BM-MSCs were obtained from the femurs and tibias of 4-week-old Wistar rats by flushing the bone marrow cavity with complete culture medium as previously described (23). The extract was centrifuged, resuspended and cultured in a 75-cm² culture flask containing Dulbecco's modified Eagle's medium with low glucose (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), and 1% penicillin/streptomycin at 37°C and 5% CO₂. One-half of the medium was replaced following 24 h of culture, and the entire culture medium every 2–3 days. Cells were subcultured when the adherent cells reached 70–80% confluence.

Cells were identified as BM-MSCs by their morphology, adherence and surface markers. BM-MSCs of 3–4 passages were identified by antibodies against cluster of differentiation (CD)29-phycoerythrin (PE)-A, CD34-PE-A, CD44-PE-A, CD45-fluorescein isothiocyanate (FITC)-A, and CD90-PE-A (eBioscience; Thero Fisher Scientific, Inc.). Briefly, resuspended cells were incubated with CD29-PE-A (1:2; cat. no. 12-0291-82), CD34-PE-A (1:40; cat. no. MA1-10205), CD44-PE-A (1:40; cat. no. MA5-16908), CD45-FITC-A (1:20; cat. no. 11-0461-82) and CD90-PE-A (1:40; cat. no. MA1-80650) at room temperature. After 20 min, the cells were washed with three times PBS and a flow cytometer was used (FACS Aria II; BD Biosciences, San Jose, CA, USA). Software was used for data analysis (FlowJo software 7.6; FlowJo LLC, Ashland, OR, USA).

Cells were additionally identified by their multilineage differentiation potential, as previously reported (24). For adipogenic differentiation, adipogenic induction medium (Cyagen Biosciences, Co., Ltd., Suzhou, China) was added to the BM-MSCs at 37°C for 3 days, followed by maintenance medium (Cyagen Biosciences, Co., Ltd.) at 37°C for 1 day. Following three cycles, the cells were cultured in maintenance medium for a further 7 days. The cells were subsequently stained with Oil Red O (Cyagen Biosciences, Co., Ltd.) at room temperature for 30 min. The sections were washed and examined using a light microscope (magnification, ×40, Olympus Corporation, Tokyo, Japan). For osteogenic differentiation, the BM-MSCs were cultured with rat BM-MSC osteogenic differentiation medium (Cyagen Biosciences, Co., Ltd.) when subconfluent at 37°C. Subsequent to 3 weeks of differentiation, the calcium depositions were stained with Alizarin red (Cyagen Biosciences, Co., Ltd.) at room temperature for 3–5 min. The sections were washed and examined using a light microscope (magnification, ×40, Olympus Corporation).

A total of 30 healthy 4–6-week-old male Wistar rats weighing ~200 g were purchased from the Laboratory Animal Center of The Affiliated Drum Tower Hospital of Nanjing University Medical School (Nanjing, China), and housed under specific pathogen-free conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee of The Affiliated Drum Tower Hospital of Nanjing University Medical School, under the National Institutes of Health (Bethesda, MD, USA) Guide for the Care and Use of Laboratory Animals. All experiments were conducted under isoflurane anesthesia, and all efforts were made to minimize suffering.

The rats were randomly divided into five groups as follows: The sham group, the I/R group, the I/R+MSCs group, the 3-methyladenine (3-MA) group and the zinc protoporphyrin IX (ZnPP) group. A hepatic warm I/R model was used as previously described (25). The rats were anesthetized with isoflurane followed by laparotomy. A sterile microvascular clamp was placed around all structures in the portal triad for the left and median liver lobes to interrupt the blood supply in all groups except the sham group, which underwent the same procedure without vascular occlusion. Reperfusion was initiated via removal of the clamp after 1 h. In addition, 1×10⁶ MSCs suspended in 0.5 ml PBS were injected via the penis dorsal vein 30 min prior to hepatic warm I/R (26) in all the groups, except the sham group and the I/R group. In the 3-MA group, the autophagy inhibitor 3-MA was administered (30 mg/kg; intraperitoneal; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) 0.5 h prior to ischemia. The rats in the ZnPP group were treated twice with ZnPP (10 mg/kg; intraperitoneal; Sigma-Aldrich; Merck KGaA) at 16 and 3 h prior to ischemia to inhibit HO-1 in vivo. All the rats were sacrificed following 6 h of reperfusion, and blood samples and liver tissues were obtained. Blood samples were stored at 4°C overnight. A portion of the liver tissues were fixed in 10% formalin solution at room temperature for ≥24 h, and then prepared for HE staining and immunohistochemistry (5 µm) as described below. The remaining liver tissues were stored at −80°C for further use.

In order to detect the implantation of BM-MSCs in the liver following I/R injury, the cells were stained with lipophilic membrane dyes (DiO; Invitrogen; Thermo Fisher Scientific, Inc.) prior to intravenous administration of BM-MSCs. The BM-MSCs were suspended at a concentration of 10⁶ cells/ml and incubated with 10 mM DiO for 20 min at 37°C. Frozen sections were fixed at 4°C for 10 min in acetone and air-dried. The nuclei were stained with DAPI (Invitrogen; Thermo Fisher Scientific, Inc.). The sections were washed and examined using a Leica TCS SP8 confocal laser-scanning microscope (magnification, ×200; Leica Microsystems GmbH, Wetzlar, Germany).

Blood samples was harvested via the postcava and centrifuged at 3,000 × g at 4°C for 10 min. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an automatic analyzer (Fujifilm Global, Tokyo, Japan), as previously described (27).

Following fixation of the liver tissues in 10% buffered formalin at room temperature for at least 24 h, paraffin embedding was performed using a standard protocol. Paraffin sections (5 µm) were stained with hematoxylin at room temperature for 3–5 min and stained with eosin at room temperature for 2 min, then examined under a light microscope (magnification, ×100) for histological evidence of liver injury. Immunohistochemical staining for HO-1 was performed on the paraffin sections which were blocked with blocking serum (OriGene Technologies, Inc., Beijing, China) at room temperature for 10 min, incubated with primary antibodies against HO-1 (1:200; cat. no. ab13243, Abcam, Cambridge, UK) overnight at 4°C and developed using a horseradish peroxidase-conjugated secondary antibody (PV-6000, undiluted, OriGene Technologies, Inc., Beijing, China) at 37°C for 30 min and a 3,3-diaminobenzidine substrate kit (ZLI-9018, undiluted; OriGene Technologies, Inc.) at room temperature for 5–20 min, according to standard methods in routine pathology. The sections were washed and examined using a light microscope (×100; Leica Microsystems GmbH, Wetzlar, Germany).

Western blotting was conducted as previously described (22). Equal amounts (40 µg) of the proteins were separated on 15% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight at 4°C with primary antibodies against HO-1 and microtubule-associated proteins 1A/1B light chain 3B (LC3B; 1:2,000; cat. no. ab192890; Abcam). GAPDH (1:1,000; cat. no. ab9484; Abcam; incubated at 4°C overnight) expression served as a loading control. The membrane was treated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:8,000; KGAA37; Nanjing KeyGen Biotech Co,. Ltd., Nanjing, China) or horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:8,000; KGAA35; Nanjing KeyGen Biotech Co,. Ltd.) at room temperature for 2 h. Blots were developed using enhanced chemiluminescence (ECL) western blotting substrate (EMD Millipore, Billerica, MA, USA) and visualized on a Tanon 5200 Multi Image Station (Tanon Science & Technology Co., Ltd., Shanghai, China). The results were quantified by Image Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Statistical analysis was performed using SPSS version 19.0 (IBM Corp., Armonk, NY, USA) and data are expressed as the mean ± standard error of the mean; results were repeated in triplicate. Differences between the groups were evaluated for significance using one-way analysis of variance combined with Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

At passage 3 or passage 4, the cells from the rat bone marrow existed as a monolayer of typical fibroblastic, spindle-shaped and plastic-adherent cells (Fig. 1A). As presented in Fig. 1B, identification of BM-MSCs was performed using flow cytometry. These cells exhibited little or no expression of hematopoietic markers (CD34 and CD45) and positive expression of stromal markers (CD29, CD44 and CD90). In addition, the BM-MSCs had the potential to differentiate into osteoblastic or adipogenic lineages (Fig. 1C). Based on these results, the cells from rat bone marrow were confirmed to be BM-MSCs. The DiO-labeled BM-MSCs were used to clarify whether BM-MSCs migrated to the liver following I/R. BM-MSCs were observed in the I/R+MSCs group, although not in the sham group or the I/R group (Fig. 1D).

In order to determine whether BM-MSCs attenuate hepatic I/R injury, the rats were pretreated with BM-MSCs prior to ischemia. Serious liver damage was observed following 6 h of reperfusion, as previously reported (25). The rats were sacrificed 6 h post-insult, and liver tissues and blood were collected for further research. Compared with the rats in the I/R group, which had the highest ALT and AST levels, a significant improvement was noted in the I/R+MSCs group (Fig. 2A and B). In addition, the liver samples from the I/R group exhibited marked abnormalities, including severe hepatocellular necrosis and cytoplasmic vacuolization, following 6 h of reperfusion, which were improved in the I/R+MSCs group (Fig. 2C).

Autophagy is part of the adaptive stress response to infection or lipopolysaccharide (LPS) exposure to support cell survival. In order to clarify the effects of pretreatment with BM-MSCs on autophagy, the expression of the autophagy associated marker LC3B in the rat liver was examined in the sham group, the I/R group and the I/R+MSCs group by western blotting. Upon the induction of autophagy, LC3B is a terminal autophagic protein used as a classic marker to monitor alterations in autophagy (28). As presented in Fig. 3, the expression of the autophagic signaling factor LC3B was markedly increased in the I/R+MSCs group compared with the other study groups. These findings confirmed that autophagy increased following pretreatment with BM-MSCs.

To evaluate whether autophagy contributes to BM-MSC-mediated protection against I/R injury, the rats were pretreated with 3-MA prior to ischemia, which is widely regarded as an inhibitor of autophagy. The expression of autophagy was measured using western blotting to determine the presence of LC3B. Compared with the I/R+MSCs group, treatment with 3-MA resulted in a decrease in LC3B expression in response to liver I/R injury (Fig. 3). Notably, compared with the I/R+MSCs group, the serum levels of AST and ALT and the histological abnormalities were reversed in the 3-MA group (Fig. 2). Based on the results following pretreatment with 3-MA, it was concluded that autophagy served a notable role in BM-MSC-mediated protection against cell death following I/R injury.

The present study subsequently aimed to analyze how BM-MSCs promote autophagy following I/R injury. A previous study reported that upregulation of HO-1 was able to mitigate I/R injury (29). Furthermore, HO-1 has been demonstrated to be associated with autophagic activity (22,30). Consistent with these findings, alterations in HO-1 expression were observed in the case of pretreatment with BM-MSCs in response to I/R injury. HO-1 expression was increased in the rat livers subjected to I/R injury, while in the I/R+MSCs group, HO-1 expression was even more significantly increased (Fig. 4). Since BM-MSCs increased HO-1 expression and autophagic activity, it was preliminarily suggested that HO-1 may serve a role in the promotion of autophagy via pretreatment with BM-MSCs. Therefore, in order to identify the association between HO-1 and autophagy, HO-1 was inhibited by ZnPP. ZnPP significantly decreased the expression of LC3B (Fig. 5). Furthermore, inhibition of HO-1 by ZnPP significantly inhibited the protective effect provided by BM-MSCs following I/R insult. The serum levels of AST and ALT in the ZnPP-treated rats were increased compared with those in the I/R+MSCs group (Fig. 2). The I/R injury-associated histopathological alterations in the livers of rats were more severe in the ZnPP group compared with the I/R+MSCs group (Fig. 2C). Taken together, these data demonstrate that HO-1, increased by pretreatment with BM-MSCs, promoted autophagy, which protected against I/R injury in vivo.