MiR-26b-5p-modified hUB-MSCs derived exosomes attenuate early brain injury during subarachnoid hemorrhage via MAT2A-mediated the p38 MAPK/STAT3 signaling pathway
Zunwei Liu, Bo Wang, Qihang Guo
a Department of Renal Transplantation, Nephropathy Hospital, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710061, Shaanxi, China
b Center for Brain Science, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710061, Shaanxi, China
A B S T R A C T
Early brain injury (EBI) is a major cause of adverse outcomes following subarachnoid hemorrhage (SAH). There is evidence that mesenchymal stem cells (MSCs) – derived exosomes are involved in the repair of SAH. EXosomes were extracted from human umbilical cord mesenchymal stem cells (hubMSCs) and identified. OXyHb treated PC12 cells were transfected with exosomes alone or together with miR-26b-5p inhibitor. Hub-MSCs derived exosomes promote cell proliferation, inhibit apoptosis and reduce inflammatory mediator expression. Trans- fection of miR-26b-5p inhibitor abolished the promoting effect of exosomes on the proliferation of PC12 cells, as well as the inhibitory effect on cell apoptosis. In addition, methionine adenosyltransferase II alpha (MAT2A) was one target gene of miR-26b-5p. OXyHb treated PC12 cells were transfected with exosomes alone or together with pcDNA-MAT2A and observed that the promoting effect of exosomes on PC12 cell proliferation was abolished by pcDNA-MAT2A, which was the same as the effect of miR-26b-5p inhibitor. OXyHb treated PC cells incubated with exosomes were transfected with miR-26b-5p inhibitor alone or together with si-MAT2A, respectively, and it was observed that exosomes decreased the phosphorylation levels of p38 MAPK and STAT3 proteins, inhibited cell apoptosis and inflammatory mediator expression, and miR-26b-5p inhibitor abrogated the effects of exo- somes, while transfection of si-MAT2A reversed the effects of miR-26b-5p inhibitor. Moreover, injection of miR- 26b-5p inhibitor resulted in increased MAT2A and pathway protein expression, increased inflammatory medi- ators, and aggravated neurological symptoms in the brain tissues of SAH rats.
1. Introduction
Subarachnoid hemorrhage (SAH) refers to a clinical syndrome caused by rupture of blood vessels at the base or surface of the brain and homeopathic flow of blood into the subarachnoid space, accounting for about 10 % of acute stroke (Qin et al., 2019). The pathophysiological mechanisms of SAH are mainly early brain injury (EBI) and delayed brain injury (DBI) after subarachnoid hemorrhage. Increasing evidence suggests that EBI is an important factor affecting the poor prognosis of SAH patients, so in-depth study of EBI will be helpful to propose treat- ment methods to improve the prognosis of SAH patients.
Stem cells are known as seed cells for repair and reconstruction of damaged tissues. Among them, mesenchymal stem cells (MSCs) are adult stem cells derived from the early stage of mesoderm development with the potential of self-repair and multi-differentiation (Faal et al., 2019; Zhang et al., 2014). Studies have found that intraventricular transplantation of human umbilical cord mesenchymal stem cells (hUC-MSCs) significantly alleviates SAH-induced chronic hydrocepha- lus, inflammatory cytokine secretion and behavioral disorders, and knockdown of transforming growth factor-beta 1 expression in hUC-MSCs enhances these effects (Chen et al., 2020). In addition, one study found that SAH rats transplanted with bone marrow mesenchymalstem cells (BMSCs) showed reduced neurological dysfunction, decreased expression of pro-inflammatory factors including interleukin (IL)-1β, IL-6 and TNF-α in the left hemisphere, and increased secretion ofanti-inflammatory factor IL-10 (Liu et al., 2019). When BMSCs were co-incubated with oXyhemoglobin-activated BV-2 microglia, it was observed that oXyHb-induced BV2 activation and polarization were inhibited, and the level of inflammatory genes was strongly down- regulated (Zhang et al., 2020).
Withing the deepening of research, exosomes derived from MSCs have attracted extensive attention and are considered as potential drug carriers, which are expected to replace MSCs transplantation. EXosomes are microvesicle-like structures produced and secreted by cells, con- taining a variety of proteins, mRNAs, miRNAs and other bioactive sub- stances, which not only participate in cell transport and cell communication, but also participate in cell composition as components of organelles (Fan et al., 2020; Li et al., 2020b; Song and Zhang, 2020). Increased evidence suggests that MSCs-derived exosomes are involved in the repair of brain injury. One study showed that traumatic brain injury animals treated with MSCs-derived exosomes at the same time exhibited shorter neurological recovery cycles and less brain damage than saline alone (Williams et al., 2019). After systemic administration of exosomes derived from MSCs to Wistar rats with experimental traumatic brain injury, it was observed that the sensorimotor function of rats recovered, the number of neonatal endothelial cells in the lesion boundary zone increased, the number of neonatal mature neurons in the dentate gyrus increased, and the neuroinflammation disappeared (Zhang et al., 2017). Other studies showed that BMSCs-exosomes significantly improved neurological function, decreased brain water content, maintained the integrity of the blood-brain barrier, and attenuated neuroinflammation in SAH rats, which was mediated by the inhibition of HMGB1-TLR4 pathway activity by miR-129-5p in exosomes (Xiong et al., 2020). Recently, there is evidence that exosomes miR-26b-5p derived from hUC-MSCs can inhibit M1 polarization of microglia by inactivating toll-like receptor pathway by targeting CH25H, thereby alleviating neurological injury after cerebral ischemia/reperfusion (Li et al., 2020a). However, the regulatory role of exosome miR-26b-5p derived from hUC-MSCs in early brain injury was still unclear.
S-adenosylmethionine, the biological methyl donor required for methylation of major nucleic acids, phospholipids, histones, biogenic amines and proteins and the precursor of polyamines and glutathione, is biosynthesized by methionine adenosyltransferase (MATs) and is essential for cell growth and differentiation. Mammalian cells express three genes, MAT1A, MAT2A, and MAT2B, with distinct expression and function. A study called decreased NRF2 expression and increased MAFG expression in astrocytes during the course of chronic inflamma- tory disease in the CNS, which synergized with MAT2A to promote DNA(injection of saline into the cisterna magna), SAH (tail vein injection, 100 μg/mL PBS), SAH exosomes (tail vein injection, 100 μg/mL), SAH exosomes NC inhibitor, SAH exosomes miR-26b-5p in-hibitor. This study was approved and supervised by the Committee of Xi’an Jiaotong University Laboratory Animal Center, and the experi- mental process conformed to the principles of the use and protection ofexperimental animals.
2. Materials and methods
2.1. Animals
The healthy adult SD rats used in this study were provided by the Laboratory Animal Center of China Medical University, weighing300 320 g. All experimental animals were raised by professionals under conditions of 12 h/12 h light and dark cycles per day, and con- stant indoor temperature and humidity with free access to food and water. Establishment of SAH animal model by classical occipital cistern secondary blood injection method (Pu et al., 2019). Thirty SD rats were randomly divided into five groups, siX in each group, namely: ShamChina). Real-time PCR was conducted by using SYBR PremiX EX TaqTM Kit (Applied Biosystems, Foster City, CA, USA). The reaction was run inABI7500 Real-time PCR system (Applied Biosystems, Carlsbad, CA). U6 were used as an endogenous control. Briefly, 2 μL of cDNA was added to 10 μL of the 2 × SYBR green PCR master miX with 0.4 μL of Taq poly- merase enzyme (RiboBio, China), 0.8 μL of each primer and 6 μL ddH2O to a final volume of 20 μL. The RT-qPCR cycling conditions consisted of:95 ◦C for 3 min; then 35 cycle amplification for 20 s at 95 ◦C, 30 s at55 ◦C, 15 s at 72 ◦C; followed by 1 min at 72 ◦C. The primers used in this study were synthesized by Sangon Biotech (Shanghai, China). The2—ΔΔCt method was used for quantitative analysis.
2.2. Cell culture and transfection
HUC-MSCs and PC12 cells were purchased from the American Typical Culture Collection (ATCC, Manassas, VA, USA). Cells were incubated in containing 10 % fetal bovine serum, 100 U/mL penicillinand 100 μg/mL streptomycin (Sigma,St. Louis, MO, USA) in RPMI 1640medium (Gibco, Rockville, MD). When the cells grew vigorously and the fusion degree was about 80 %, they were digested with 1 mL of 0.25 % trypsin. When most of the cells were observed to be shrunken and rounded and floating on the liquid surface in a flowing state, 3 mL DMEM was added to terminate digestion and passaged at a ratio of 1:2. The miR-26b-5p inhibitor and negative control inhibitor were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Transfection wasperformed by using Lipofectamine 2000 transfection reagent (Invi- trogen, Carlsbad, CA, USA) according to the manufacturer’s in-structions. The pcDNA3.1 and pcDNA-MAT2A were purchased from RiboBio Co., Ltd (Guangzhou, China) and transfected into cells accord- ing to the instructions.
2.3. Isolation and extraction of exosomes
The exosomes derived from hUC-MSCs were extracted by using the exosomes extraction kits, and the specific operation was as follows. The P2 generation hUC-MSCs were incubated in DMEM medium without exosomes for 24 h, and 20 mL cell culture supernatant was collected, centrifuged at 3,000 g for 30 min, and the precipitation was discarded. The supernatant was taken and the extraction reagent was added at aratio of 5:1. The supernatant was placed at 4℃ for 12 h, and thencentrifuged at 100,000 g for 60 min to obtain exosomes precipitation. The PBS was resuspended and repeatedly blown with a pipette gun until the precipitate was dissolved. The concentration was adjusted to beabout 50 and 100 μg/mL. The samples were packaged and stored at-80℃ for subsequent experiments.
2.4. RT-qPCR
Total RNA was isolated from PC12 cell by using the TRIzol (Invi- trogen, Carlsbad, CA, USA). Then the first strand of cDNA was synthe-methylation and suppress antioXidant and anti-inflammatory tran-sized by using MMLV Reverse Transcriptase kit (TaKaRa, Da Lian,scriptional programs, aggravating neuroinflammation (Wheeler et al., 2020), suggesting that MAT2A may play an important role in brain injury.
In conclusion, this study deeply explored the specific protective mechanism of miR-26b-5p-modified hUB-MSCs derived exosomes in SAH, aiming to provide new ideas for alleviating early brain injury.
2.5. MTT assay
When the density of cells reached 80 %, cells were digested with 0.25% trypsin, and single cell suspension was made with DMEM medium containing 10 % serum. 200 μL of culture medium containing 2 103 cells were added into each well of 96-well plate, and incubated in a 5% CO2 incubator at 37℃ for 72 h. According to the time point, add 20 μL of5 mg/mL MTT solution into each hole, continue to culture for 4 h, then stop the culture, discard the culture supernatant; add 150 μL DMSO into each hole and shake for 10 min to dissolve the crystals fully, and mea-sure the absorbance value of each hole at 490 nm wavelength. Each experimental procedure was processed at least three times.
2.6. Flow cytometry
Cells were digested with 0.25 % trypsin, rinsed with PBS three times, and all collected cells were transferred to a 15 mL centrifuge tube, centrifuged at 1,500 rpm for 5 min, and the supernatant was discarded.
The cells were washed with PBS, and the supernatant was discarded by centrifugation and repeated twice. 400 μL PBS was added to resuspend the cells, while 2 mL of pre-cooled ethanol was added, and the cells were rested at -20℃ for 30 min. Next, the cells were centrifuged at 1,500 rpm for 5 min, resuspended with 1000 μL PBS, separated at 1,500 rpm for5 min, and incubated for 30 min in the dark after staining, then apoptosis was detected by flow cytometry.
2.7. Luciferase reporter gene assay
StarBase (http://starbase.sysu.edu.cn/) was used to predict potential target genes of miR-26b-5p, and MAT2A was screened as the research object. The wild type and mutant 3′-UTR sequences of MAT2A werecloned into the PGL3 vector (Promega, USA) fused luciferase gene sequence, respectively. HEK293 cells were seeded in 24-well plates, and when growing to approXimately 70 % confluence, co-transfected with luciferase plasmids and miR-26b-5p mimic or NC mimic by using Lip- ofectamine 2000. After 48 h of transfection, the luciferase reporter ac-tivity was measured by Dual-Luciferase Reporter Assay System (Promega, Madison, WI) under the manufacturer’s instructions.
2.8. Western blotting
Total protein was extracted from cells or brain tissues by using Pro- prep TM protein EXtraction Solution (Daejeon, Korea). The protein content of each sample was determined by using the BCA Protein Assay Kits (Thermo Scientific). Then, equal amounts of proteins (15 μg/lane) were separated on a 12 % sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to polyvinylidenedifluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA).The membranes were blocked in 5% (w/v) nonfat dry milk in TBST (Tris-buffered saline-0.1 %Tween) at 25 ◦C for 3 h and then incubated with the following primaryantibodies: rabbit polyclonal anti-GAPDH antibody (1:1000, Abcam, ab8245), rabbit monoclonal anti-CD63 antibody (1:1500, Abcam, ab217345), mouse monoclonal anti-CD81 antibody (1:1200, Abcam, ab79559), rabbit monoclonal anti-TSG101 antibody (1:2500, Abcam, ab125011), rabbit monoclonal anti-AliX antibody (1:2000, Abcam, ab186492), Rabbit polyclonal anti-MAT2A antibody (1:2500, Abcam, ab154343), Rabbit monoclonal anti-COX-2 antibody (1:2000, Abcam, ab15191), Rabbit monoclonal anti-MCP-1 antibody (1:1000, Abcam, ab214819), Rabbit monoclonal anti-iNOS antibody (1:800, Abcam, ab178945), rabbit monoclonal anti-STAT3 antibody (1:1500, Abcam, ab68153), rabbit monoclonal anti-AKT (phospho Y705) antibody (1:5000, Abcam, ab76315). Then, the membranes were incubated with horseradish peroXidase (HRP)-COnjugated goat anti-rabbit IgG (1:3000, Abcam, ab6721) or goat anti-mouse IgG (1:3000, Abcam, ab6728) for 1 h at room temperature. The bands were visualized by using an ECL Plus Chemiluminescence Reagent Kit (Pierce, Rockford, IL, USA) and were photographed by a chemiluminescence imaging system. Image J software was used to quantify the band densities.
2.9. Statistical analysis
All statistical analyses were performed by using the SPSS software (ver.22.0; SPSS, Chicago, IL). All data were shown as mean ± SD.
Comparisons between two groups were made by the student’s t-test. Data between multiple groups were performed with one-way analysis of variance (ANOVA) followed by post hoc analysis with LSD test. P＜0.05 was considered statistical significance.
3. Results
3.1. Identification of exosomes derived from hUC-MSCs
HUC-MSCs culture supernatant was collected, and then cell-derived exosomes were extracted with a kit and purified by ultracentrifugation. Westem Blotting was used to detect the expression of exosomes marker proteins in the precipitates isolated from hUC-MSCs. The results showed that the expression of exosomes marker proteins including CD63, CD81, AliX and TSG101 was strongly positive in the extracts isolated and pu- rified from hUC-MSCs, which confirmed that the obtained extracts were exosomes (Fig. 1A&B). The conditioned medium (CM) supplemented with exosomes inhibitor GW4869 was used as the control.
3.2. Effects of exosomes derived from hUC-MSCs on oxyHb-induced PC12 cells
It is well known that neuronal apoptosis and inflammatory response are the main causes of early brain injury caused by SAH. Here, we used oXyHb to induce PC12 neurons to simulate the SAH model in vitro, and further explored the effects of exosomes derived from hUC-MSCs on injured cells. We found that oXyHb induction significantly inhibited cell proliferation, promoted cell apoptosis, and increased the expression of inflammatory mediators including COX-2, MCP-1 and iNOS in cells compared with the control group. Moreover, oXyHb-induced PC12 cellswere treated with exosomes at concentrations of 50 and 100 μg/mL,respectively, and we observed enhanced cell proliferation (Fig. 2A), decreased apoptosis (Fig. 2B), and decreased expression of inflamma- tory mediators (Fig. 2C) in the cells. Similarly, we found that exosomeswith a concentration of 100 μg/mL had a more significant regulatoryeffect on cells.
3.3. Effects of exosomes derived from hUC-MSCs modified by miR-26b- 5p on oxyHb-induced PC12 cells
Studies have reported that exosomes miR-26b-5p derived from hUC- MSCs has an inhibitory effect on neuroinflammation, but its potential regulatory role in SAH is still worth studying. Next, we explored the effect of exosomes derived from hUC-MSCs on oXyHb-induced expres- sion of miR-26b-5p in PC12 cells. We found that the expression of miR- 26b-5p was significantly down-regulated in oXyHb-induced PC12 cells compared with the control group, however, the expression of miR-26b- 5p was restored when oXyHb-induced PC12 cells were treated withexosomes at a concentration of 100 μg/mL. Interestingly, transfection ofthe inhibitor of miR-26b-5p in injured cells incubated with exosomes, we observed that the expression of miR-26b-5p was decreased again (Fig. 3A). Furthermore, oXyHb inhibited cell proliferation, promoted apoptosis and secretion of inflammatory mediators, while exosomes treatment significantly reversed the effect of oXyHb on cells, but with the intervention of miR-26b-5p inhibitor, we found that the protective effect of exosomes on injured cells was counteracted, manifested by the weakened cell proliferation ability (Fig. 3B), increased apoptosis (Fig. 3C) and increased secretion levels of inflammatory mediators (Fig. 3D).
3.4. MAT2A was a direct target of miR-26b-5p
StarBase3.0 (http://starbase.sysu.edu.cn/) was used to predict po- tential target genes of miR-26b-5p, and MAT2A was screened as the research object. The wild type and mutant 3′-UTR sequences of MAT2Awere shown in Fig. 4A. Next, the luciferase reporter gene analysisshowed that miR-26b-5p mimic significantly reduced the luciferase ac- tivity of wild-type MAT2A, but had no significant effect on the luciferase activity of mutant-type MAT2A (Fig. 4B). To further verify the target relationship between miR-26b-5p and MAT2A, we bi-directionally regulated the expression of miR-26b-5p in cells. Western blotting re- sults showed that miR-26b-5p mimic significantly downregulated the protein expression of MAT2A. Conversely, transfection of miR-26b-5p inhibitor promoted the expression of MAT2A (Fig. 4C).
3.5. Effect of regulating MAT2A expression on oxyHb-induced PC12 cells
Previous studies found that oXyHb-induced expression of miR-26b- 5p was significantly downregulated in PC12 cells, and we confirmed that miR-26b-5p directly targeted the 3′ UTR of MAT2A. Here, wedetected the expression of MAT2A in PC12 cells induced by oXyHb and further explored the effect of regulating MAT2A expression on cells. As expected, oXyHb alone induced an increase in the protein expression of MAT2A in PC12 cells. In the combination treatment group, we observed that the protein expression of MAT2A was inhibited, but this inhibition was counteracted after overexpression of MAT2A (Fig. 5A). Moreover, overexpression of MAT2A in injured cells incubated with exosomes significantly inhibited cell proliferation (Fig. 5B), promoted apoptosis (Fig. 5C) and upregulated the secretion levels of inflammatory media- tors including COX-2, MCP-1 and iNOS (Fig. 5A&D).
3.6. MiR-26b-5p-modified exosomes inhibited p38 MAPK/STAT3 signaling pathway to alleviate oxyHb-induced cell injury
In this study, we found that the phosphorylation levels of p38 MAPK and STAT3 proteins, apoptosis and secretion of inflammatory mediators were decreased in exosomes-treated cells compared with oXyHb alone induction (Fig. 6A). As mentioned earlier, inhibition of miR-26b-5p expression in exosomes-treated cells significantly reversed the protec- tive effect of exosomes on oXyHb-induced cell injury. Furthermore, we simultaneously inhibited the expression of miR-26b-5p and MAT2A incells treated with oXyHb and exosomes. The results showed thatknockdown of MAT2A expression decreased the phosphorylation levels of p38 MAPK and STAT3 proteins, decreased apoptosis (Fig. 6B), and inhibited the secretion of inflammatory (Fig. 6C) mediators compared with transfection of miR-26b-5p inhibitor alone. To further validate the specific role of p38 MAPK, we introduced the p38 MAPK inhibitor SB20358 in oXyHb treated PC12 cells. The results showed that consistent with the effects of MAT2A siRNA, SB20358 decreased the phosphory- lation levels of p38 MAPK and STAT3, decreased the expression of in- flammatory mediators COX-2, MCP-1 and iNOS in cells, and inhibited cell apoptosis (supplementary Fig. 1).
3.7. MiR-26b-5p-modified exosomes attenuate early brain injury in SAH rats
Next, we used the classical occipital cistern secondary bloodinjection method to establish an animal model of SAH and validate the neuroprotective effect of miR-26b-5p-modified exosomes in in vivo. Compared with sham group, the expression of miR-26b-5p was down- regulated, the expression of MAT2A protein was increased, and the phosphorylation levels of pathway-related proteins were increased in brain tissue of SAH rats. Compared with SAH group, exosomes signifi- cantly upregulated the expression of miR-26b-5p (Fig. 7A), decreased the expression of MAT2A protein, and decreased the phosphorylation levels of pathway-related proteins (Fig. 7B), but the regulatory effect of exosomes was significantly reversed by miR-26b-5p inhibitor. Further- more, 24 h after modeling, exosomes significantly alleviated neurolog- ical symptoms (Fig. 7C) and brain edema (Fig. 7D) in SAH rats, but transfection of miR-26b-5p inhibitor reversed the neuroprotective effect of exosomes. Similarly, we observed that miR-26b-5p inhibitor reversed the inhibitory effect of exosomes on the production of inflammatory mediators (Fig. 7E).
4. Discussion
Numerous studies have shown that neuroinflammation and blood- brain barrier disruption are the two main pathological mechanisms ofearly brain injury after SAH. After SAH, blood rapidly enters the sub- arachnoid space, resulting in increased intracranial pressure, decreased cerebral blood flow, and transient cerebral ischemia. Stimulated by erythrocyte lysates and transient cerebral ischemia, intracellular in- flammatory pathways are activated, and a large number of inflamma-tory mediators are synthesized and released, ultimately leading to brain edema (Liu et al., 2018; Xie et al., 2018). What’s worse, neuronal cells undergo apoptosis under stress conditions, which directly affects various neural activities. After the blood-brain barrier is destroyed, the perme- ability increases, which promotes the infiltration of inflammatory cells, especially neutrophils, and the occurrence of brain edema, further aggravating the inflammatory response, leading to neurological deficits after SAH (Xu et al., 2017). Therefore, alleviating the inflammatory response after SAH is expected to improve the prognosis of SAH patients. To promote functional recovery in patients with nerve injury, re- searchers have attempted to apply MSCs transplantation to restore normal nerve function. With the deepening of research, more evidence points out that transplanted cells promote endogenous nerve remodeling and enhance angiogenesis by paracrine way, and then improve the re- covery of nerve function after brain injury. At present, exosomes or extracellular vesicles derived from MSCs have been widely used inanimal models including traumatic brain injury, cerebral ischemia and cerebral hemorrhage.
Studies have shown that MSCs-derived exosomes significantly in- crease cerebrovascular density and the number of new neurons, inhibit the activation of astrocytes and microglia, and alleviate neurological symptoms in traumatic brain injury rats (Zhang et al., 2015). In a rat striatal hemorrhage model, systemic administration of MSCs-derived exosomes improved spatial memory, learning, motor and sensory memory functions in rats, possibly by promoting angiogenesis and axonal remodeling (Han et al., 2018). In addition, compared with PBS treatment, BMSCs-derived exosomes significantly inhibited the expres- sion of pro-apoptotic proteins and pro-inflammatory cytokines, while down-regulating the expression of iNOS and up-regulating Arg1 expression to regulate microglia/macrophage polarization, thereby reducing neuroinflammation in mice with craniocerebral injury (Ni et al., 2019).
In this study, we observed that hUC-MSCs derived exosomes reduced oXyHb-induced apoptosis and production of inflammatory mediators. In-depth study revealed that the protective effect of exosomes on PC12 neurons was mediated by miR-26b-5p. Mechanistic studies showed that exosomes derived from hUC-MSCs modified by miR-26b-5p blocked the MAPK/STAT3 signal transduction system to promote neurological re- covery by inhibiting MAT2A expression. MAT2A belongs to the adeno- sylmethionine transferase family, whose members, under the action of ATPase, can catalyze methionine to generate important methyl donors in organisms, and then regulate the methylation of DNA and histones (Zhao et al., 2018). Unfortunately, the study of MAT2A in cerebral hemorrhagic diseases has not been reported yet, but the related studiesin other diseases can still support our conclusion. Some studies have found that MAT2A is up-regulated in TGF-β1-induced hepatic stellate cell, induces p65 phosphorylation, activates the NF-κB transduction system, and aggravates liver fibrosis. Inhibiting the expression ofMAT2A with NPLC0393, a small molecule inhibitor of TGF-β1, decreased the phosphorylation level of p65, but overexpressing MAT2 abolished the therapeutic effect of NPLC0393 (Wang et al., 2019).
Early brain injury caused by subarachnoid hemorrhage is mainly related to neuronal apoptosis and inflammatory response. A large number of studies have confirmed that MAPK and STAT3 signal trans- duction systems play important roles in inflammation, stress response, cell survival and apoptosis. The expression of MMP-9 protein and phosphorylated p38 MAPK in the sciatic nerve has been found to be positively correlated with the severity of the disease. Treatment with p38 MAPK inhibitor (SB203580) significantly reduced the levels of MMP-9 mRNA and protein in the sciatic nerve at the peak of autoim- mune neuritis, and alleviated the severity of the nerve lesion (Sun et al., 2015).In the progression of intracerebral hemorrhage, Leucine-rich repeat kinase 2 has been reported to mediate inflammatory response, cognitive dysfunction, blood-brain barrier damage, and neuronal apoptosis via the p38 MAPK/Drosha pathway, thereby accelerating secondary brain injury (Cao et al., 2018). Similarly, a large body of evidence suggests that the STAT3 pathway is involved in the develop- ment of early brain injury after SAH. Recent studies have shown that recombinant human erythropoietin inhibits brain cell apoptosis, neuronal necrosis, albumin exudation and brain edema in experimental SAH by downregulating the phosphorylation levels of JAK2 and STAT3 proteins in the cortex (Wei et al., 2017).
In conclusion, our findings further support the positive role of MSCs- derived exosomes in brain injury repair, which provides a new reference for improving the prognosis of SAH patients.
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