The Effect of Bay 11-7082 Inhibits Rat BMSCs to Hepatocyte-Like Cells via NF-κB Signaling in Vitro

Bone marrow stem cells (BMSCs) could be induced into hepatocytes by different cytokines, which has a great potential in tissue engineering. However, the differentiation was not so efficient mainly due to the less understanding of molecular mechanism. Our objective was to investigate the effect of BAY 11-7082 on the differentiation of BMSCs into hepatocyte-like cells. We observed the majority of differentiated cells were spherical or ovate in shape, indocyanine green (ICG) positive, α-1-antitrypsin (AAT) expression increased and NF-кB transferred into nuclear. Whereas the BMSCs additionally added with BAY 11-7082, which were showed fusiform or polygonal morphologies unchanged in shape, ICG uptake and AAT expression reduced, NF-кB scattered in the cytoplasm. These data suggest the activation of BAY 11-7082 may inhibit the differentiation of BMSCs into hepatocyte-like cells via NF-кB signaling.


Introduction
Bone marrow stem cells (BMSCs) are multipotent stem cell population that can be derived from bone marrow and induced to differentiate into a variety of cell types in vitro [1] . However, the differentiation of BMSCs was too complicated to demonstrate clearly the biochemical and molecular signals of these phenomenons .
In previous studies showed NF-κB participates in the differentiation of BMSCs into neurons or osteoblast.
NF-κB usually stays in the cytoplasm and was a trimer. Such as P50, P60 and inhibit-protein IκBα [2] .
Then IκBα will dissociate from the trimer by phosphorylation after stimulated by other signals. At the same time, NF-κB consist of P50 and P65 transfers into nuclear and combines with the common κB sequence in the enhancer domain of κB reaction gene to promote the expression of downstream genes [3] .
Our work recently showed BMSCs could be induced to hepatocyte-like cells that ingested ICG. While ICG-uptake is a useful marker to identify differentiated hepatocytes in vitro, which was used to illustrate the degree of differentiation [4] . And we mainly stimulated BMSCs by  (Beyotime) to investigate the variety of BMSCs into hepatocyte-like cells.

Reagents and animals
The DMEM-low glucose medium was from Hyclone.
The fetal bovine serum (FBS) was obtained from Gibco. HGF and β-NGF were from Sigma. Adult SD rats (weight from 200~300g, six weeks) were provided from the Medical-Experimental Animal Center of Qingdao University.

Separation and culture of rat BMSCs
By whole bone marrow adherence method for separation of SD rats BMSCs, we took adult rats bone marrow aspirates from bilateral shinbones of normal donors, flushed with DMEM-low glucose medium supplemented with 10% FBS, and seeded directly in 25 cm 2 culture flasks at a density of 1×10 7 cells/cm 2[5] . The cultures were maintained at 37 ºC in a humidified atmosphere of 5% CO2, while the non-adhered cells were removed via the culture medium changing at 24 th h during primary culturing.
When BMSC reached to 80% confluency, we dissociated them with Accutase enzymes (Mpbio) and replated for subculture. Subsequently, the third passage cells were collected and used in this experiment.

Preparation of LTF
The neonatal rat liver homogenate was supplemented with 10% FBS DMEM-low glucose medium (100 mg:1 ml), which was centrifugalized for 20 min at 10000 rpm after standing at 4 ºC, then the supernatant fluid after 0.22 μm membrane filter reserved at -20 ºC. The effective concentration of LTF was 100 mg/ml.

Hepatocyte-like cells induction and group design
The subjects were divided into eight groups in total, each group induced by different stimuli ( Table 1 ).
The final concentration of HGF, β-NGF, LTF and BAY 11-7082 was 20 ng/ml [6] , 40 ng/ml, 1 mg/ml, 10nmol/ml [7] , respectively. Culture media was changed every 3 days and the induction maintained for 20 days. In addition, all of BAY groups were treated with BAY 11-7082 in the presence of 10% FBS DMEM-low glucose medium for one day before the formal induction.

ICG uptake assay
ICG was dissolved in sterile phosphate buffered saline (PBS) to produce a fresh 5 mg/ml stock solution and was diluted in 10% FBS DMEM-low glucose medium to a final concentration of 1 mg/ml [4] , added to differentiated BMSCs induced for 1 day and 20 days, which were incubated at 37 ºC for 2 h for the uptake of ICG by the BMSCs. Then culture flask

Immunofluorescence.
BMSCs induced for 20 days were fixed and permeabilized using 100% cold acetone for 10 min.
After washing with PBS, the samples were blocked  and used to assess protein content [9] . The relative protein levels were assessed by the chemiluminescent signal compared with GAPDH or TBP protein.

Statistical analysis
The results were expressed as means ± SD. Statistical significance was determined using GraphPadPrism 5 software, Analysis followed a Student's t test was used to determine significant difference between two groups [10] . For analysis of multiple groups, the P values were adjusted using the Bonferroni method after analysis of variance (ANOVA) and P values < 0.05 were considered to be statistically significant.

BMSCs
The primary cells of BMSCs during three times subculture presented fibroblast-like, fusiform or polygonal morphologies closely spaced growth in shape (Figures 1 A). After stimulated for 20 days.

Some
BMSCs were differentiated into hepatocyte-like cells and became round or oval shaped (Figures 1 B)

ICG uptake of differentiated BMSCs
After continuously induced for 20 days. None of cells within control group and BAY control group could ingest ICG and the percentage of positive cells was 0% (Figures 1 C-D). The cells appeared green in HGF induced group, β-NGF induced group and LTF induced group (Figures 1 E-G

NF-κB transfer of differentiated BMSCs
After induced for 20 days (Figures 2 A-B)

NF-κB and AAT expression of differentiated BMSCs
The ANOVA and multiple comparisons for data by which has demonstrated again the BAY 11-7082 will decrease the preference that differentiation of BMSCs into hepatocyte-like cells.
ICG is clinically used as a test substance to evaluate liver function because it is eliminated exclusively by hepatocytes. Yamada T et al [4] focused on molecular analyses that liver-specific protein genes such as AAT , CPSase I (the first enzyme of the urea cycle indicate that ICG positive cell differentiation is specified toward hepatocytes and that these cells possess the genetic characteristics of almost terminally differentiated hepatocytes. Furthermore, [15] . But in this paper, we just observed whether NF-кB signals transfer into nuclear.
Only by altering the expression of genes or stimulating with cytokines can we induce the differentiation of BMSCs into hepatocytes.
Levinson-Dushnik et al [16] examined embryonic stem (ES) cells expressed albumin which specific protein come from hepatocytes after transfected HNF3α or HNF3β genes into the ES cells. Liu X P et al [17] demonstrated albumin and AAT were found from primordial germ cells which were cultured in the presence of hepatocyte abstract, β-NGF, HGF and ingested ICG. In the present study, LTF as a positive control provided an appropriate microenvironment that supports differentiation of BMSCs into hepatocytes. However LTF is a multiple factor inducer, for this reason, HGF (an essential factor in the development and regeneration of the liver) [18] and β-NGF were used for inducing in order to explore distinctly.
NF-κB as one of the most important factor for cell proliferation and differentiation. Previous studies had indicated inhibiting the activation of NF-κB could promote the differentiation of BMSCs into neurons [19,20] . Interestingly, studies of osteoblast emphasized the activation of NF-κB enhanced the expression of BMP2 genes, which promote the osteogenic differentiation [21,22]  However, it was still higher than control (Figures 2   H). This implied that some other factors except NF-κB signaling regulate cell differentiation.

Conclusions
In this paper, we showed the cells of BAY group reduced ICG uptake and AAT expression, NF-кB scattered in the cytoplasm. While the induce groups showed ICG uptake and AAT expression increased, NF-кB signals transferring into nuclear. Therefore