by OGD/R-exposed BV-2 cells. Our study was concordant with a previous study showing that MSC-exos could inhibit microglia-mediated neuroinflammation in perinatal brain injury [36].
Previous studies revealed that microglial pyroptosis plays an important role in the progression of neonatal HIBD [16]. Additionally, it has been shown that MSC-exos protect against H/I-induced injury by inhibiting pyroptosis in various cells, such as endothelial cells, cardiomyocytes, and neuronal cells [19,35,38,39]. In the present study, MSC-exos significantly increased cell viability, inhibited the release of IL-1β and IL-18, and downregulated the expression levels of the key proteins associated with pyroptosis in OGD/Rexposed BV-2 cells. These findings suggested that the protective effect of MSC-exos against I/R-induced neuronal injury was associated with a reduced microglial pyroptosis. To our knowledge, the present study is the first to report that MSC-exos attenuate I/R-induced neuronal injury by preventing microglial pyroptosis.
We then investigated how MSC-exos inhibited microglial pyroptosis. Mitophagy is a process in which damaged mitochondria are selectively removed by autophagy mechanisms to maintain the stability of the intracellular environment [40]. Mitophagy has been shown to inhibit the activation of the NLRP3 inflammasome by reducing the release of ROS, which is an important activator of the NLRP3 inflammasome [41][42][43]. Recently, activation of mitophagy has been demonstrated to be a vital protective mechanism of MSC-exos against different diseases [24,25]. Importantly, the inhibitory effect of mitophagy on pyroptosis has been demonstrated in several reports [23,29,30]. In accordance with these results, the present study showed that  (e, f) SH-SY5Y cells were cultured in CM collected from BV-2 cells treated as indicated in (b); after 24 h of culture, the viability of SH-SY5Y cells was determined by the CCK-8 assay (e), and LDH release was measured by ELISA with a specific kit (f). * * P < 0:01 versus the control group; ## P < 0:01 versus the OGD/R group; @@ P < 0:01 versus the siNC+PBS group; && P < 0:01 versus the siNC+Exos group; ▲▲ P < 0:01 versus the PBS+CM-NC group; ▼▼ P < 0:01 versus the PBS+CM-si3a group.
MSC-exos treatment significantly upregulated the levels of mitophagy-related proteins in OGD/R-exposed BV-2 cells. When mitophagy was blocked with 3-MA or mdi-1, the inhibitory effect of MSC-exos against OGD/R-induced pyroptosis was markedly reversed in BV-2 cells. These data strongly indicated that MSC-exos prevent OGD/R-induced pyroptosis of microglial BV-2 cells by enhancing mitophagy.
We also examined the potential activators of mitophagy in our system. FOXO3a, a transcription factor of the O subclass of the forkhead family, has been shown to promote the activation of mitophagy by regulating the expression levels of key proteins associated with mitophagy, such as Parkin and BNIP3 [31,32]. FOXO3a has also been reported to inhibit pyroptosis by regulating the inflammatory response [33,34]. Recently, FOXO3a was demonstrated to be an important downstream target of MSC-exos [17]. Our results showed that MSC-exos significantly upregulated FOXO3a expression in OGD/R-exposed BV-2 cells. FOXO3a siRNA not only reversed MSC-exo-induced mitophagy and pyroptosis inhibition in OGD/R-exposed BV-2 cells but also inhibited the protective effect of MSC-exos against BV-2 cell-mediated injury in SH-SY5Y cells. These findings suggested that MSC-exos ameliorate OGD/R-induced injury of SH-SY5Y cells by inhibiting pyroptosis of microglial BV-2 cells through promoting FOXO3a-dependent mitophagy. However, the mechanism by which FOXO3a regulates mitophagy in our system remains unclear. Mitophagy has been demonstrated to be mediated by two distinct pathways: one involves mitophagy receptors such as BNIP3, FUNDC1, and NIX and the other pathway is dependent on Parkin/ PINK1 pathway-mediated ubiquitination [40]. Previous studies showed that FOXO3a could regulate Parkin expression at the transcriptional level [32,44]. The expression level of BNIP3 was also reported to be regulated by FOXO3a [31,45]. Whether the Parkin and/or BNIP3 pathway is involved in FOXO3a-mediated mitophagy in our system is an interesting point for us to investigate in the near future.
It is well known that MSC-exos contain various micro-RNAs (miRNAs), circular RNAs (circRNAs), long noncoding RNAs (lncRNAs), proteins, and mRNAs that can be stably transferred to recipient cells [46,47]. Recent studies have shown that abnormally expressed circRNAs may be involved in the pathogenesis of neonatal HIBD [48]. Furthermore, exosome-shuttled circRNAs have been shown to improve ischemic brain injury [49]. Therefore, it is worthwhile to investigate which circRNAs in MSC-exos inhibit microglial pyroptosis and ameliorate neuronal damage by regulating FOXO3a in our system.

Conclusions
In conclusion, our findings revealed that MSC-exos upregulated FOXO3a expression to enhance mitophagy and repress I/R-induced pyroptosis in microglia, therefore alleviating subsequent neuronal injury ( Figure 6). Although the effect and mechanism of MSC-exos against cerebral I/R injury found in the present study will be verified in further in vivo studies, the salient findings from the present study provide a microglia-centric view of the neuroprotective effect of MSC-exos. MSC-exos, as a new candidate therapeutic strategy, have excellent application prospects for neonatal HIBD.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.