Specific-pathogen-free (SPF) adult inbred Brown-Norway (BN) rats and Lewis rats were purchased from the Academy of Military Medical Sciences, Beijing, China. Male BN rats (4-5 wk old; 100-120 g) were inbred for the extraction and characterization of BMMSCs. Inbred male Lewis rats (6-8 wk old; 200-220 g) were the liver transplantation donors, and the inbred male BN rats were the recipients. The experimental animals were kept at 23 °C, with 50% humidity, and a 12 h light and dark cycle for 2 wk, with free access to water and food, and regular replacement of cage and clean bedding before the experiments. All experimental procedures were performed in accordance with the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health, 8^th ed. 2011). All protocols were approved by the Animal Care and Research Committee of Tianjin First Central Hospital. All the rats were anesthetized with chloral hydrate to minimize their pain.

The following instruments and reagents were used: Dulbecco’s modified Eagle medium (DMEM)/F12, penicillin-streptomycin solution, and trypsin/EDTA solution (Gibco, Carlsbad, CA, United States); fetal bovine serum (FBS; Biowest, Nuaillé, France); dexamethasone phosphate sodium (5 mg/mL), sodium glycerophosphate (216 mg/mL), insulin (40 U/mL), 1-methyl-isobutyl-xanthine, vitamin C and indomethacin (Sigma Aldrich, St. Louis, MO, United States); Oil Red O powder (Dingguo Changsheng Biotechnology, Beijing, China); von Kossa cell staining kit (Genmed, Shanghai, China); recombinant adenovirus expressing rat HO-1 (Genechem Co., Ltd., Shanghai, China); phosphate buffer solution (PBS), highly sensitive radio immunoprecipitation assay (RIPA), bicinchoninic acid (BCA) protein assay kit (Solarbio, Beijing, China); Western blotting-associated reagents (Boster, Wuhan, China); normal goat serum (Minhai Biotechnology, Lanzhou, China); SuperPicture™ Polymer Detection Kit (Thermo, Waltham, MA, United States); diaminobenzidine (Dako, Glostrup, Denmark); flow cytometry-related antibodies (anti-rat CD34-fluorescein isothiocyanate (FITC), CD29-phycoerythrin (PE), CD45-PE, CD90-FITC, RT1A-PE, and RT1B-FITC; Biolegend, San Diego, CA, United States); rabbit antibodies for inducible nitric oxide synthetase (iNOS), endothelial nitric oxide synthetase (eNOS), von Willebrand factor (vWF), and mouse antibodies for endothelin (Abcam, Cambridge, United Kingdom); rabbit antibodies for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SAB, College Park, MD, United States); goat anti-rabbit IgG labeled with horseradish peroxidase (HRP) and goat anti-mouse IgG labeled with HRP (Invitrogen, Carlsbad, CA, United States); ELISA kits (Biovalue, Shanghai, China); ATP assay kit (Jiancheng Biotechnology, Nanjing, China); inverted fluorescent microscope (Olympus, Japan); and FACSCalibur flow cytometric analysis (BD FACSAria III, Franklin Lakes, NJ, United States).

BMMSCs were isolated aseptically from the femur and tibia of BN rats after sacrifice by cervical dislocation. After cutting off both ends of the epiphyseal plate, the marrow cavity was rinsed by DMEM/F12 containing 10% FBS. Red blood cells (RBCs) were lysed using 0.1 mol/L NH₄Cl, and the remaining cells were washed, resuspended as a single cell suspension, and cultured in T75 culture flask at 37 °C with 5 mL/L CO₂ in an incubator. Well-grown third-passage cells were resuspended and then labeled fluorescently with antibodies (anti-CD29-PE, anti-CD34-FITC, anti-CD45-PE, anti-CD90-FITC, anti-RT1A-PE and anti-RT1B-FITC) for 30 min for flow cytometric analysis.

Adipogenic differentiation: Well-grown third-passage BMMSCs were inoculated into 6-well plates at 2 × 10⁵ cells/well. After complete adherence, BMMSCs were cultured in adipogenic differentiation medium (DMEM/F12 containing 10% FBS, 1 μmol/L dexamethasone, 10 μg/mL insulin, 0.5 mmol/L 1-methyl-3-isobutyl xanthine, and 0.1 mmol/L indomethacin). The medium was changed every 72 h. After induction for 8-10 d, BMMSCs were fixed by 4% paraformaldehyde, and stained with Oil Red O for 30 min. The BMMSCs were then rinsed with PBS and positive cells showed orange lipid droplets.

Osteogenic differentiation: Well-grown third-passage BMMSCs were also inoculated into 6-well plates at 2 × 10⁵ cells/well. After complete adherence, BMMSCs were cultured in osteogenic differentiation medium (DMEM/F12 containing 10% FBS, 0.1 μmol/L dexamethasone, 10 mmol/L sodium glycerophosphate, and 50 μg/mL vitamin C). The medium was changed every 72 h. After induction for 13-15 d, BMMSCs were stained using a von Kossa staining kit, and positive cells showed black calcium deposits.

HO-1-bearing recombinant adenovirus (Adv/HO-1) was diluted to 10 pfu/cell with complete culture medium, which was used to replace the original medium of well-grown third-passage BMMSCs. After 6-8 h, the Adv/HO-1 culture medium was replaced with complete culture medium for continued cultivation of the BMMSCs. After 48 h, the proportion of cells containing green fluorescence was observed under a fluorescence microscope.

A 50% RLT rejection model was established with Lewis donor rats and BN recipient rats, using the two-cuff technique by a single operator. The donor rats were anesthetized with 5% chloral hydrate, and incised by abdominal median incision. After dividing the perihepatic ligaments, the portal vein (PV) and infrahepatic vena cava (IHVC) were isolated. The anterior wall of the common bile duct was cut off and a stent was implanted, the hepatic artery was ligated and cut off, the PV was punctured and infused with 4 °C lactated Ringer’s solution, and the IHVC and suprahepatic vena cava (SHVC) were both cut off as the outflow tract. When the liver turned yellowish and the outflow of perfusion fluid became clear, the PV was dissected and the donor liver was removed. The SHVC was trimmed, the PV and IHVC were prepared as vascular cuffs, and then the harvested graft was preserved at 4 °C. The perihepatic ligaments were divided in the recipient rats, the PV and IHVC were blocked, and the PV was punctured and infused with 1 mL normal saline (NS). The PV, SHVC, and IHVC were then dissected, and the liver was removed. The donor liver was placed orthotopically in the abdominal cavity of the recipient. The SHVC was anastomosed using an 8-0 nylon suture. Cuff anastomosis of the PV and IHVC was then performed. The graft was reperfused by opening the PV, SHVC, and IHVC in turn. The bile duct was connected by a stent suture. After checking that all the cuff tubes were not distorted and no leak occurred in the SHVC and IHVC, the abdomen was washed and closed. The detailed surgical procedure was described previously[36].

Experimental rats were divided into three groups: the control group (receiving 1 mL NS), the BMMSCs group (receiving 5 × 10⁶ BMMSCs resuspended in 1 mL), and the HO-1/BMMSCs group (receiving 5 × 10⁶ HO-1/BMMSCs resuspended in 1 mL). All injections in the three groups were administered via the superficial dorsal veins immediately after RSL. Five rats in each of the three groups were euthanized on postoperative day (POD) 0, 1, 5, 7, or 14, respectively, and their peripheral venous blood and transplanted liver tissues (cooled in liquid nitrogen and stored at -80 °C) were collected for further analysis.

The rat serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and mitochondrial AST (ASTm) were measured using an automatic biochemical analyzer (Hitachi, Japan) according to the manufacturer’s instructions.

The rat serum levels of ET-1, eNOS, iNOS, nitric oxide (NO), and hyaluronic acid (HA) were detected using the ELISA kits according to the manufacturer’s instructions.

The pathological injuries were most obvious on POD 7; therefore, POD 7 was chosen as the time point to measure the PVP. The rats were anesthetized by intraperitoneal injection of phenobarbital, and fixed in the supine position, the abdominal cavity was opened, and the PV was exposed. An ileum mesenteric vein branch in the right side of anterior mesenteric vein near the PV was isolated, its distal end was ligated, a suitable nick to insert a catheter was made, and then the catheter was inserted along the anterior mesenteric vein upstream. After fixing the catheter and connecting it to the pressure transducer, the PVP was measured using a Power Lab ML880 (AD Instrument, Australia).

Liver tissues collected from different POD were treated with RIPA lysis buffer to extract total proteins, and the concentrations of the total proteins were detected using a BCA protein assay kit. The proteins were separated electrophoretically and then transferred to nitrocellulose membranes. After blocking with 5% skimmed milk for 2 h, ET-1 (1:250), eNOS (1:200), iNOS (1:250), vWF (1:250) and internal reference protein GAPDH (1:3000) antibodies were added and incubated at 4 °C overnight. The membranes were then rinsed with Tris Buffered Saline with Tween-20 (TBST), incubated with secondary antibodies (1:5000) for 2 h at room temperature, rinsed with TBST again, the chemiluminescence HRP substrate was added, and the membranes were exposed in a gel imaging analysis system (Alpha Innotech FluorChem FC2, CA, United States). The images were analyzed using the AlphaView SA 3.4.0 software (San Jose, CA, United States) to determine the grey scale. The relative abundance of a target protein was calculated as target protein band brightness value - background brightness value/internal reference protein GAPDH band brightness value - background brightness value. The resulting ratio was the relative abundance of the target protein. The samples were replicated three times in different batches at each time point.

The transplanted livers on POD 0, 1, 5, 7, and 14 were sectioned, fixed, paraffin embedded, sliced, and stained with hematoxylin and eosin (HE). The histopathological changes in the liver tissues were observed in five randomly selected fields under a light microscope. Acute rejection was graded according to the Banff criteria[37].

The transplanted liver was sliced, dewaxed by xylene, dehydrated by gradient ethanol, subjected to antigen retrieval, and blocked by normal goat serum at 37 °C for 1 h. After incubation with primary antibodies (1:500) at 37 °C for 1 h and at 4 °C overnight, the slides were incubated with secondary antibodies at 37 °C for 40 min, developed with DAB and stained with hematoxylin, differentiated using 1% hydrochloric acid ethanol, dehydrated using gradient ethanol, and clarified and encapsulated using xylene. The tissue slices were observed to determine the presence of ET-1, eNOS, iNOS and vWF in the transplanted liver. The immunohistochemical results were analyzed using the Image-Pro Plus 6.0.0.260 software (IPP, Media Cybernetics, Rockville, MD, United States).

The activity of Na⁺-K⁺-ATPase in the liver tissues was detected using the chemical colorimetry method according to the instruction manual of the ATPase test kit.

The transplanted liver was double-fixed by glutaraldehyde and osmic acid, dehydrated by gradient acetone, immersed in embedding medium, ultrathin sectioned using an automatic microtome (LeicaRM2235, Leica, Germany), and stained with 1% uranyl acetate. The tissue slices were observed and filmed under a transmission electron microscope (Hitachi, Japan) to observe the status of mitochondria in the hepatocytes.

SPSS 17.0 (SPSS GmbH, Munich, Germany) was used for the statistical analysis. All data were presented as means ± SD. Different groups of data were compared by analysis of variance (ANOVA). Differences were considered to be statistically significant at P < 0.05.

Morphology and phenotype identification of BMMSCs: MSCs are spindle-shaped in morphology like fibroblasts in which the cytoplasm can stretch peripherally and maintain the ability to differentiate into a variety of other cell lines when cultured ex vivo. BMMSCs grew adherently when observed under microscope, and the cells were long spindle-shaped and partially vortexed or chrysanthemum-like, with typical morphological characteristics of MSCs (Figure 1A). Flow cytometry analysis showed that the positive rates of CD29, CD90 and RT1A in the third generation BN rat BMMSCs were 99.5%, 97.4%, and 96.9%, respectively; and the negative rates of CD34, CD45, and RT1B were all above 95% (Figure 1E-G).

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Figure 1 Morphology, transformation, and flow cytometric analysis of bone marrow-derived mesenchymal stem cells. A: The third generation of bone marrow-derived mesenchymal stem cells (BMMSCs) (normal field, × 100): BMMSCs grew to complete adherence, and the cells were spindle-shaped, some were in a vortex or daisy-likearrangement; B: HO-1/BMMSCs (fluorescence field, × 100): more than 80% of the BMMSCs expressed green fluorescence after transduction with HO-1; C: Adipogenic induction of BMMSCs, with orange lipid droplets stained by Oil red O in cytoplasm (× 200, shown by an arrow); D: Osteogenic induction of BMMSCs, with black calcium deposits in cytoplasm stained by von Kossa’s reagent (× 200, shown by an arrow); E: The percentage of CD29+CD34- cells was 99.5%; F: The percentage of CD90+CD45- cells was 97.4%; G: The percentage of RT1A+RT1B- cells was 96.9%.

After induction by adipogenic differentiation medium for 8-10 d, BMMSCs showed multiple orange lipid droplets in their cytoplasm, which was consistent with the characteristics of adipocytes (Figure 1C). Similarly, BMMSCs showed black granular or lumpy calcium deposits in their cytoplasm after induction by osteogenic differentiation medium for 13-15 d, which was consistent with the characteristics of osteoblasts (Figure 1D). These results showed that the extracted BMMSCs had the potential to differentiate into adipocytes and osteoblasts in vitro.

BMMSCs were infected with recombinant adenovirus expressing HO-1 gene for 48 h, and the cells were observed under a fluorescence microscope. The rate of positive cells expressing green fluorescent protein was more than 80% (Figure 1B).

Histopathological manifestations of the transplanted liver in the NS group included obvious congestion in the hepatic sinus, red blood cell deposition in the sinus, peri-central vein and portal area, hepatocyte swelling, eosinophilic degeneration, and punctate necrosis. The infiltration of a large volume of mixed lymphocytes in the central vein and the portal area was accompanied by necrosis of hepatocytes on POD5. Infiltration of inflammatory cells and necrosis of hepatocytes increased on POD7. The mixed lymphocyte infiltration became significant on POD14, with severe hepatic sinus congestion, hepatocyte destruction, disappearance of lobular structure, and significant liver fibrosis. The transplanted livers in the BMMSCs group showed no obvious hepatic sinus congestion, and the endothelial swelling and hepatocyte necrosis was less severe than those in the NS group. The histological changes showed disordered hepatic lobules on POD5, with slightly infiltrated inflammatory cells and mild necrosis of hepatocytes. Inflammatory cell infiltration and hepatocyte necrosis progressed on POD7. On POD14, lymphocyte infiltration became obvious, liver sinus congestion increased, accompanied by liver fibrosis and incomplete hepatic lobule structures. The HO-1/BMMSCs group showed less rejection, and fewer injuries of the liver allografts compared with the BMMSCs and NS groups at all time points, without obvious hepatic sinus congestion, large volume of inflammatory cell infiltration, or disordered lobular structures; only mild hepatic fibrosis was observed (Figure 2A).

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Figure 2 Histological changes of the rat liver and grading of acute cellular rejection after reduced size liver transplantation. A: Hematoxylin and eosin staining of the transplanted liver (× 200): Rejection injuries were aggravated with increased post-operative time in all three groups. Histopathologically, the transplanted liver in the NS group showed obvious congestion in the hepatic sinus, hepatocyte swelling, mainly eosinophilic degeneration, and punctate necrosis. The infiltration of a large volume of mixed lymphocytes in the central vein and the portal area was accompanied by disordered hepatic lobules and necrosis of hepatocytes on postoperative day (POD) 5. Infiltration of inflammatory cells and necrosis of hepatocytes deteriorated on POD7. The mixed lymphocyte infiltration became significant on POD14, with severe hepatic sinus congestion, hepatocyte destruction, and disappearance of the lobular structure. Inflammatory cells infiltration, hepatocyte destruction, and lobular destruction of the transplanted liver in BMMSCs group were less severe than those in the NS group. HO-1/BMMSCs group showed less rejection, and fewer injuries of liver allografts compared with the BMMSCs and NS groups at each time point, without obvious hepatic sinus congestion, large volume of inflammatory cells infiltration, or disorder of the lobular structure; B: Grading of acute cellular rejection (ACR): The rejection injuries tended to deteriorate with increasing post-operative time in all groups. Rejection in the HO-1/BMMSCs group was significantly less severe than that in the BMMSCs and NS groups at all time points (P < 0.05). POD1: HO-1/BMMSCs group vs BMMSCs group vs NS group: 1.40 ± 0.55 vs 2.40 ± 0.55 vs 3.20 ± 0.45 (P < 0.05). POD5: HO-1/BMMSCs group vs BMMSCs group vs NS group: 2.20 ± 0.45 vs 3.40 ± 0.55 vs 5.60 ± 0.55 (P < 0.01). POD7: HO-1/BMMSCs group vs BMMSCs group vs NS group: 3.00 ± 0.71 vs 4.20 ± 0.45 vs 6.40 ± 0.55 (P < 0.01). POD14: HO-1/BMMSCs group vs NS group: 6.60 ± 0.55 vs 8.60 ± 0.55 (P < 0.01); HO-1/BMMSCs group vs BMMSCs group: 6.60 ± 0.55 vs 8.00 ± 0.71 (P < 0.01). ^aP < 0.05 vs NS group, ^cP < 0.05 vs HO-1/BMMSCs group.

The rejection injuries tended to increase in all the groups with time after operation. Rejection in the HO-1/BMMSCs group was the least severe at all time points, and was significantly less severe than that in the BMMSCs and NS groups (P < 0.05). Rejection in the BMMSCs group was significantly less severe than that in the NS group on POD1, 5, and 7 (P < 0.05). There was no significant difference between the BMMSCs group and the NS group on POD14 (Figure 2B).

The serum ALT and AST in the three groups increased initially and then decreased gradually after operation, while TBIL tended to increase with time. The serum levels of liver enzymes and TBIL in the HO-1/BMMSCs group were significantly lower than in the BMMSCs and NS groups on POD1, 5, 7, and 14 (P < 0.05). The serum liver enzymes and TBIL in the BMMSCs group were significantly lower than in the NS group (P < 0.05; Figure 3).

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Figure 3 The level of liver function indexes after liver transplantation. A: Alanine aminotransferase (ALT); B: Aspartate aminotransferase (AST); C: Total bilirubin (TBIL). The serum ALT and AST in the three groups increased initially and then decreased with increasing post-operative time. The serum liver enzymes in the HO-1/BMMSCs group were the lowest, and NS group were the highest on postoperative day (POD) 1, 5, 7, and 14 (P < 0.05). TBIL showed a trend to increase with time. TBIL of the HO-1/BMMSCs group were the lowest, and were highest in the NS group on POD5, 7 and 14 (P < 0.05). ^aP < 0.05 vs NS group, ^cP < 0.05 vs HO-1/BMMSCs group.

The PVP correlates with the blood flow and resistance of the PV, and plays an important role in maintaining the blood flow in the liver. The PVP was measured on POD7 after RLT. The PVP in the HO-1/BMMSCs and BMMSCs groups was significantly lower than that in the NS group (P < 0.05; Figure 4).

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Figure 4 Portal vein pressure on the 7^th d after liver transplantation. A: Portal vein pressure (PVP) of normal BN rats; B: PVP of normal saline (NS) group on postoperative day (POD) 7; C: PVP of bone marrow-derived mesenchymal stem cells (BMMSCs) group on POD7; D: PVP of HO-1/BMMSCs group on POD7. PVP of both the HO-1/BMMSCs group and the BMMSCs group was significantly lower than that of NS group on POD7 (P < 0.05). HO-1/BMMSCs group vs NS group: 10.67 ± 0.35 vs 21.26 ± 0.20 (P < 0.01); BMMSCs group vs NS group: 10.95 ± 0.22 vs 21.26 ± 0.20 (P < 0.01).

Effects of HO-1/BMMSCs on ET-1 expression in the transplanted liver: ET-1 positive cells were expressed in the hepatic sinusoids of transplanted livers surrounding the Glisson system. The proportions of ET-1 positive cells in the BMMSCs and HO-1/BMMSCs groups were significantly lower than that in the NS group, and HO-1/BMMSCs group showed fewer positive cells than the BMMSCs group. There were relatively more ET-1-positive cells in the NS group, which increased with time after operation (Figure 5A and B).

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Figure 5 Endothelin-1 expression in the liver after liver transplantation. A: Immunohistochemistry (IHC) of endothelin-1 (ET-1); B: IHC integrated optical density (IOD) of ET-1; C: ET-1 protein levels. ET-1 protein levels: postoperative day (POD) 1: HO-1/BMMSCs group vs BMMSCs group vs NS group: 0.04 ± 0.02 vs 0.07 ± 0.01 vs 0.27 ± 0.02 (P < 0.05). POD5: HO-1/BMMSCs group vs BMMSCs group vs NS group: 0.29 ± 0.01 vs 0.35 ± 0.01 vs 0.45 ± 0.01 (P < 0.01). POD7: HO-1/BMMSCs group vs BMMSCs group vs NS group: 0.28 ± 0.03 vs 0.34 ± 0.01 vs 0.42 ± 0.02 (P < 0.05). POD14: HO-1/BMMSCs group vs BMMSCs group vs NS group: 0.27 ± 0.04 vs 0.51 ± 0.01 vs 0.71 ± 0.01 (P < 0.01). ^aP < 0.05 vs NS group, ^cP < 0.05 vs HO-1/BMMSCs group.

The levels of ET-1 in the transplanted liver decreased initially and then increased with time after operation. The level of ET-1 was the lowest in the HO-1/BMMSCs group, while the NS group showed the highest levels on POD1, 5, 7, and 14 (P <0.05). The results indicated that BMMSCs could inhibit the secretion of ET-1 by SECs, thereby reducing sinusoidal contraction, decreasing sinusoidal vascular resistance, and improving hepatic sinus perfusion. The sinusoidal vasoconstriction alleviating effects of HO-1/BMMSCs were superior to those of BMMSCs (Figure 5C).

The serum ET-1 levels after RLT decreased initially and then increased gradually after operation. The level of ET-1 in the HO-1/BMMSCs group was lower than that in the NS group on POD1 (P < 0.01). The level of ET-1 in the HO-1/BMMSCs group was the lowest among the three groups, while the NS group showed the highest levels on POD5 and POD7 (P < 0.01). The level of ET-1 in the HO-1/BMMSCs group was significantly lower than that in the BMMSCs and NS groups on POD14 (P < 0.01). The results indicated that BMMSCs could inhibit the secretion of ET-1 in rats after liver transplantation, and the inhibitory effects of HO-1/BMMSCs were better than those of BMMSCs (Figure 6A).

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Figure 6 Endothelin-1/nitrous oxide related factors expression in serum after liver transplantation. A: Expression of endothelin-1 (ET-1); B: Expression of endothelial nitric-oxide synthase (eNOS); C: Expression of inducible nitric-oxide synthase (iNOS); D: Expression of NO. The serum ET-1 after RLT decreased initially and then increased with the extension of post-operative time. Postoperative day (POD) 1: HO-1/BMMSCs group vs NS group: 24.53 ± 1.22 vs 29.29 ± 2.97 (P < 0.01). POD5: HO-1/BMMSCs vs BMMSCs vs NS group: 28.84 ± 0.17 vs 32.18 ± 0.30 vs 36.27 ± 0.92 (P < 0.01). POD7: HO-1/BMMSCs group vs BMMSCs group vs NS group: 29.92 ± 0.28 vs 31.87 ± 0.50 vs 36.25 ± 0.54 (P < 0.01). POD14: HO-1/BMMSCs group vs NS group: 33.82 ± 1.44 vs 40.03 ± 0.17 (P < 0.01); HO-1/BMMSCs group vs BMMSCs group: 33.82 ± 1.44 vs 38.35 ± 2.09 (P < 0.01). The serum levels of eNOS increased with the extension of post-operative time. POD1: HO-1/BMMSCs group vs NS group: 5.86 ± 0.30 vs 4.30 ± 0.07 (P < 0.01); HO-1/BMMSCs group vs BMMSCs group: 5.86 ± 0.30 vs 4.74 ± 0.54 (P < 0.01). POD5: HO-1/BMMSCs vs BMMSCs vs NS group: 7.39 ± 0.29 vs 6.36 ± 0.07 vs 5.27 ± 0.16 (P < 0.01). POD7: HO-1/BMMSCs group vs BMMSCs group vs NS group: 7.43 ± 0.19 vs 6.17 ± 0.12 vs 5.37 ± 0.12 (P < 0.01). POD14: HO-1/BMMSCs group vs NS group: 6.72 ± 0.30 vs 5.40 ± 0.15 (P < 0.01); HO-1/BMMSCs group vs BMMSCs group: 6.72 ± 0.30 vs 5.26 ± 0.20 (P < 0.01). The serum levels of iNOS increased with the extension of post-operative time, and was most significant in NS group. POD1: HO-1/BMMSCs group vs NS group: 8.57 ± 0.07 vs 10.24 ± 0.42 (P < 0.01); BMMSCs group vs NS group: 8.56 ± 0.20 vs 10.24 ± 0.42 (P < 0.01). POD5: HO-1/BMMSCs vs BMMSCs vs NS group: 9.06 ± 0.05 vs 9.97 ± 0.08 vs 15.66 ± 0.13 (P < 0.01). POD7: HO-1/BMMSCs group vs NS group: 9.87 ± 0.42 vs 15.96 ± 0.74 (P < 0.01); BMMSCs group vs NS group: 10.85 ± 0.90 vs 15.96 ± 0.74 (P < 0.01). POD14: HO-1/BMMSCs group vs NS group: 10.04 ± 0.61 vs 16.85 ± 0.31 (P < 0.05); BMMSCs group vs NS group: 10.73 ± 0.64 vs 16.85 ± 0.31 (P < 0.01). The serum levels of NO tended to increase initially and then decrease with the extension of post-operative time. POD1: HO-1/BMMSCs group vs NS group: 10.64 ± 0.36 vs 7.92 ± 0.59 (P < 0.01); HO-1/BMMSCs group vs BMMSCs group: 10.64 ± 0.36 vs 8.56 ± 0.44 (P < 0.01). POD5: HO-1/BMMSCs vs BMMSCs vs NS group: 9.17 ± 0.08 vs 9.65 ± 0.09 vs 10.50 ± 0.07 (P < 0.01). POD7: HO-1/BMMSCs group vs NS group: 9.19 ± 0.12 vs 10.00 ± 0.10 (P < 0.01); BMMSCs group vs NS group: 9.41 ± 0.39 vs 10.00 ± 0.10 (P < 0.01). ^aP < 0.05 vs NS group, ^cP < 0.05 vs HO-1/BMMSCs group.

Effects of HO-1/BMMSCs on eNOS expression in the transplanted liver: The immunohistochemical results showed that eNOS positive cells were present in the hepatic sinusoids around the Glisson system. The ratios of eNOS positive cells in the BMMSCs and HO-1/BMMSCs groups were significantly higher than in the NS group. HO-1/BMMSCs group showed more eNOS-positive cells than the BMMSCs group, and the number of eNOS-positive cells was low in the NS group (Figure 7A and B).

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Figure 7 Endothelial nitric-oxide synthase expression in liver after liver transplantation. A: Immunohistochemistry (IHC) of endothelial nitric-oxide synthase (eNOS); B: IHC integrated optical density (IOD) of eNOS; C: Western blotting and eNOS protein levels. eNOS protein levels: postoperative day (POD) 1: HO-1/BMMSCs group vs BMMSCs group vs NS group: 0.79 ± 0.14 vs 0.42 ± 0.02 vs 0.25 ± 0.02 (P < 0.05). POD5: HO-1/BMMSCs vs NS group: 0.84 ± 0.04 vs 0.50 ± 0.01 (P < 0.01); HO-1/BMMSCs group vs BMMSCs group: 0.84 ± 0.04 vs 0.50 ± 0.01 (P < 0.05). POD7: HO-1/BMMSCs group vs BMMSCs group vs NS group: 1.00 ± 0.12 vs 0.84 ± 0.06 vs 0.50 ± 0.01 (P < 0.05). POD14: HO-1/BMMSCs group vs BMMSCs group vs NS group: 0.90 ± 0.09 vs 0.77 ± 0.07 vs 0.48 ± 0.02 (P < 0.01). ^aP < 0.05 vs NS group, ^cP < 0.05 vs HO-1/BMMSCs group.

The level of eNOS in the transplanted liver tended to increase with time after operation. The level of eNOS was the highest in HO-1/BMMSCs group, and the lowest in the NS group on POD1, 7, and 14 (P < 0.05). The level of eNOS in the HO-1/BMMSCs group was higher than that in the BMMSCs and NS groups on POD5 (P < 0.05). These results suggested that BMMSCs could promote the synthesis of eNOS in the transplanted liver, and more significantly in the HO-1/BMMSCs group (Figure 7C).

The serum levels of eNOS increased from POD1 to POD14 after RLT, but were lower than that on POD0. The levels of eNOS in the HO-1/BMMSCs group were significantly higher than those in the BMMSCs and NS groups on POD1 and POD14 (P <0.01). The level of eNOS was highest in HO-1/BMMSCs group and lowest in the NS group on POD5 and POD7 (P <0.01). This suggested that BMMSCs could promote the synthesis of eNOS in rats after RLT, and the promotive effects of HO-1/BMMSCs were more significant than those of BMMSCs (Figure 6B).

Effects of HO-1/BMMSCs on iNOS expression in the transplanted liver: The immunohistochemical results showed that iNOS positive cells were present in hepatic sinusoids around the Glisson system, and that iNOS was expressed in the cytoplasm of hepatocytes. The ratio and staining intensity of iNOS positive cells in the BMMSCs and HO-1/BMMSCs groups were significantly lower than those in the NS group, and those in the HO-1/BMMSCs group were lower than in the BMMSCs group. The number and staining intensity of iNOS positive cells in the NS group were relatively high (Figure 8A and B).

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Figure 8 Inducible nitric-oxide synthase expression in the liver after liver transplantation. A: Immunohistochemistry (IHC) of inducible nitric-oxide synthase (iNOS); B: IHC integrated optical density (IOD) of iNOS; C: iNOS protein levels. iNOS protein levels: postoperative day (POD) 1: HO-1/BMMSCs group vs NS group: 0.21 ± 0.02 vs 0.35 ± 0.07 (P < 0.05). POD5: HO-1/BMMSCs vs NS group: 0.48 ± 0.03 vs 0.30 ± 0.05 (P < 0.01); HO-1/BMMSCs group vs BMMSCs group: 0.48 ± 0.03 vs 0.34 ± 0.02 (P < 0.01). POD7: HO-1/BMMSCs group vs BMMSCs group vs NS group: 0.38 ± 0.01 vs 0.52 ± 0.04 vs 0.83 ± 0.03 (P < 0.01). POD14: HO-1/BMMSCs group vs BMMSCs group vs NS group: 0.45 ± 0.06 vs 0.73 ± 0.08 vs 0.86 ± 0.03 (P < 0.05). ^aP < 0.05 vs NS group, ^cP < 0.05 vs HO-1/BMMSCs group.

The level of iNOS in the transplanted liver tended to increase with the extension of post-operative time. The level of iNOS in the HO-1/BMMSCs and BMMSCs groups was lower than that in the NS group at all time points. The level of iNOS in the HO-1/BMMSCs group was significantly higher than that in the NS group on POD1 (P < 0.05), and was significantly higher than that in BMMSCs and NS groups on POD5 (P < 0.05). The expression of iNOS was highest in the HO-1/BMMSCs group and lowest in the NS group on POD7 and POD14 (P < 0.05). This suggested that BMMSCs could inhibit the synthesis of iNOS in the transplanted liver, and that the inhibitory effects of HO-1/BMMSCs were more significant than those of BMMSCs (Figure 8C).

The serum expression of iNOS increased with the extension of post-operative time. The expression of iNOS in HO-1/BMMSCs group and BMMSCs group was significantly lower than that in NS group on POD1, 7, and 14 (P < 0.01). The expression of iNOS was the lowest in HO-1/BMMSCs group and highest in NS group on POD5 (P < 0.01). This suggested that BMMSCs could inhibit the synthesis of iNOS in rats after RLT, and the inhibitory effects of HO-1/BMMSCs were more significant than that of BMMSCs (Figure 6C).

Effects of HO-1/BMMSCs on NO production in rats after RLT: The serum expression of NO tended to increase initially and then decrease with time after operation; however, the decreasing tendency was not significant in all groups. The serum NO in the HO-1/BMMSCs group was higher than that in the BMMSCs and NS groups on POD1 (P < 0.01). The serum NO on POD5 was lowest in the HO-1/BMMSCs group and highest in the NS group (P < 0.01). The serum NO in the HO-1/BMMSCs and BMMSCs groups was significantly lower than that in the NS group on POD7 (P < 0.01). This suggested that BMMSCs could inhibit the synthesis of NO in the transplanted liver, and the inhibitory effects of HO-1/BMMSCs were more significant than those of simple BMMSCs (Figure 6D).

Effects of HO-1/BMMSCs on vWF expression in hepatic sinusoids: vWF was expressed in sinusoidal endothelial cells and SECs of transplanted liver. Immunohistochemical staining for liver vWF showed that the ratios of vWF positive cells in the BMMSCs and HO-1/BMMSCs groups were significantly higher than that in the NS group, and the level in the HO-1/BMMSCs group was higher than that in the BMMSCs group. The number of vWF positive cells in the NS group was relatively low and the cells were scattered. Whereas the vWF positive cells in the BMMSCs and HO-1/BMMSCs groups were arranged regularly and were consistent with hepatic sinusoids. These results showed that HO-1/BMMSCs could promote the proliferation of SECs and the remodeling of hepatic sinusoids more significantly than BMMSCs. The results suggested that HO-1 could enhance the ability of BMMSCs to promote the regeneration of hepatocytes (Figure 9A and B).

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Figure 9 von Willebrand factor expression in the liver after liver transplantation. A: Immunohistochemistry (IHC) of von willebrand factor (vWF); B: IHC integrated optical density (IOD) of vWF; C: Western blotting and vWF protein levels.vWF protein levels: Postoperative day (POD) 1: HO-1/BMMSCs group vs NS group: 0.36 ± 0.05 vs 0.20 ± 0.00 (P < 0.01); HO-1/BMMSCs group vs BMMSCs group: 0.36 ± 0.05 vs 0.25 ± 0.01 (P < 0.01). POD5: HO-1/BMMSCs vs BMMSCs group vs NS group: 0.43 ± 0.08 vs 0.30 ± 0.01 vs 0.19 ± 0.01 (P < 0.05). POD7: HO-1/BMMSCs group vs BMMSCs group vs NS group: 0.46 ± 0.04 vs 0.33 ± 0.03 vs 0.26 ± 0.03 (P < 0.05). POD14: HO-1/BMMSCs group vs NS group: 0.28 ± 0.03 vs 0.18 ± 0.01 (P < 0.01). ^aP < 0.05 vs NS group, ^cP < 0.05 vs HO-1/BMMSCs group.

Intrahepatic expression of vWF tended to increase initially and then decrease after POD7; however, the NS group showed no significant increasing trend. The expression of vWF in the HO-1/BMMSCs group was significantly higher than in the BMMSCs and NS groups on POD1 (P < 0.05). The level of vWF was highest in the HO-1/BMMSCs group and lowest in the NS group on POD5 and POD7 (P < 0.05). The level of vWF in the HO-1/BMMSCs group was higher than that in NS group on POD14 (P < 0.05). These results suggested that BMMSCs could promote the synthesis of vWF in the transplanted liver graft, and that the effect of HO-1/BMMSCs was more significant than that of BMMSCs. However, the effects of BMMSCs decreased with time after operation (Figure 9C).

Effects of HO-1/BMMSCs on the degradation of HA by SECs: The serum HA in all groups tended to increase initially, then decrease, and later increase again with increasing post-operative time. Serum HA was lowest in the HO-1/BMMSCs group and highest in the NS group on POD5 and POD7 (P < 0.01). Serum HA in the HO-1/BMMSCs group was lower than that in the NS group on POD14 (P < 0.01). This suggested that BMMSCs could promote the degradation of HA in rats after RLT, and the effect of HO-1/BMMSCs was more significant than that of simple BMMSCs. However, the effects of BMMSCs decreased with increasing post-operative time (Figure 10).

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Figure 10 Hyaluronic acid expression in serum after liver transplantation. The serum hyaluronic acid (HA) level tended to increase initially, then decrease, and later increase again with the extension of post-operative time. Serum HA in the HO-1/BMMSCs group was lower than that of the BMMSCs and NS groups on postoperative day (POD) 5: HO-1/BMMSCs vs BMMSCs group vs NS group: 4.31 ± 0.07 vs 4.66 ± 0.24 vs 5.99 ± 0.15 (P < 0.01). POD7: HO-1/BMMSCs group vs BMMSCs group vs NS group: 4.78 ± 0.23 vs 5.73 ± 0.11 vs 6.72 ± 0.14 (P < 0.01). POD14: HO-1/BMMSCs group vs NS group: 11.43 ± 0.52 vs 12.52 ± 0.51 (P < 0.01). ^aP < 0.05 vs NS group, ^cP < 0.05 vs HO-1/BMMSCs group.

Effects on mitochondrial morphology under electron microscope: There were various degrees of damage to mitochondria in hepatocytes after RLT. The damage was more severe on POD7 in the NS group, with obvious mitochondrial swelling, vacuolization, and disturbed structures of mitochondrial cristae (some even disappeared). Mitochondria in the HO-1/BMMSCs and BMMSCs groups showed mild swelling, no vacuolization, and integrated structures of the mitochondrial ridge. These results suggested that HO-1/BMMSCs could ameliorate the damage to mitochondria (Figure 11).

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Figure 11 Mitochondrial ultrastructure changes on the 7^th d after liver transplantation. A: Postoperative day (POD) 0; B: Normal saline (NS) group on POD7; C: Bone marrow-derived mesenchymal stem cells (BMMSCs) group on POD7; D: HO-1/BMMSCs group on POD7 (× 25000). The mitochondrial damage to hepatocytes was relatively severe in the NS group, with obvious swelling and vacuolized mitochondria and disturbed structures of mitochondrial cristae (some even disappeared). Mitochondria in the HO-1/BMMSCs and BMMSCs groups showed mild swelling, no vacuolization, and the integrated structures of the mitochondrial ridge were present.

ATPase can degrade ATP to produce ADP and inorganic phosphorus, thus the amount of inorganic phosphorus reflects the activity of ATPase. The activities of ATPase in the transplanted liver tended to increase initially and then decrease after POD 7. The activities of ATPase in the HO-1/BMMSCs group were higher than those of the BMMSCs and NS groups on POD1, 7, and 14 (P < 0.05). The activities of ATPase in the HO-1/BMMSCs and BMMSCs groups were both higher than that in NS group on POD5 (P < 0.05). The results suggested that HO-1/BMMSCs could improve the activity of hepatic Na⁺-K⁺-ATPase and promote hepatic energy metabolism (Figure 12A).

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Figure 12 Liver Na⁺-K⁺-ATPase activity and mitochondrial aspartate aminotransferase level after liver transplantation. A: Na⁺-K⁺-ATPase activity; B: Serum mitochondrial aspartate aminotransferase (ASTm). Na⁺-K⁺-ATPase activity (μmoLPi/mgprot per hour): The degradation of ATP per mg of protein per hour produces 1 μmol of inorganic phosphorus was defined as one unit of ATPase activity. The activities of ATPase in the HO-1/BMMSCs group were higher than those of the BMMSCs and NS groups. Postoperative day (POD) 1: HO-1/BMMSCs group vs NS group: 0.79 ± 0.04 vs 0.42 ± 0.02 (P < 0.01); HO-1/BMMSCs group vs BMMSCs group: 0.79 ± 0.04 vs 0.57 ± 0.04 (P < 0.01). POD5: BMMSCs vs NS group: 0.59 ± 0.01 vs 0.40 ± 0.03 (P < 0.05); HO-1/BMMSCs vs NS group: 0.64 ± 0.04 vs 0.40 ± 0.03 (P < 0.01). POD7: HO-1/BMMSCs group vs NS group: 0.79 ± 0.03 vs 0.46 ± 0.02 (P < 0.01); HO-1/BMMSCs group vs BMMSCs group: 0.79 ± 0.03 vs 0.58 ± 0.03 (P < 0.01). POD14: HO-1/BMMSCs group vs NS group: 0.63 ± 0.05 vs 0.40 ± 0.03 (P < 0.01); HO-1/BMMSCs group vs BMMSCs group: 0.63 ± 0.05 vs 0.52 ± 0.03 (P < 0.05). ASTm showed a tendency to increase initially and then decrease with the extension of post-operative time. The expression of ASTm was the lowest in the HO-1/BMMSCs group and highest in the NS group on POD0, 1, 5, 7 and 14 (P < 0.05). ^aP < 0.05 vs NS group, ^cP < 0.05 vs HO-1/BMMSCs group.

ASTm exists in the mitochondria of hepatocytes, and is released into the blood when hepatocytes are severely damaged. Thus, the serum ASTm level can indicate whether the liver function is normal or not, and is also a sign of mitochondrial damage. In the present study, ASTm showed a tendency to increase initially and then decrease with increasing post-operative time. The expression of ASTm was lowest in the HO-1/BMMSCs group and highest in the NS group on POD0, 1, 5, 7 and 14 (P < 0.05; Figure 12B).

Graft injury after reduced-size liver transplantation (RLT) can affect the quality of donor liver seriously. Disturbance of the hepatic microcirculation and disorder of the hepatic energy metabolism are important factors affecting liver function after LT. Bone marrow mesenchymal stem cells (BMMSCs) can alleviate hepatic ischemia-reperfusion injury, accelerate liver regeneration, and have anti-inflammatory and immunoregulatory effects; however, their survival rate is low and survival time is short. Heme oxygenase-1 (HO-1) can regulate BMMSCs by enhancing the regulatory effects of BMMSCs under the state of hypoxia and oxidative stress, and can prolong the survival time of BMMSCs. The aim of this study was to explore the effects of HO-1/BMMSCs on hepatic microcirculation and energy metabolism of the transplanted liver in a rat model of rejection following RLT.

BMMSCs have the potential for multipotent differentiation, regeneration promotion, and anti-inflammatory and immunoregulatory effects, and have been used widely in a variety of cell therapy research. However, the survival rate and survival time of BMMSCs in the diseased tissue are low. Therefore, how to improve the survival time of BMMSCs in vivo, and improve the microcirculation and energy metabolism of the transplanted liver at the same time, is a research hotspot.

HO-1/BMMSCs could improve the hepatic microcirculation after RLT significantly, and they decreased SECs injury and restored the energy metabolism of the damaged hepatocytes, showing a good protective effect on the transplanted liver.

In this study, BMMSCs modified by HO-1 could prolong the survival time and improve the activity of BMMSCs, and further demonstrated the protective effect of HO-1/BMMSCs on the transplantated liver after RLT. These results provided the basis for improving the quality of reduced-size transplanted livers by gene therapy combined with stem cells transplantation, and provide a reliable method to expand the source of donor livers.

Sinusoids are central structures of the hepatic microcirculation, and the sinusoidal wall consists of SECs, Kupffer cells, hepatic stellate cells, and crypt cells, which play important roles in maintaining sinusoidal microcirculation and intrahepatic homeostasis. BMMSCs are non-hematopoietic stem cells derived from bone marrow, with multi-directional differentiation potential, and function in tissue repair, paracrine signaling, anti-inflammation, and immunoregulation. HO-1 is a microsomal oxidase in mammals, with protective effects, including anti-inflammation, anti-oxidative stress, anti-apoptosis, anti-ischemia reperfusion injury, and microcirculatory modulation.

This article demonstrates the protective effects of HO-1/BMMSCs on the transplanted liver in a rat model of rejection following RLT, which was studied from hepatic microcirculation and energy metabolism, hepatic microcirculation is very important and not well studied in liver transplantation. This study will be of interest and the paper is clearly written.