Effect of Lipopolysaccharide and 4-(Methylnitro-samino)-1- (3-pyridyl)-1-butanone on the Proliferation of Mouse Bone Marrow Stem Cells

BMSCs were isolated, cultured and proliferated in vitro. The proliferation of mouse BMSCs treated with different concentrations of LPS and NNK were detected by MTT assay, the expression of NF-κB and CUGBP1 in the BMSCs were analyzed by Immunocytochemistry assay and Western Blot. MTT results shown that different concentrations of LPS and NNK could promote the proliferation of BMSCs and the promotion effect of LPS12.5μg/ml, NNK10μg/ml and LPS12.5μg/ml combined NNK 10μg/ml was much more significant than others. Immunocytochemistry and Western Blot revealed that the location and the expression level of NF-κB and CUGBP1 in BMSCs treated with LPS and NNK were changed with different incubation time. The highest expression of NF-κB was detected at the LPS48h, NNK24h and LPS combined NNK24h and significant difference can be seen in-group compared. The highest expression of CUGBP1 was LPS96h, NNK48h and LPS combined NNK24h and significant difference can be seen in-group compared. The irritation of LPS and NNK on the proliferation of mouse BMSCs may mediate by up-regulating the expression of NF-κB and CUGBP1.


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one of the most harmful components of cigarette smoke and can cause a variety abnormal change of immune function. It can result in the production of toxins that bind DNA to form adducts that causes the mutation of oncogene and anti-oncogenewhen it is metabolized in body. One study suggests that NNK can bind with acetylcholine receptors to promote cell proliferation, survival and migration [3] .
Nuclear transcription factor (NF-κB) has the function of transcriptional activation, occurrence of inflammation, anti-apoptosis, directly involving in cell cycle regulation, and the activation of NF-κB can promote cell proliferation [4 ～ 5] , suppresses NF-ĸB activation causing reduced proliferation and induction of apoptosis in cell culture [6 ～ 7] . CUG linking protein1 (CUGBP1) is a kind of RNA-binding protein that acts in the nucleus and cytoplasm to regulate alternative splicing, deadenylation, mRNA stability and translation [8] . CUGBP1 expression is associated with the proliferation of cells and its down-regulated expression inhibits cell proliferation [9] . Previous studies showed that LPS combined NNK can significantly increase the incidence of lung cancer in vivo and stimulate the proliferation of cancer cells [10] . However, the effect of LPS and NNK on the proliferation and NF-κB,

The isolation, purification and amplification of mouse BMSCs
After sacrificed, the femur and tibia ofmouse were separated in clean bench and the cells in bone marrow cavities were rinsed by DMEM-high glucose medium containing 10% FBS, the cell suspension was transferred into 25ml flask and cultured in incubator with 37℃, 5%CO2, and saturated humidity.
BMSCs were purified by differential adhesion method.

Statistical Analyses
Data were represented as mean±standard deviation (mean±SD) and analyzed by SPSS 17.0.The difference was considered significant when P< 0.05.

The effect of LPS, NNK and LPS combined NNK on the proliferation of mouse BMSCs
The proliferation activities of BMSCs incubated with LPS (12.5μg/ml, 25μg/ml and 50μg/ml), NNK (5μg/ml, 10μg/ml and 20μg/ml) and LPS combined NNK for 96h were detected by MTT assay and compared.
There was no significant difference between the group of LPS50 and control (tcontrol, LPS50=3.578, P ＞ 0.05).

CUGBP1 in mouse BMSCs
The expression of NF-κB and CUGBP1in BMSCs incubated with LPS (12.5μg/ml) for 24h, 48h and 96h were detected and compared.

CUGBP1 in mouse BMSCs
The expression of NF-κB and CUGBP1 in BMSCs incubated with NNK (10μg/ml) for 24h, 48h and 96h were observed and compared.
After incubation with NNK for 24h, 48h, 96h respectively, the positive expression of NF-κB were significantly increased in both cytoplasm and nucleus.
Upon stimulation, IκBα undergoes phosphorylation and ubiquitination-dependent degradation leading to p65 nuclear translocation and binding to a specific consensus sequence in the DNA [12~13] . Activated NF-κB binds to specific DNA sequences and regulates the expression of its target genes, leads to the expression of the downstream signal which may be caused the variation of cell proliferation [14] . Recently studies showed that the migration and proliferation ability of breast cancer cells would be suppressed after NF-κB signaling pathways were inhibited [15] , miR-451 over expression inhibits cell proliferation by inhibiting the NF-κB signaling pathway through the direct suppression of IKK-β [16] .
In this paper, we demonstrated that LPS at the Chronic inflammation is accompanied by increased production of tissue reactive oxygen and nitrogen intermediates. The pre-neoplastic activity of reactive oxygen species is mainly due to their ability to cause DNA damage. Proteins and lipids are also significant targets for oxidative attack, and modification of these molecules in a developing tumor microenvironment can increase the risk of mutagenesis [24~26] .
The results showed that peak expression and nuclear transfer of NF-κB in BMSCs stimulated by LPS and NNK respectively were in a time; but the peak expression of CUGBP1 was lag than that of NF-κB and its nuclear transfer. When BMSCs treated with LPS combined NNK, the peak of NF-κB expression and its nuclear transfer was consistent with the peak of CUGBP1 expression. The relationships between NF-κB and CUGBP1 in the process of BMSCs proliferation need a further study.