Antioxidant treatment enhances human mesenchymal stem cell anti-stress ability and therapeutic efficacy in an acute liver failure model

One of the major problems influencing the therapeutic efficacy of stem cell therapy is the poor cell survival following transplantation. This is partly attributed to insufficient resistance of transplanted stem cells to oxidative and inflammatory stresses at the injured sites. In the current study, we demonstrated the pivotal role of antioxidant levels in human umbilical cord mesenchymal stem cells (hUCMSCs) dynamic in vitro anti-stress abilities against lipopolysaccharide (LPS)/H2O2 intoxication and in vivo therapeutic efficacy in a murine acute liver failure model induced by D-galactosamine/LPS (Gal/LPS) by either reducing the antioxidant levels with diethyl maleate (DEM) or increasing antioxidant levels with edaravone. Both the anti- and pro-oxidant treatments dramatically influenced the survival, apoptosis, and reactive oxygen species (ROS) production of hUCMSCs through the MAPK-PKC-Nrf2 pathway in vitro. When compared with untreated and DEM-treated cells, edaravone-treated hUCMSCs rescued NOD/SCID mice from Gal/LPS-induced death, significantly improved hepatic functions and promoted host liver regeneration. These effects were probably from increased stem cell homing, promoted proliferation, decreased apoptosis and enhanced secretion of hepatocyte growth factor (HGF) under hepatic stress environment. In conclusion, elevating levels of antioxidants in hUCMSCs with edaravone can significantly influence their hepatic tissue repair capacity.

green fluorescent light visualization. Quantification of green fluorescence was analyzed by using ImageJ (Version 1.48, National Institutes of Health, Bethesda, MD).

RNA extraction and quantitative PCR assay. Total RNA of cells was extracted by using illustra TM
RNAspin mini kit (GE healthcare, UK). The preparation of the first-strand cDNA was conducted following the instruction of the SuperScript TM First-Strand Synthesis System (Invitrogen, Calsbad, CA). The mRNA expression levels of Bcl-2, Bax1, NAD(P)H:quinone oxidoreductase-1 (NQO-1), malic enzyme-1 (ME-1), oncostatin M (OSM) and epidermal growth factor (EGF) (for sequence information, see Supplementary Table 1) were measured by Takara SYBR premix Taq quantitative PCR system (Takara Bio Inc, Shiga, Japan) and in MyiQ2 real-time PCR machine (Bio-Rad, Hercules, CA). Parallel amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. Relative quantification was done by using the 2 −ΔΔCt method. The relative expression of the specific gene to the internal control was obtained and then expressed as percentage of the control value. All real-time PCR procedures including the design of primers, validation of PCR environment and quantification methods were performed according the MIQE guideline 18 . Cellular protein extraction, Western blotting, and Nrf2 activity assay. At each treatment time-point, cells were washed with sterile PBS for 3 times and then subjected to cytosolic and nuclear protein extraction by using a NE-PER Nuclear and Cytoplasmic Extraction System (Pierce, Rockford, IL). Protein samples were then quantified with BCA method from Bio-Rad. Western blot analyses of cell lysates were performed as described 19 . Parallel blotting of β -actin was used as the internal control.
To further investigate the mechanism of endogenous antioxidant level change, the nuclear protein of each sample was subjected to the measurement of Nrf2 transcription factor activity assay by using a commercial kit from Cayman Chemical Company (Ann Arbor, MI).

GSH/GSSG ratio measurements.
To measure the intracellular oxidative status of stem cells, the ratio between reduced glutathione (GSH) to oxidized glutathione (GSSG) of each cellular protein sample was measured by using a GSH/GSSG detection assay kit from Abcam (Cambridge, England).

Inhibition of the ERK, PKC, and Keap1 pathways.
To further investigate the underlying mechanisms contributing to the endogenous antioxidant level of hUCMSCs after edaravone or DEM pre-treatment, we firstly transiently silenced the expression of Keap1, the repressor of Nrf2 activation using its specific siRNA combination (Santa Cruz BioTechnology, Santa Cruz, CA). Transfection of Keap1 siRNA (100 nM) was conducted 1-day before the pre-treatment with DEM by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA). Given the function of the ERK and PKC pathway in oxidative stress progression, the role of ERK/MAPK or PKC signaling following edaravone treatment was evaluated. That is, 1-hour before the edaravone (20 μ M) incubation, cells were treated with 25 μ M PD98059 (specific ERK/MAPK inhibitor) or 10 nM staurosporine (specific PKC inhibitor). Then cells were subjected to LPS/H 2 O 2 challenge as previously described.
Animal experiments. All animal experiments, including procedures, sampling and animal cares, in the current study were approved by and in accordance with guidelines and regulations from the ethical committee of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. Male 6-week old (~20 g) non-obese diabetic severe combined immune-deficient (NOD/SCID) mice were bought from Guangdong Experimental Animal Center (Guangzhou, China). Mice were randomly divided into 8 groups (n = 12): (1) control group: mice were intraperitoneally (i.p.) injected with PBS only; (2) Gal/ LPS group: mice were i.p. injected with 600 mg/kg Gal and 8 μ g/kg LPS dissolved in PBS simultaneously; (3)(4)(5) vehicle-stem cell groups: mice were injected through tail-vein (t.v.) with 2 × 106 hUCMSCs (untreated, 20 μ M edaravone-pretreated, and 50 μ M DEM-pretreated, respectively) at passage 2; (6-8) Gal/LPS-stem cell groups: mice received 600 mg/kg Gal and 8 μ g/kg LPS via i.p. injection, followed 6-hour later by 2 × 10 6 hUCMSCs (untreated, 20 μ M edaravone-pretreated, and 50 μ M DEM-pretreated, respectively) at passage 2 through t.v. injection. The dosage combination of Gal and LPS, as well as the delivery route of stem cells were selected based on our previous study 20 . Murine serum was collected at day 1, 3, and 7 post-transplantation. Liver samples were collected at the end of the 7-day experiment and stored at -80 °C until further processing.
Serum and liver tissue analysis. Serum was collected by centrifugation from whole blood sample at 1,000 xg for 10 min at 4 °C and stored at -80 °C. Liver tissue samples were fixed in 10% phosphate-buffered formalin, processed for histology and embedded in paraffin blocks. Five-micrometer tissue sections were cut and stained with hematoxylin and eosin (H&E).

Serum ALT and AST assay.
To evaluate the hepatic injury at the enzymatic level, serum ALT and AST levels were measured by using ALT (SGPT) and AST (SGOT) reagent sets (Teco diagnostics, Anaheim, CA) according to manufacturer's instructions.
Scientific RepoRts | 5:11100 | DOi: 10.1038/srep11100 Genomic DNA extraction and quantitative real-time PCR. To quantify the transplanted hUCM-SCs and i-Heps that homed at the mice liver, a recently established real-time PCR quantification system has been used in the current study 21 . Briefly, genomic DNA at day 7 post-treatment was extracted from mouse livers using QIAamp genomic DNA extraction kit (Qiagen, Hilden, Germany). A pair of primers (see Supplementary Table 1 for sequence information) that generate a 141-bp fragment of human Down syndrome region at chromosome 21 were used to quantify the human-derived cells. The real-time PCR reaction was performed using an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA) for 40 cycles with denaturing at 95 °C for 30 seconds and annealing at 63 °C for 34 seconds, with a SYBR-Green Realtime PCR mix (Takara, Dalian, China). PKH labeling and fluorescent microscopy. The fluorescent dye PKH26 has been used as the cell tracer to locate the transplanted stem cells in host animal 22 . Before transplantation, hUCMSCs at passage 2 were labeled with the PKH26 MINI kit (Sigma-Aldrich) according to the manufacture's suggestions and previously reported protocol 23 . When animals were sacrificed, liver tissues were cryopreserved in optimal cutting temperature medium (OCT, TissueTek, SakuraAmericas, Torrence, CA). Frozen tissue sections (5 μ m) were collected on glass slides and fixed with 100% methanol for 10 min at 4 °C, then washed in PBS for 10 min at room temperature. Local hepatic cells were mounted using DAPI mounting solution (Beyotime Biotechnology, Jiangsu, China) and used to normalize the expansion percentage of transplanted stem cells.
hUCMSCs proliferation and apoptosis following transplantation. After 7-day post-transplantation to the injured NOD/SCID mice liver, donor hUCMSCs proliferation was quantified by immunohistochemical staining of PCNA. Fresh liver tissues were embedded with OCT medium and "snap-frozen" in dry ice. Frozen sections of 10-μ m thickness were prepared and subjected to permeabilization in acetone at -20 °C for 10 min. To reduce non-specific signal, slides were incubated with goat serum blocking buffer (Boster, Wuhan, China) at room temperature for 1-hour. Subsequently, the slides were incubated with primary antibodies PCNA (1:100, Cell Signaling). After washing thrice with PBS, slides were incubated with mouse antibody against mouse IgG conjugated with Alexa flour (1:1000, Cell Signaling). Sections were co-stained with human cytokeratin-18 (hCK-18; 1:100, Abcam HK, NT, HK) and goat antibody against rabbit IgG conjugated with FITC (1:1000, Abcam HK). Apoptosis was quantified by terminal dUPT nick end-labeling (TUNEL) using ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Billerica, MA) after 3-day post-transplantation. The number of PCNA cells or apoptotic cells was quantified in 3 microscopic fields at × 40 magnification using ImageJ software. Serum ELISA assay. Serum level of TNF-α and IL-6 from each mouse was measured by using ELISA kits from PeproTech (Rocky Hill, NJ) according to the manufacturer's instructions.
In vitro and in vivo secretion of hepatocyte growth factor (HGF) by hUCMSCs. For the evaluation of HGF secretion by hUCMSCs in vitro, cells at passage 3 were received pre-treatment with edaravone or DEM as described above and then washed thrice with Ca2 + and Mg2 + -free PBS (Sigma-Aldrich), and cultured in 10 ml DMEM supplemented with 0.05% bovine serum albumin (BSA, Sigma-Aldrich) for 24 hours. After that, collected and concentrated conditioned medium was subjected to HGF ELISA measurement (RayBioteck, Norcross, GA). For in vivo measurements, paraffin embedded liver tissues at day 7 post-challenge were prepared for immunohistochemical staining of HGF. HGF-secreting cells were labeled with HRP/DAB system (Zhongqiao, Beijing, China). Hematoxylin was used as the counterstain of cellular nuclei. Statistical analysis. Data from each group were expressed as means ± SEM. Statistical comparison between groups was done using the Kruskal-Wallis test followed by Dunn's post hoc test to detect differences in all groups. A value of p < 0.05 was considered to be statistically significant (Prism 5.0, Graphpad software, Inc., San Diego, CA).  Increased endogenous antioxidant level attenuated stem cell apoptosis. To investigate the effects and mechanisms of endogenous antioxidant level on the pathogenesis of apoptosis under oxidative/inflammatory stress, the apoptotic ratio and corresponding caspase-3/7 activity were examined at each time point after LPS/H 2 O 2 challenge. It was shown that the basal apoptotic ratio slightly increased from the beginning of the experiment to 72-hour post-treatment (~2.0% vs. ~3.6%, P < 0.05). LPS/H 2 O 2 challenge significantly increased the cellular apoptosis as time prolonging (P < 0.001). Pre-treatment with edaravone (10 μ M and 20 μ M) or DEM significantly decreased or further increased the apoptosis of hUCMSCs, respectively (P < 0.05 or < 0.001; Fig. 2A). The activity change of cellular caspase-3/7 was consistent with the change of cellular apoptotic ratio (Fig. 2B). To confirm the involvement of intrinsic apoptotic pathway in this study, the mRNA expressional changes of Bcl-2 and Bax1 were then quantified by quantitative PCR. Results exhibited that, when compared with the control group, LPS/H 2 O 2 challenge significantly down-regulated the mRNA level of anti-apoptotic molecule Bcl-2, while up-regulated the mRNA level of pro-apoptotic molecule Bax1 at 12, 24, 36, 48, 60, 72-hour post-treatment. Pre-treatment with both concentrations of edaravone abolished such effects. DEM pre-incubation further reduced the level of Bcl-2 and increased the level of Bax1 after LPS/H 2 O 2 challenge, indicating an exacerbated status of stem cell apoptosis (Fig. 2C,D).

Edaravone improved hUCMSCs viability and morphology after oxidative/inflammatory chal-
Edaravone attenuated cellular ROS production after oxidative/inflammatory challenge. To directly demonstrate the antioxidant effects of edaravone on LPS/H 2 O 2 -caused oxidative stress, DCFH-DA staining of hUCMSCs was applied to show the change of cellular ROS production. From 24-hour post-treatment afterwards, LPS/H 2 O 2 -induced obvious DCFH-DA positive signals in the culture medium, which was attenuated by edaravone pre-treatment but worsened by DEM pre-treatment (Fig. 3A,B). In cells, a decreased ratio of GSH/GSSG is an indication of oxidative stress, since when cells are exposed to increased levels of oxidative stress, oxidized glutathione (GSSG) accumulates and the reduced form (GSH) decreases 24 . In line with the ROS production, LPS/H 2 O 2 challenge caused significant decrease of cellular GSH/GSSG ratio when compared with that of the control group at 12, 24, 36, 48, 60, and 72 hours post-treatment. Such decreases were evidently restored by edaravone (10 μ M and 20 μ M) but further reduced by DEM (Fig. 3C). It should be noted that the restoration of GSH/GSSG by  Edaravone restored levels of endogenous antioxidant enzymes impaired by oxidative/inflammatory challenge. CAT and SOD1 are important endogenous antioxidant enzymes against intracellular and extracellular oxidative stress 25 . Western blotting results suggested that at 12, 24, 36, 48, 60, and 72 hours after LPS/H 2 O 2 treatment, the protein expression levels of both CAT and SOD1 were significantly down-regulated (particularly for SOD1; Fig. 4). Edaravone pre-treatment significantly restored + DEM. "*" "**" "***" mean significant changes (P < 0.05, 0.01, 0.001) between control and treatments, respectively; "#" "##" "###" mean significant changes (P < 0.05, 0.01, 0.001) between edaravone treatment group (10 μ M or 20 μ M) and LPS/H 2 O 2 group, respectively; "@" "@@" "@@@" mean significant change (P < 0.05, 0.01, 0.001) between DEM-treated group and LPS/H 2 O 2 group, respectively.
Scientific RepoRts | 5:11100 | DOi: 10.1038/srep11100 their expression levels while DEM pre-incubation reduced them to lower levels (P < 0.05; Fig. 4), indicating that the redox regulating effects of edaravone and DEM were directly associated with the modulation of endogenous antioxidant enzymes.

Edaravone and DEM influence stem cell antioxidant level through regulating MAPK, PKC and Nrf2 pathways.
Under oxidative stress, MAPK and PKC pathways are activated to degrade Keap1, the repressor of transcription factor Nrf2, leading to the activation of Nrf2 and downstream antioxidant processes in the cell 26 . To test the involvement of these mechanisms in the antioxidant status change of hUCMSCs, we firstly measured the change of the MAPK pathway. Results showed that LPS/H 2 O 2 treatment potentiated the phosphorylation of both p38 MAPK and ERK1/2 at most time points post-treatment without influencing their total protein expression (Fig. 5). In agreement with previous results, pre-treatment with edaravone or DEM significantly abolished or further strengthened the effects of LPS/H 2 O 2 treatment, respectively (Fig. 5). Then the transcriptional activity change of Nrf2 was assessed. It was found that at 12, 24, 36, 48, 60, and 72 hours after LPS/H 2 O 2 treatment, the activity of Nrf2 was significantly decreased when compared with the control group (Fig. 6A). Application of 20 μ M, but not 10 μ M edaravone partially restored the activity. Pre-incubation with DEM, as expected, further reduced the activity of Nrf2 (Fig. 6A). In addition, the mRNA expression of Nrf2 downstream antioxidant genes, NQO-1 and ME-1 was also down-regulated by LPS/H 2 O 2 challenge at most of the treatment time points, which was in agreement with the results of CAT/SOD1 protein expression. Edaravone and DEM further differentiately regulated their expressions (Fig. 6B,C). Under oxidative/inflammatory stress conditions (LPS/H 2 O 2 exposure), the inhibition of the ERK or PKC pathway, using PD98059 or staurosporine, respectively, caused a significant decrease in the cell survival of edaravone-treated hUCMSCs compared to uninhibited edaravone-treated hUCMSCs (Fig. 6D). Vehicle-PD98059 or staurosporine treatment in hUCMSCs only slightly reduced their survival rates when compared to rates of uninhibited, hUCMSCs, indicating that the decrease of cell viability was not attributed to the a direct toxicity of these agents (data not shown). Then we examined the effects of Keap1 silence (which causes the enhancement of Nrf2 activity) on DEM-treated hUCMSCs. Twenty four hours after the Keap1 siRNA transfection, the activity of Nrf2 in hUCMSCs was significantly higher than that of un-transfected or transfected with control siRNA cells (P < 0.001; Fig. 6E). After the LPS/H 2 O 2 exposure, the inhibition of Keap1 partially restored the cell viability that impaired by the pre-incubation with DEM (Fig. 6F), indicating an essential involvement of Nrf2 in the antioxidant properties of hUCMSCs.

In vivo hUCMSCs engraftment and survival in an acute liver injury model. Seven days after
the Gal/LPS intoxication of NOD/SCID mice, 50% of mice tested (n = 12) survived. For those mice with co-injection of hUCMSCs, only 2 mice were dead. Edaravone-pretreatment successfully rescued all mice. In the group of DEM-pretreated prior to Gal/LPS intoxication, however, 3 mice died during the experiment (Fig. 7A). Gal/LPS treatment is known to induce hepatocyte necrosis and inflammatory responses. In the current study, Gal/LPS treatment alone caused evident hepatic necrosis in the NOD/ SCID mice in 24-hour (Fig. 7B). Administration of edaravone-pretreated stem cells partially alleviated such hepatic injury (Fig. 7B,C). Seven days after the treatment, edaravone-pretreated stem cells showed the best ameliorative effects while DEM-pretreated stem cells exhibited only minimal therapeutic effects, when compared to the non-stem cell injected Gal/LPS group (Fig. 7B,C). In the vehicle control groups, injection of either hUCMSCs or edaravone/DEM pre-treated hUCMSCs showed no significant changes on the liver morphology throughout the experiment (data not shown).
To quantify the human-derived cells engrafted in the NOD/SCID mice liver, the ratio between human gene (Down Syndrome Region Sequence) and host genome at the end of the experiment (7 days) was determined by using quantitative real-time PCR. It was found that vehicle stem cell groups (no pre-treatment, edaravone or DEM pre-treated) only generated a small amount of human gene (Fig. 7D). Transplantation with edaravone-pretreated hUCMSCs following the Gal/LPS intoxication showed the highest abundance of human gene, while DEM-pretreated hUCMSCs showed significantly less human gene than that of non-treated hUCMSCs after Gal/LPS intoxication (Fig. 7D). These results suggested that administration of hUCMSCs after acute liver injury could accelerate the host hepatic regenerative process. Edaravone pre-treatment significantly improved the therapeutic effects and engraftment efficacy of stem cells while DEM pre-treatment impaired these effects. In addition, recruitment of hUCMSCs into the mice liver was also examined by PKH immunofluorescence (IF) and further proved that edaravone improved the expansion efficiency of stem cells after acute liver failure (Fig. 7E).

Transplantation of hUCMSCs improved serum biochemistry.
To examine the influence of antioxidant status in stem cell proliferation and apoptosis after transplantation, we quantified the number of PCNA+ cells or apoptotic cells (with co-staining of hCK-18) per × 40 high-powered field. It was shown that, when compared with untreated hUCMSCs, pre-treatment with edaravone significantly increased the PCNA+ cell number but reduced the apoptotic cell number, indicating a potentiated resistance of transplanted hUCMSCs against host liver stresses (Fig. 8A,B).

Infused hUCMSCs ameliorated host hepatic injury partly through secreting HGF.
To elucidate the possible mechanisms of the ameliorated role of transplanted stem cells in the host liver, we firstly evaluated the secretion level change of HGF by cultured hUCMSCs. It was shown that naïve hUCM-SCs secreted measurable level of HGF to the culture medium. Pre-treatment with edaravone slightly promoted such secretion while DEM showed opposite effects (Fig. 9A). Furthermore, we found that after transplantation, infused stem cells were capable of secreting HGF in the host liver. DEM treatment impaired such paracrine actions (Fig. 9B,C).

Discussion
Stem cell-based therapy has been recognized as a promising treating strategy of a variety of diseases, including liver disorders. For example, bone marrow MSCs provide protection against liver injury by antioxidative process, vasculature protection, hepatocyte differentiation, and trophic effects 27 . However, the efficacy of these therapies is below expectations 28 . One of the main reasons for this is the low survival ratio of transplanted stem cells at injured sites because of harsh oxidative stress and inflammatory environment 29 . To counter these effects, a number of pre-treatment methods began to emerge. For example, it was reported that pre-treatment with antioxidant N-acetylcysteine (NAC) significantly improved cell survival ratio of muscle-derived stem cells (MDSCs) and cardiac function in an acute murine model of myocardial infarction 14 . A recent study also found that treatment with melatonin, a common antioxidant, further improved adipose-derived mesenchymal stem cell (ADSC) therapy for acute interstitial cystitis in rat 30 . Thus, enhancement of endogenous antioxidant level of stem cells before transplantation seems to be a strategy to improve the therapeutic efficacy.
We have previously identified that hUCMSCs, which are easily accessible and multipotent, exhibited evident repairing effects on an acute liver failure murine model induced by Gal/LPS 15 . Although these hUCMSCs and trans-differentiated i-Heps rescued mice with improved hepatic functions, the repair process remains limited. To increase the repair efficacy, in the current study, we firstly used edaravone as antioxidant and DEM, a nontoxic chemical that binds to GSH and inactivates it, to reduce antioxidant levels 31 , as pro-oxidant to examine the dynamic changes of cellular endogenous antioxidant level, viability, and apoptosis. From 24-hour post-treatment, hUCMSCs started to show significant loss of viability, with increased ROS production and cellular apoptosis. Edaravone significantly counteracted such effects while DEM further exacerbated them, including the regulation of antioxidant enzymes and apoptotic genes. This result is consistent with other studies using NAC or melatonin 14,30 . It was extensively studied that MAPK and PKC pathways could directly modulate the transcriptional activity of Nrf2, which plays antioxidant roles by controlling the transcription of downstream genes 32,33 . We also found that the beneficial effects of edaravone on hUCMSCs were through decrease of phosphorylated p38 MAPK and ERK1/2, as well as increase of Nrf2 activity and its downstream antioxidant enzyme expression (Figs 5 and 6). Application of MAPK inhibitor PD98059 or PKC inhibitor staurosporine reversed the improvement by edaravone. Silence of Nrf2 inhibitory protein -Keap1 partially abolished the detrimental effects of DEM. These results confirmed that the modulation of stem cell endogenous antioxidant level was, at least partly, through the MAPK-PKC-Nrf2 pathway. This is confirmed by a very recent study showing that dynamic changes in intracellular ROS levels regulate stem cell homeostasis through Nrf2-dependent signaling 34 . It should be noted that in the current study, expression of antioxidant enzymes (CAT, SOD1, NQO-1 and ME-1) and Nrf2 activity were down-regulated by oxidant treatment, which was in line with several recent studies in stem cells and hepatocytes [35][36][37] . Some other reports, however, found that antioxidant genes are increased to survive when cells are exposed to oxidative stress inducers 30,38,39 . This difference might be attributed to the defensive nature of human MSCs to oxidative stress through expressing high basal level of active forms of CAT, glutathione peroxidase (GPx), and SOD, which confers the resistance against acute ROS-mediated cellular damage 40 . Addition of toxin or oxidant may impair this defense wall to maintain the relatively low level of antioxidant enzymes and Nrf2 activity, resulting in the oxidative stress status of the stem cells.
In line with the in vitro findings, when compared with untreated hUCMSCs, edaravone pre-treated stem cells exhibited improved therapeutic properties, including enhanced survival ratio of the mice, improved hepatic histology, reduced serum aminotransferases/cytokines and promoted liver regeneration (Figs 7 and 8). This can be from increased transplanted stem cell number, which is probably from induced cell proliferation and inhibited apoptosis by the pre-treatment with edaravone. In contrast, DEM-treated hUCMSCs showed minimal therapeutic effects on acute liver failure. These results strongly suggested that the antioxidant status of hUCMSCs before transplantation is vital for the functional tissue repair of acute liver failure 14 .
Indeed, there are several limitations of the current study. Firstly, we only investigated the involvement of MAPK, PKC and Nrf2 in the regulation of stem cell antioxidant status. Functions of other oxidative stress-related pathways, such as PI3K/Akt and FoxO/TXNIP need further studies. Secondly, mechanisms that influence the enhanced repairing efficacy of stem cell after transplantation are only partly examined. We proved that increased number of transplanted hUCMSCs secreted more HGF to promote the recovery of adjacent host liver cells and this action was also influenced by the antioxidant and pro-oxidant treatments (Fig. 9), which was consistent with previous reports 41,42 . Thirdly, regardless of the fact that MSCs are proven with minimal immunogenicity and low tumorigenesis 43 , the safety of drug-treated stem cell transplantation needs long-term observation in animal models and clinical trials. In a long-term approach in NOD/SCID mice, the safe and efficient use of MSCs by injection not revealed side effect 44 .
ROS are increasingly recognized as important signaling molecules involved in gene regulation of stem cells 45 . Since oxidative stress, inflammation, and necro-apoptosis are typical consequences of acute liver failure which holds high possibility to cause death 46 , transplantation with enhanced anti-oxidative ability stem cells may significantly improve the therapeutic efficacy in clinical trials. Thus, reducing the in vitro culture duration 15 and maintaining relatively high antioxidant endogenous level of isolated stem cells (e.g. by clinically proven drug edaravone) before transplantation are possible strategies for future regenerative medicine applications. Furthermore, this study (1) provided useful information of the dynamic cellular changes at different time points after in vitro oxidative/inflammatory stress induction, which will assist future stem cell handling in both basic study and clinical trials; and (2) implied a new potent and widely used clinical drug, edaravone, as an useful antioxidant treating agent in stem cell preparation before transplantation.