Isolation and Purification of Sea Cucumber Peptide and Its Impact on the Secretion of Collagen by NIH/3T3 Cells
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Abstract:
A sea cucumber peptide, SP12, was isolated and purified from Stichopus japonicus, and its impact on NIH/3T3 cell proliferation and secretion of type I collagen was studied. SP12 was purified by ion exchange chromatography and gel filtration chromatography from fresh Stichopus japonicus using a cell activity tracking method. The effect of SP12 on NIH/3T3 cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and its impact on cell cycle progression was determined by flow cytometry. The effect of SP12 on collagen secretion by NIH/3T3 cells was measured by hydroxyproline kit, and its impact on the expression of type I collagen, matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinases-1 (TIMP-1) in NIH/3T3 cells was measured at the protein and gene levels by western blot and reverse-transcription polymerase chain reaction (RT-PCR). The results clearly demonstrate that SP12 can considerably enhance the proliferation rate of NIH/3T3 cells and promote progression from G1 phase to S phase. In a range of 0~20 μg/mL, SP12 enhanced collagen secretion by NIH/3T3 cells in a dose-dependent manner. At the protein and genetic levels, SP12 considerably promoted the expression of type I collagen and TIMP-1, and inhibited the expression of MMP-1; this might be the mechanism underlying SP12 promotion of collagen secretion by NIH/3T3 cells.
