Intraportal mesenchymal stem cell transplantation prevents acute liver failure through promoting cell proliferation and inhibiting apoptosis

Jian-Feng Sang, Xiao-Lei Shi, Bin Han, Tao Huang, Xu Huang, Hao-Zhen Ren and Yi-Tao Ding

Nanjing, China

Intraportal mesenchymal stem cell transplantation prevents acute liver failure through promoting cell proliferation and inhibiting apoptosis

Jian-Feng Sang, Xiao-Lei Shi, Bin Han, Tao Huang, Xu Huang, Hao-Zhen Ren and Yi-Tao Ding

Nanjing, China

BACKGROUND: Transplantation of mesenchymal stem cells (MSCs) has been regarded as a potential treatment for acute liver failure (ALF), but the optimal route was unknown. The present study aimed to explore the most effective MSCs transplantation route in a swine ALF model.

METHODS: The swine ALF model induced by intravenous injection of D-Gal was treated by the transplantation of swine MSCs through four routes including intraportal injection (InP group), hepatic intra-arterial injection (AH group), peripheral intravenous injection (PV group) and intrahepatic injection (IH group). The living conditions and survival time were recorded. Blood samples before and after MSCs transplantation were collected for the analysis of hepatic function. The histology of liver injury was interpreted and scored in terminal samples. Hepatic apoptosis was detected by TUNEL assay. Apoptosis and proliferation related protein expressions including cleaved caspase-3, survivin, AKT, phospho-AKT (Ser473), ERK and phospho-ERK (Tyr204) were analyzed by Western blotting.

RESULTS: The average survival time of each group was 10.7 ± 1.6 days (InP), 6.0±0.9 days (AH), 4.7±1.4 days (PV), 4.3± 0.8 days (IH), respectively, when compared with the average survival time of 3.8±0.8 days in the D-Gal group. The survival rates between the InP group and D-Gal group revealed a statistically significant difference (P＜0.01). Pathological and biochemical analysis showed that liver damage was the worst in the D-Gal group, while less injury in the InP group. Histopathological scores revealed a significant decrease in the InP group (3.17±1.04, P＜0.01) and AH group (8.17±0.76, P＜0.05) as compared with that in the D-Gal group (11.50±1.32). The apoptosis rate in the InP group (25.0%±3.4%, P＜0.01) and AH group (40.5%±1.0%, P＜0.05) was lower than that in the D-Gal group (70.6%±8.5%). The expression of active caspase-3 was inhibited, while the expression of survivin, AKT, phospho-AKT (Ser473), ERK and phospho-ERK (Tyr204) was elevated in the InP group.

CONCLUSIONS: Intraportal injection was superior to other pathways for MSC transplantation. Intraportal MSC transplantation could improve liver function, inhibit apoptosis and prolong the survival time of swine with ALF. The transplanted MSCs may participate in liver regeneration via promoting cell proliferation and suppressing apoptosis during the initial stage of ALF.

(Hepatobiliary Pancreat Dis Int 2016;15:602-611)

mesenchymal stem cells;

stem cell transplantation;

acute liver failure;

apoptosis;

regeneration

Introduction

Liver damage caused by viruses, drugs, toxins or alcohol could lead to acute liver failure (ALF) with indications of hepatic encephalopathy, hepatorenal syndrome, severe infection, multiple organ failure, and even death.[1]The key strategy for the treatment of ALF is to reduce hepatocyte necrosis and stimulate hepatocyte regeneration. Current therapies including drug therapy and artificial liver therapy may reduce mortality, but the therapeutic efficacy is still limited.[2,3]Though liver transplantation is the most effective treatment for ALF, the difficulties including severe donor shortage, numerous complications, immune rejection, requirements of immunosuppressive agents and high medical costs greatly

limit the clinical application of liver transplantation.[4]

Stem cell transplantation is a new way in recent years for ALF treatment due to its sufficient source, low immunogenicity and the potential to differentiate into hepatocyte-like cells.[5,6]Mesenchymal stem cells (MSCs) have the potential to differentiate into hepatocyte-like cells in vitro and in vivo with partial hepatic functions under appropriate environmental conditions.[7-11]MSCs can be regarded as the seeding cells for transplantation in relation to liver diseases.[12]Though autologous cell transplantation may prevent immunological rejection, it still has some problems in its application.

The mechanism of ALF involves various inflammatory factors and cytokines, and cellular proteins such as the Fas family and caspase signal activated apoptosis of hepatocytes.[7,13-16]It has been indicated that the therapeutic efficacy of MSC transplantation is not only associated with the purity of MSCs, but also with the administration routes.[17]However, preclinical studies in large animal models for identifying the most effective and practical administration of MSCs are not characterized sufficiently.[18-20]Most studies are focused on sole route, thus, it is hard to compare and identify which route is the most effective and practical, and which route may be attributed to future clinical application of MSCs.

In this study, a D-Gal induced swine ALF model was established to explore the therapeutic efficacy of MSC transplantation during the treatment of ALF. In particular, the therapeutic efficacy of different MSC transplantation routes such as peripheral vein transplantation, intraportal transplantation, arteria hepatica transplantation and intrahepatic transplantation in D-Gal induced ALF was compared before and after bone MSC transplantation. The therapeutic outcome in the present study might contribute to the future clinical application of MSCs.

Methods

Animals

Chinese experimental miniature swine (15±3 kg, aged approximately 5 to 8 months) were obtained from the Laboratory Animal Center of the Affiliated Drum Tower Hospital of Nanjing University Medical School and maintained under conventional conditions. All animal experimental procedures were approved by the Animal Care Ethic Committee of Nanjing Drum Tower Hospital.

Isolation, culture and characterization of MSCs

MSCs were isolated and cultured according to the previous report.[21]In brief, porcine MSCs were isolated by bone marrow aspirates from the iliac crests of the animals. MSCs were collected by density gradient centrifugation over a Ficoll histopaque layer (20 minutes, 400 g, density 1.077 g/mL) (TBD, China) and cultured in lowglucose Dulbecco’s modified Eagle’s medium (DMEMLG; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Gibco). The nonadherent cells were removed after the first 24 hours and changed every 3-4 days thereafter. When the cells reached up to 80% confluence, the cells were detached using 2.5 g/L Trypsin-EDTA (Gibco) and re-plated at a density of 1×104cells/cm2for expansion. Surface markers of the cultured MSCs were identified by flow cytometric analysis (FACScan, Becton Dickinson, Franklin Lakes, NJ, USA) using fluorescein isothiocyanate (FITC)-labeled monoclonal antibody for staining to CD45 (Antigenix America, Huntington Station, NY, USA) and phycoerythrin (PE)-conjugated antibodies against CD29 (VMRD, Pullman, WA, USA), CD44 and CD90 (Becton Dickinson). Isotypic antibodies served as controls.

Swine ALF model establishment and treatment

Under general anesthesia with mechanical ventilation via an endotracheal tube, animals received a single intravenous injection of 0.3 g/kg D-Gal (Sigma, St. Louis, MO, USA) dissolved in 0.9% saline solution, via the external jugular vein.[11]Thirty-six swine were randomly divided into 6 groups including normal control group, D-Gal group, peripheral vein MSC transplantation (PV) group, intraportal MSC transplantation (InP) group, arteria hepatica MSC transplantation (AH) group and intrahepatic MSC transplantation (IH) group. D-Gal group was administrated with 0.3 g/kg D-Gal, and normal control group was administrated with the same volume saline. All groups were administrated with laparotomy except normal control group. Liver injured animals in the PV group were subjected to slow administration of 1×107MSCs suspended in 2 mL normal saline via the external ear vein after D-Gal induction for 24 hours. The abdomens of liver injured animals in the InP and AH groups were opened to expose the portal vein and arteria hepatica, respectively, and approximately 1×107MSCs suspended in 2 mL normal saline were slowly injected into the portal vein and arteria hepatica, respectively, after D-Gal induction for 24 hours. Liver injured animals from the IH group were opened to expose the liver and approximately 1×107MSCs suspended in 2 mL normal saline were slowly injected into the liver directly after D-Gal induction for 24 hours. A 30-gauge needle was used for the procedure. The pinhole at the injection site was pressed for hemostasis. Thereafter, the laparotomy incision was enclosed in layers.

Blood and serum analysis

Blood sampling was performed before and after MSC transplantation. Venous blood samples were drawn at day -1, 1, 3 and 5 after MSC transplantation (day 0) for biochemical analysis. Serum levels of ALT, AST, ALP, LDH, total bilirubin (TB), direct bilirubin (DBIL), and γ-GT were monitored to reflect the liver function.

Histological assessment

After cell transplantation for 3 days, animal liver tissues were surgically collected under general anesthesia. Parts of the liver tissues were snap-frozen in liquid nitrogen and stored at -70 ℃ until use. For histological analysis, liver tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections with 5 μm thickness were affixed to slides, de-paraffinized, and stained with hematoxylin and eosin (HE) to determine morphological changes. The histology of liver injury was interpreted and scored in terminal samples. Histopathological characteristics were evaluated by 3 pathologists blinded to the animals’ treatment and scored for steatosis, necrosis, and inflammation as follows: 0, normal; 1, mild change; 2, mild to moderate severity; 3, moderate severity; 4, serious severity and 5, maximum severity. The scores were summed for each animal to obtain comprehensive scores. The sections were photographed with a Leitz Aristoplan microscope (Wetzlar, Germany).

TUNEL assay

TUNEL assay was completed according to the previous report.[22]Hepatic apoptosis was detected by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay kit (Roche Applied Science, Sweden). Paraffin sections from histological assessment were routinely de-paraffinized, rehydrated, and then rinsed by PBS. After blocking endogenous peroxidase activity by H2O2in methanol, permeability liquid (1 g/L Triton X-100 was dissolved in 0.1% sodium citrate), TUNEL reaction solution and Converter-POD were added. Each slice was further stained by 3, 3-diaminobenzidine (DAB), and hepatocyte apoptosis was observed under a microscope. The brown particles in the nucleus were considered as apoptosis-positive cells. Three fields were randomly selected in each slice under high magnification field (400 ×). The percentage of TUNEL positive cells relative to the total cell count was used to estimate the apoptosis rate. Counts were performed in 3 fields for each group.

Western blotting

For Western blotting analysis,[23]frozen samples were lysed in lysis buffer containing 20 mmol/L Tris (pH 7.4), 250 mmol/L NaCl, 2 mmol/L EDTA (pH 8.0), 0.1% Triton X-100, 0.01 mg/mL aprotinin, 0.005 mg/mL leupeptin, 0.4 mmol/L PMSF, and 4 mmol/L NaVO4. The equal amount of proteins was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were electronically transferred onto nitrocellulose membrane, and probed with various primary antibodies (1:1000) such as AKT, phospho-AKT (Ser473), ERK, phospho-ERK (Tyr204), survivin, active caspase-3 and internal control antibody GAPDH (EnoGene, New York, NY, USA). The blot was washed, exposed to horseradish peroxidase-conjugated secondary antibodies for 1 hour, and finally detected by ECL reagent (GE Healthcare, USA). The band densitometric analysis of the scanned blots was conducted using ImageJ software and the results were expressed as fold change relative to the internal control.

Statistical analysis

The data were expressed as mean±standard deviation (SD). The significance of the results obtained from the control and treated groups was determined by Student’s unpaired t test and one way analysis of variance (ANOVA). The comparison of survival time of swine was conducted by the Kruskal-Wallis test. A statistically significant difference was considered as two-tailed P value less than 0.05. Analyses were performed with SPSS 17.0 software (Chicago, IL, USA).

Results

MSC phenotype

After first seeding for 24 hours, MSCs could be observed in newly formed colonies; MSCs rapidly grew into fibroblast-like cells with a single nucleus. After the first passage, they looked like spindles or asters with a slim body. At passage 4, however, most of the miscellaneous cells were eliminated, and the remaining uniform fibroblastlike cells were MSCs. The expression of different cell surface markers including CD29, CD44 and CD90 in MSCs from passage 4 was determined by flow cytometry. The results showed that more than 90% MSCs after passage 4 were positive for CD29, CD44 and CD90, but negative for CD45 (Fig. 1).

Survival of swine after ALF

The swine ALF model clinically presented listlessness, appetite loss and xanthochromia. Piebald change was observed in the liver, indicating that the hepatonecrosis was induced by D-Gal, which was further confirmed by histological examination (Fig. 2A). All swine in the D-Gal

group and IH group died within 5 days after D-Gal injection. However, the survival rates were 33.3%±3.5%, 66.7%±7.2% and 100.0%±0.0% in the PV group, AH group and InP groups on the 5th day after D-Gal injection, respectively. Until the swine were killed at 12 days, the survival rate of the InP group was 50.0±5.3%. The average survival time in the InP group, AH group, PV group and IH group were 10.7±1.6, 6.0 ±0.9, 4.7±1.4 and 4.3±0.7 days, respectively. While the average survival time of the D-Gal group was 3.8±0.8 days. The survival rates between the InP group and D-Gal group exhibited a statistically significant difference (P＜0.01) (Fig. 2B).

Fig. 1. A: Morphology of MSCs at passage 4 (original magnification ×200). B: Surface markers of cultured MSCs were identified by flow cytometric analysis. More than 90% of MSCs at passage 4 were positive for CD29 (low right), CD44 (up right) and CD90 (middle right), but negative for CD45 (left). Isotypic antibodies served as the control.

Fig. 2. Hepatic necrosis was induced by D-Gal (A) and the survival rate of D-Gal-induced acute liver failure swine after MSC transplantation at different transplantation routes (B). The animals from the intraportal MSC transplantation (InP) group showed a significantly higher overall survival rate as compared to the D-Gal group (P＜0.01). Bar, 1 cm.

Liver function

Serum levels of ALT, AST, ALP, LDH, TB and DBIL were significantly and progressively elevated after D-Gal induction for 1 day (Fig. 3, P＜0.05 or P＜0.01), suggesting that acute liver injury is successfully achieved by D-Gal induction. The InP group showed most significant improvement of liver function after treatment of MSCs. The serum levels of ALT, ALP and TB in the InP group were significantly lower than those in the D-Gal group within 1-5 days (P＜0.05). The serum levels of AST and LDH in the InP group were significantly lower than those in the D-Gal group (P＜0.05) at day 3. The serum level of DBIL in the InP group was significantly lower than that in the D-Gal group (P＜0.05) at day 1 and 5. Some improvements were also observed in the AH group. The serum level of ALT in the AH group was significantly lower than that in the D-Gal group (P＜0.05) within 1-5 days. The serum level of ALP in the AH group was significantly lower than that in the D-Gal group (P＜0.05) at day 3 and 5. The serum level of DBIL in the AH group was significantly lower than that in the D-Gal group (P＜0.05) at day 1. Although the improvement of some biochemical indices was observed in the PV and IH groups, no statistically significant difference was achieved.

Histological analysis

Histopathological studies of swine liver tissues from the normal control group revealed normal liver lobules with the central vein, normal hepatocytes and hepatic si-

nusoid (Fig. 4A). However, liver tissue samples from the injured model group after D-Gal injection demonstrated severe hepatic necrosis in most of the lobules, sinusoidal congestion, vacuolization, trabecular fragmentation and granulocytic infiltration in the portal space and septa. Extensive neutrophil infiltration, lobular architecture collapse and mild fibrotic septa formation were observed. Vesicular lipid droplets were observed in hepatocytes, and spotty necrosis of hepatocytes was shown in lobules (Fig. 4B). Liver damage was most obvious in the D-Gal group, while less inflammatory cell infiltration and relatively complete lobular architecture were observed in the InP group. The most significant improvement was observed in the InP group when compared with other MSC transplantation routes. The lobular architecture and slight inflammatory cell infiltration could be recognized (Fig. 4D). The InP group (3.17±1.04, P＜0.01) and AH group (8.17±0.76, P＜0.05) revealed a significant decrease in histopathological scores when compared with the D-Gal group (11.50 ±1.32). However, no statistically significant difference was achieved in the PV group (9.50±0.50) and IH group (10.67±0.58) (Fig. 5).

Apoptosis analysis

The number of apoptotic cells revealed a quick increase after D-Gal induction. It is obvious that the number of apoptotic cells was significantly smaller in the liver from the InP group, indicating the anti-apoptotic role of MSCs (Fig. 6). The apoptotic rates in the InP group (25.0%±3.4%, P＜0.01) and AH group (40.5%±

1.0%, P＜0.05) were lower than those in the D-Gal group (70.6% ±8.5%), although there was no significant difference between the PV group (60.3%±6.2%) and IH group (55.1%±4.4%) (Fig. 6).

Fig. 3. Animals were received a single intravenous injection of D-Gal at the dose of 0.3 g/kg. Serial serum samples of day -1, 1, 3 and 5 after MSC transplantation (day 0) were collected during the follow-up period. Serum levels of ALT, AST, ALP, LDH, DBIL and TB were monitored to reflect liver function. D-Gal: model group; InP: intraportal MSC transplantation group; PV: peripheral vein MSC transplantation group; AH: arteria hepatica MSC transplantation group; IH: intrahepatic MSC transplantation group. *: P＜0.05, #: P＜0.01, compared with the D-Gal group.

Fig. 4. HE staining of liver tissues after MSC transplantation for 3 days. A: Normal control group. B: D-Gal model group. Hepatic lobules were filled with necrosis and inflammatory cells. The black star indicated large areas of hemorrhage and necrosis in liver lobule. The black arrow indicated the atty degeneration of hepatocytes. The black triangle indicated peripheral lobular fibrous tissue as well as inflammatory cell infiltration inside. C: PV group. The black triangle indicated interlobular connective tissue. The black star indicated hepatic sinus hemorrhage and necrosis of hepatocytes. D: InP group. The black triangle indicated interlobular connective tissue. The black arrow indicated portal area. E: AH group. The black triangle indicated interlobular connective tissue. The black star indicated the centrilobular vein. Hepatic sinus dilation and hemorrhage were observed in the peripheral lobular. F: IH group. The black arrow indicated portal area. The black triangle indicated interlobular connective tissue. The black star indicated a lobuli hepatis, hepatic sinus hemorrhage and necrosis of hepatocytes (original magnification ×100).

Fig. 5. The histopathological scores of liver tissues after MSC transplantation for 3 days. D-Gal: model group; InP: intraportal MSC transplantation group; PV: peripheral vein MSC transplantation group; AH: arteria hepatica MSC transplantation group; IH: intrahepatic MSC transplantation group. *: P＜0.05, #: P＜0.01, compared with the D-Gal group.

Fig. 6. Anti-apoptotic effect of MSCs in D-Gal induced ALF. After cell transplantation for 3 days, animal liver tissues were surgically prepared under general anesthesia for the detection of apoptotic cells by TUNEL assay for DNA strand break labeling (up). TUNEL-positive (brown nuclei) and -negative cells were further counted for apoptosis rate (down). A: Normal control group; B: D-Gal model group; C: PV group; D: InP group; E: AH group; F: IH group (original magnification ×100).

Western blotting analysis

According to above results, InP had the best efficiency among all MSC transplantation routes in preventing acute liver failure, which attracted our interest to explore the potential mechanisms. The expression of active caspase-3 was obviously elevated and survivin was significantly decreased in the D-Gal group, which may be contributed to the apoptosis of hepatocytes during ALF. However, the expression of active caspase-3 revealed significant decrease and the expression of survivin exhibited a significant elevation in the InP group when compared with the D-Gal group (P＜0.01). The relative expression of AKT, phospho-AKT (Ser473), ERK, and phospho-ERK (Tyr204) was significantly lower in the D-Gal group than that in the normal control group (P＜0.01). MSC transplantation could lead to the increased expression of AKT, phospho-AKT (Ser473), ERK, phospho-ERK

(Tyr204) (P＜0.01), which may play an important role in liver regeneration (Fig. 7).

Fig. 7. Western blotting for the expression of AKT, phospho-AKT (Ser473), ERK, phospho-ERK (Tyr204), survivin and cleaved caspase-3. Liver tissues were collected after intraportal MSC transplantation for 3 days. The expression of AKT, phospho-AKT (Ser473), ERK, phospho-ERK (Tyr204), survivin, cleaved caspase-3 and GAPDH were detected by immunoblotting (A). The band densitometric analysis was conducted and expressed as fold change relative to the internal control (B and C). Representative gels of 6 livers/group were shown. #: P＜0.01, compared with the D-Gal model group.

Discussion

Bone marrow-derived MSC may be a potential therapeutic choice for ALF because of its mesodermal origin multipotent adult stem cell character with the potential for self-renewal.[24]Petersen[25]and Schwartz et al[26]have demonstrated that MSCs could differentiate into hepatocytes in vitro and in vivo. Because of its ability to differentiate in multiple organs, MSC has gained considerable interest for the potential application in liver diseases.[27]Cho et al[28]have confirmed that MSC transplantation is an ideal candidate for liver disease treatment because of its involvement in both liver repair and reconstruction. Furthermore, MSC transplantation has been confirmed to be able to reduce CCl4-induced liver fibrosis in mice.

MSC may be a suitable candidate for hepatocyte transplantation, which holds a promising future in the treatment of acute or chronic liver failure. The efficacy of MSC transplantation is also dependent on the administration route of MSCs.[29-31]Sun et al[29]have studied BMSC transplantation via four routes for the treatment of ALF in rats. Liver function in rats with ALF achieves an obvious improvement following BMSC transplantation through the hepatic artery, portal vein and vena caudalis. These 3 methods are effective in transplanting BMSCs for the treatment of ALF. However, the transplantation via intraperitoneal injection reveals no therapeutic effect. Porada et al[32]have evaluated transplantation therapy in two pediatric hemophilia A animals and proved that the nonablative MSC transplantation with a porcine FVIII-encoding lentivector is straightforward, safe, and converted life-threatening, debilitating HA to a moderate phenotype in a large animal model. Cao et al[19]have studied the effect of placental mesenchymal stem cells (PMSCs) in treating Chinese miniature pigs with ALF by transplantation via the jugular vein, X-raytreated PMSCs transplantation via the portal vein, and PMSC transplantation via the portal vein. Histological data have demonstrated that the transplantation of PMSCs via the portal vein could reduce liver inflammation, decrease hepatic denaturation and necrosis, and promote liver regeneration. The 7-day survival rates suggest that PMSC transplantation via the portal vein is able to significantly prolong the survival of ALF pigs when compared with other three groups. Li et al[33]have explored the safety, effectiveness, and underlying mechanisms of BMSC transplantation for treating fulminant hepatic failure (FHF) in pigs via intraportal route or the peripheral vein immediately after D-Gal injection. All animals in the peripheral vein and control groups died of FHF within 96 hours. In contrast, 13 of 15 animals in the intraportal route group have a long-term survival period (＞6 months). Shi et al[30]have identified that portal vein MSC transplantation after D-Gal induction for 24 hours is useful in ALF, and combinatorial therapy with MSC transplantation and IL-1R antagonist (IL-1Ra) is a promising treatment for ALF. Xiao et al and Shi et al[15,34]have also explored the synergistic effect of IL-1Ra

administration and stem cell transplantation in swine suffering from ALF to find that combinatorial therapy with IL-1Ra chitosan nanoparticles and portal vein MSC transplantation exhibits great synergistic effect on paracrine function and inflammation suppression.

In our study, InP is able to reduce liver injury biomarkers, improve hepatic functional parameters, and increase survival, which is the most efficient route among all MSC transplantation routes. InP could improve liver serologies of ALT, AST, ALP, TB, LDH and DBIL and provide long-term survival benefit. The swine survival time in the InP group is significantly longer when compared with that of untreated animals. Furthermore, the histopathological scores in the InP group reveal a significant decrease when compared with those in the D-Gal group (P＜0.01). Although the AH group could improve ALT, ALP and DBIL, decrease the histopathological scores and number of apoptotic cells (P＜0.05), no statistically significance can be achieved in survival rate. Taken together, intraportal MSC transplantation could inhibit the process of hepatocellular apoptosis in swine with D-Gal-induced ALF significantly, thereby improving the survival rate.

Furthermore, we have explored the potential mechanisms of intraportal MSC transplantation on hepatic protection. Caspase-3 is a key mediator of apoptotic cells and its activation indicates the intrinsic apoptotic pathway.[35]Our western blotting data have shown that the expression of active caspase-3 is nearly undetectable after sham operation, but significantly elevated in the D-Gal group, which suggests that procaspase 3 activation plays a prominent role in D-Gal induced severe liver apoptosis, a major cause of further hepatic failure. Intraportal MSC transplantation could inhibit caspase 3 activation and increase survivin expression level (P＜0.01), correspondingly indicating the protective effect on D-Gal induced liver apoptosis.

D-Gal induced liver injury contains not only the increased hepatocellular apoptosis but also the decreased cell proliferation. Effective MSC transplantation may induce up-regulated secretion of growth factors, thus participating in the priming phase of liver regeneration, making hepatocytes responsive to growth factors such as hepatocyte growth factor (HGF), EGF and transforming growth factor-α (TGF-α) and promote hepatocyte replication and liver growth in vivo.[36]Accumulating evidence has revealed that MSCs secreted trophic factors which play key therapeutic roles in hepatocyte survival and regeneration. MSCs secreted trophic factors include the anti-apoptotic factors such as stromal cell-derived factor 1, HGF, insulin-like growth factor 1 (IGF-1) which correlate with reduced inflammation, the mitogenic factors such as EGF, HGF, nerve growth factor (NGF), TGF which are primarily associated with hepatocyte proliferation, and angiogenic factors (VEGF) which are responsible for liver regeneration.[37-39]In recent years, adipose tissue-derived mesenchymal stem cells (AT-MSCs) have also been explored to be used in mice with carbon tetrachloride CCl4-caused liver injury because of their high accessibility with minimal invasiveness.[40-42]It has been reported that after transplantation, AT-MSCs can improve liver functions and promote tissue repair, ATMSCs secrete interleukin 1 receptor alpha (IL-1Ralpha), IL-6, IL-8, granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein 1, NGF, and HGF.[41]Zagoura et al[42]have also confirmed that human spindle-shaped amniotic fluid (AF)-MSCs or hepatic progenitor-like (HPL) cells might be valuable tools to induce liver repair and support liver function by cell transplantation. The presence of anti-inflammatory factors such as interleukins IL-10, IL-1Ra, IL-13 and IL-27 may play an important role in cell treatment in diseases of the liver.

Pathways including AKT and ERK signaling are important in hepatocyte protection from apoptosis and in enhancement of hepatic repair after liver injury as well as the inactivation of Akt pathway resulted from the delayed liver regeneration in mice.[43,44]In the present study, D-Gal could cause a marked decrease in AKT, phospho-AKT (Ser473), ERK and phospho-ERK (Tyr204) expressions. MSC transplantation significantly increased the expressions of AKT, phospho-AKT (Ser473), ERK, phospho-ERK (Tyr204), which may play an important role in liver regeneration after D-Gal injury. It is supposed that live regeneration after MSC transplantation may mainly be contributed to the PI3K pathway and ERK pathway. Increased apoptosis and suppressed liver regeneration is suspected to be the major factor contributing to ALF. The inhibition of apoptosis and the promotion of cell proliferation may be a possible contribution during intraportal MSC transplantation; however, the clear underlying mechanisms remain to be further elucidated.

In summary, intraportal MSC transplantation is superior to other MSC transplantation pathways due to its advantages of improving liver function, inhibiting apoptosis, and prolonging survival time of ALF swine. The transplanted MSCs may quickly participate in liver regeneration through the signal pathways promoting cell proliferation and inhibiting apoptosis during the initial stage of ALF. Therefore, intraportal MSC transplantation could possibly be applied in clinical therapy in the future. An increased understanding of liver regeneration cascade in MSC transplantation could lead to improved clinical therapeutic outcomes for acute or chronic liver failure.

Contributors: DYT proposed the study. SJF and SXL designed the research. SJF, HB, HT, HX and RHZ performed the research. SJF and HB analyzed the data and wrote the paper. All authors contributed to the design and interpretation of the study and to further drafts. DYT is the guarantor.

Funding: This study was supported by grants from the National Natural Science Foundation of China (81300338), 863 National Science and Technology Plans (2013AA020102) and Project Funding of Clinical Medical Center of Digestive Disease in Jiangsu Province (BL2012001).

Ethical approval: All animal experimental procedures were approved by the Animal Care Ethic Committee of Nanjing Drum Tower Hospital.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received October 25, 2015

Accepted after revision May 23, 2016

Author Affiliations: Department of General Surgery, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China (Sang JF, Shi XL, Han B, Huang T, Huang X, Ren HZ and Ding YT)

Yi-Tao Ding, Professor, Department of General Surgery, the Affiliated Drum Tower Hospital of Nanjing University Medical School, No. 321 Zhongshan Road, Nanjing 210008, China (Tel: +86-25-83304616ext66866; Fax: +86-25-83317016; Email: yitaoding@hotmail. com)

? 2016, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(16)60141-8

Published online September 30, 2016.