A total of 18 of male C57BL/6 mice, aged 6–8 weeks, weighing 16–18 g, were purchased from the Academy of Military Medical Science (Beijing, China) and were housed in a pathogen-free room, with a 12 h light/dark cycle at 20–25°C. They were maintained on a normal diet or HFD obtained from TROPHIC Animal Feed High-Tech Co., Ltd. (Nantong, Jiangsu, China). The food compositions of the two dietary groups are presented in Table I. The total energy and cholesterol content in the two dietary groups are presented in Table II. The total protein, carbohydrate and total fat content within the total energy in the two dietary groups are presented in Table III. The mice were randomized into two groups: i) Normal mice; and ii) T/HFD mice. A total of 21 weeks subsequently, the T/HFD mice were randomized into two groups: i) T/HFD mice; and ii) T/HFD+MSC mice, which were intravenously injected twice with 1×10⁶ MSCs/mouse, at 21 and 23 weeks. A total of 21 weeks subsequently, the diets of the T/HFD and T/HFD+MSC mice were replaced with a normal diet. A total of 28 weeks subsequently, all of the animals were sacrificed and tissues were harvested. Mice were weighed weekly. The present study was approved by the Animal Ethics Committee of Tianjin Medical University (Tianjin, China).

MSCs obtained from murine compact bone were isolated and culture-expanded as described previously (19,21). Femurs and tibiae were collected from a total of 3 2–3-week-old female C57BL/6 mice (weighing 6–10 g) and were purchased from the Academy of Military Medical Science, Beijing, China. They were housed in a pathogen-free room, with a 12 h light/dark cycle at 20–25°C. Bone marrow was flushed with PBS or α-modified minimal essential medium (α-MEM) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using a syringe. The compact bones were excised into chips of ~1 mm into plastic culture dishes, and washed with PBS or α-MEM until the released cells were removed. The cells were incubated in α-MEM containing 10% select fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C, in an atmosphere containing 5% CO₂. The medium was replaced every 4–5 days. Adherent cells (passage 0) were confluent following 1–2 weeks of incubation and were harvested using a cell scraper. The cells were passaged by digestion with 0.25% trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.). Subsequent to 5–8 passages, the cells were used for further experiments.

The prepared MSCs, as described above, were harvested by digestion with trypsin, and stained for 30 min at 4°C with fluorescein isothiocyanate-conjugated anti-mouse CD11b, CD45, CD105 and ataxin-1 (Sca-1) antibodies, or phycoerythrin-conjugated anti-mouse CD29, CD44 and CD135 antibodies (all eBioscience, Inc.; Thermo Fisher Scientific, Inc.). Cells were analyzed using a FACSCalibur instrument using a laser at a wavelength of 488 nm (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometric data were analyzed using Flow Jo software (version 7.6; Tree Star, Inc., Ashland, OR, USA).

Livers were perfused with PBS, removed, weighed and sliced into 0.5×0.5 cm sections. The sections were embedded in paraffin subsequent to being fixed in 4% (w/v) paraformaldehyde and were cut into 6-µm-thick sections. The paraffin sections were stained with 0.5 % w/v hematoxylin for 5 min and 1% w/v eosin for 10 sec (HE) at room temperature to assess inflammation and steatosis, and picrosirius red to assess fibrosis. Evaluation of the extent of resultant NASH (3) was performed using the following scaling scores.

Hepatocytes containing fat vacuoles were subjectively visualized and graded according to the following scale: 0, Normal, no hepatocytes affected; 1, minor, <5% of hepatocytes affected; 2, mild, 5–33% of hepatocytes affected; 3, moderate, 34–66% of hepatocytes affected; and 4, severe, >66% of hepatocytes affected.

Grading of lobular inflammation was performed as follows: 0, None; 1, 1–2 foci/x20 field; 2, 2–4 foci/x20 field; and 3, >4 foci/x20 field.

Staging of NASH was performed as follows: 0, None; 1, extensive zone 3 perisinusoidal fibrosis; 2, zone 3 perisinusoidal, and portal or periportal fibrosis; 3, bridging fibrosis; and 4, cirrhosis.

Single-cell suspensions derived from spleens were prepared by mechanical disruption and filtered through a 40-µm cell strainer (BD Biosciences). The cells were placed in H₂O 30–50s, soon later added with 1/10 volume 10× PBS, and red blood cells were removed with cytolysis. The cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate, 1 µg/ml ionomycin (Enzo Life Sciences, Inc., Farmingdale, NY, USA) and 3 µg/ml brefeldin A (eBioscience, Inc.; Thermo Fisher Scientific, Inc.) for 5 h. The cells were subsequently stained for surface markers using rat anti-mouse CD4-allophycocyanin (catalog no. 17-0042-81, eBioscience, Inc., San Diego, CA, USA) for 30 min at 4°C. The cells were analyzed using the 488 nm laser of a FACSCalibur instrument and the data generated were analyzed using FlowJo software version 7.6.1 (Tree Star Inc., Ashland, OR, USA).

GraphPad PRISM software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) was used to perform a Student's t test Results are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

According to modified previously-described procedures (19), mMSCs were isolated from compact bone (from 2–3-week-old female C57BL/6 mice) and cultured. The MSCs appeared to be vortex-shaped and fibroblast-like, although not polygon-shaped (osteocytes) (Fig. 1A). In order to further identify the adherent cells, immunophenotypic features were analyzed. As presented in Fig. 1B, negative surface markers of MSCs, including CD11b, CD45 and CD135, were not expressed; however, the cells expressed surface markers characteristic of MSCs, including CD29, CD44, CD105 and Sca-1. The results of the present study indicated that these cells were MSCs.

In order to investigate the beneficial effects of MSCs on NAFLD, a mouse model of NAFLD was established using HFD, and the mice were intravenously-injected twice with 1×10⁶ MSCs/mouse at weeks 21 and 23 (Fig. 2A). A marked difference was observed between T/HFD and T/HFD+MSC mice at 28 weeks post-treatment (Fig. 2B). Compared with normal control mice, the T/HFD-fed mice exhibited an accelerated elevation of body weight between 1 and 21 weeks (Fig. 2C). Following the two intravenous injections of MSCs, the weight of the T/HFD-fed mice decreased markedly at 21–28 weeks (Fig. 2C).

Weight gain is often accompanied by the expansion of subcutaneous adipose tissue. The results of the present study demonstrated that the mass of subcutaneous abdominal adipose tissue increased in the T/HFD-fed mice, an effect which was reversed by the MSC treatment (Fig. 3).

NFLD may progress via hepatic lipid accumulation to NAS, and via lobular inflammation to NASH (3). Hepatocytes containing fat vacuoles may be visualized as clear bubbles following liver section HE staining. As presented in Fig. 4, hepatic lipid accumulation indicated by clear bubbles occurred in T/HFD-fed mice (Fig. 4A), while it was decreased in T/HFD+MSC mice (Fig. 4B). It was additionally observed that HFD-induced lobular inflammation was present within the THFD-fed mouse livers (Fig. 4A), which was suppressed in the T/HFD+MSC mice (Fig. 4B). As indicated by scores obtained from the methods described above, increases in steatosis (Fig. 4C) and lobular inflammation (Fig. 4D) induced by HFD were significantly decreased in response to MSC treatment.

NAFLD frequently progresses to fibrosis (3). Therefore, the stages of fibrosis in the liver samples were assessed by staining with picrosirius red. Fibrogenesis was observed within the T/HFD-fed mouse livers (Fig. 5A), and a reduction of hepatic fibrogenesis occurred in the T/HFD+MSC mice (Fig. 5B). Significant differences in fibrosis stage scores were observed between the T/HFD and T/HFD+MSC mice (Fig. 5C).

MSCs are able to interact with innate and adaptive immune system cells, leading to the modulation of numerous effector functions (9). Analysis of splenic leukocytes was performed in order to explore the effect of MSCs on immunological responses. It was observed that, compared with T/HFD-fed mice, the number of CD4⁺ T lymphocytes in the spleen was decreased by treatment with MSCs in the T/HFD+MSC mice (Fig. 6).