Effects Of Asiaticoside On Proliferation, Collagen And TGF-β₁mRNA For Scar Fibroblasts

Objectivescar tissue, is an inevitable result of the repair after wounds. It not only influences beautiful but also lead to organize and organs of a functional impairment. But wounds repair is a very complicated process of biology, there are lots of pathogenic factors in the whole pathophysiological process of scar, includ molecular biology, immunology and cytochemical. General concept that the formation mechanism of scar mainly by the interrelationship of repair cells, extracellular matrix, cytokines. This experiment investigate the inhibitory effect of Asiaticoside on proliferation and expression of transforming growth factor-βlmRNA (TGF-β1), TypeⅠandⅢcollagen mRNA of the cultured scar fibroblasts.Methods1. Fibroblast culture In sterile surroundings, put the scar tissue in DMEM culture solution to rinse, human scar fibroblasts were respectively isolated from scar by tissue plant methods, subcultured, purifiedand and identifiedetc.2. Cell morphological observation After the scar fibroblast are collected, inoculate them on 96-well microtiter plates serum at a density of 2×104 cells/ml with 20%DMEM, each of which is filled with 200μL cell suspension, after 24 hours, cells will adherent to the bottle, then make a blank control group and respectively add in drug. After 24 hours in the culture incubator of 37℃/5%CO2, then observe the cell morphological changes under the reverse microscope.3. The proliferative activity of cells After observe the cell morphological changes, fill each well with 5mg/mL MTT 20μL, continue culture for 4h in the culture incubator of 37℃/5%CO2, add in DMSO 150μL per well, shake for 10min, exam the absorbance value at 492nm with ELISA, determine the proliferation inhibitory effect of Asiaticoside at various final concentrations on the growth of scar fibroblast.4. Detection of mRNA by RT PCR After the scar fibroblast are collected, inoculate them on 6-well microtiter plates serum, then add different concentration of Asiaticoside(0.1mg/mL and 1. Omg/mL). The total RNA of scar fibroblast samples were purified with Trizol and submitted to cRNA synthesis, after that, PCR amplification was carried out, and then ran agarose gels electrophoresis with the products of PCR amplification. Expression of TGF-β1mRNA, collagenⅠand collagenⅢmRNA secretion in scar fibroblast were detected with reverse transcription-polymerase chain reaction method(RT-PCR).ResultsResults Compared with control group, Cell morphological observation showed that Asiaticoside has obvious influence on scar fibroblasts. It was assayed by MTT method that Asiaticoside dose-dependently inhibited the proliferation of scar fibroblasts, and the work concentration was originated at 0.1 mg/mL. With RT-PCR, at the concentration of 1. Omg/mL it could reduce TGF-β1mRNA, and that of TypeⅠandⅢcollagen mRNA expression obviously.ConelusionTraditional Chinese medicine Asiaticoside can significantly inhibited the proliferation of scar fibroblasts, inhibit the expression of TGF-β1mRNA, TypeⅠandⅢcollagen mRNA,which might be one of mechanisms of Asiaticoside inhibiting scar formation.