| Objective:Chitosan oligosaccharides (COS), a low molecular weight, water-soluble, is derived from the chemical or enzymatic decomposition of chitosan.COS consists of 2 to 10 amino-glucose molecules withβ-1,4 glycosidic bonds, and is easily absorbed by tissues and regulates multiple cellular functions. COS showed significant cellular functions and was shown to interact with a variety of cells. In the current study, effects of COS on NF-κB signaling pathway, anti-tumor activity, and promotion of cell differentiation were investigated. We also used fluorescein isothiocyanate-labeled chitosan oligosaccharides (FITC-COS) to determine its subcellular localization, which is important to help us understand the mechanism underlying its multiple functions.Methods:(1) The effects of COS on nuclear factor -kappaB (NF-κB) signaling pathway were investigated by using luciferase assay. The nuclear translocation of NF-κB was determined by laser scanning microscopy. The expression of NF-κB downstream genes (cyclin D1, TNFa and IL-6) were tested by real time PCR;(2) The appearance of COS stimulated cells were demonstrated by the inverted microscope and electronic microscope. The red dead cells were calculated after incubating with COS in dead-live cell staining experiment. The role of COS on the mitochondrial membrane potential, proliferation and apoptosis related protein in A549 cells were investigated by flow cytometry and real time PCR. We employed two-dimensional electrophoresis coupled with MAILDE-TOF MS/MS analysis to identify differentially expressed proteins among COS stimulating cells, and to test the results by western blotting.(3)The proliferation of UCB-MSC cells were demonstrated by MTT, and the expression of cyclin D1, ALP, BMP2 and RUNX2 were observed by real time PCR. The bone nodule formation in cells was test by alizarin red staining;(4) We used fluorescein isothiocyanate-labeled chitosan oligosaccharides (FITC-COS) to determine its subcellular localization in pDsRed-Mit transfected cells by using a laser confocal microscope, and to investigate the quantization of COS that entered cells by flow cytometry, which would help us understand the mechanism it interacted with cells.Results:(1) Chitosan oligosaccharides increased NF-κB-dependent luciferase gene expression, and the 0.1mg/ml have the most significant effects; (2) Five chitosan oligosaccharides, from dimer to hexamer (chitobiose, chitotriose, chitotetraose, chitopentaose,chitohexaose) increased NF-κB-dependent luciferase gene expression and NF-κB downstream genes transcription, and chitotetraose and chitohexaose were the most potent activators. Laser scanning microscopy experiments showed that chitotetraose and chitohexaose also activated the p65 subunite of NF-κB traslocating from cytoplasm to nucleus; (3) The appearance of cells was changed after stimulated with lmg/ml COS. Some cells became fusiform or round, some were floating and dead. Apoptotic body and chromatin margination were seen in some cells. (4) After cultered with 1mg/ml COS for 4 days, the dead A549 cells increased to 38.6%, significantly up-regulated compared with control(3.4%). At the same time, COS declined the mitochondrial membrane potential of the A549 cells. (5) COS down regulated the mRNA levels of cyclin D1 and bcl-xl mRNA in A549 cell. Of all the five COS fractions, COS6 was the most potent inhibitor of A549 cellular proliferation. (6) After treated with 1mg/ml COS for 4 days, two-dimensional electrophoresis and western blotting showed that the expression of LASP-1 protein decreased. (7) COS can promote the proliferation of UCB-MSC cells, and the most significant was at 100-300μg/ml, which increased cyclin D1 and RUNX2 genes transcription. (8) COS could stimulated ALP expression in later period. Some A549 cells were showed positive reaction after alizarin red staining. (9) Fluorescent particles were seen in cells after 10 minutes of incubation with FITC-COS, indicating that COS entered the cells very rapidly. Cells treated with FITC-COS reached the fluorescence equilibrium value in a period of time. When the COS was removed from culture medium, the localization of COS was not changed. (10) Cells treated with different concentrations of FITC-COS exhibited different intracellular fluorescence intensities and locations. Cells treated with low concentrations of FITC-COS showed a spotty distribution of fluorescence around nuclear. The localization was identical with that of DsRed-Mit. While in high concentration, it was also found in the cytoplasm and nucleus and was enriched in the nucleolus and karyotheca.Conclusion:COS showed significant cellular functions in NF-κB, tumor apoptosis, and osteogenic differentiation signaling pathways. COS increased the activity of NF-κB, decreased the proliferation of A549 cells, and promoted osteogenic differentiation of UCB-MSC. COS was preferentially localized in the mitochondria. However its subcellular localization was changed with the concentration, which suggested that the concentration of COS were very important on its interaction with a variety of cells. The activity of COS didn't increase with the amount of amino-glucose molecules, which showed that the structure of COS and the three dimension of the COS interacting protein was another important factor in its function.
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