The Effect Of Artesunate On The Human Skin Hypertrophic Scar Epithelial Cells Proliferation And The Expression Of TGF-β1, ITGB1 And MMP-1

Purpose:Human skin hypertrophic scar epithelial cells were cultured in vivo, evaluate the effects of artesunate(ART) on the cell proliferation, and the expression level of transforming growth factor-beta 1(TGF-β1), integrin β1(ITGB1) and matrix metalloprotein-1 (MMP-1). Explore the effect of ART on the human skin hypertrophic scar epithelial cells.Methods:1. Human skin hypertrophic scars were digested with neutral protease overnight and the epidermis was separated, the epidermis was digested to single cells with trypsin. Thereafter, collected the suspension cells and cultured in vivo. Observe the growth of cells by inverted phase microscope. Fibroblasts were removed by digested with trypsin and obtain the pure epithelial cells. Identify the origination of cells by immunocytochemistry staining using anti-vimentinmonoclonal antibody and cytokeratin antibody. The isolated and purified epithelial cells were used in follow-up experiments.2. Cell proliferation with different concentrations of ART interfered 24 hours was evaluated by MTT assay, and the morphological changes of cells were observed by microscope after the cells were cultured by ART.3. The genetic transcription level of TGF-β1, ITGB1 and MMP-1 were detected by fluorescent quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) assessment.4. The protein expression levels of TGF-β1, ITGB1 were detected by Western Blot (WB).5. The protein expression level of MMP-1 in the conditioned culture media of cells was measured by enzyme linked-immuno-sorbent assay (ELISA) kit.Results:1. Epithelial cells got from human skin hypertrophic scar can be adherent on cell culture flask. The cells were in ploygon shape, arange in cobblestone appearance when the cell culture flaskwere full of cells, and the cells were positive stained in Cytokeratin and negative in Vimentin immunocytochemistry staining, which identified that the cells were skin hypertrophic scar derived epithelial cells.2. The results, detected by MTT assay, showed the proliferation of cells can be inhibited by various concentrations of ART and in a concentration-dependent manner. The differences compared with the blank control group, except the 150μM ART group had no statistical sense (P>0.05), others showed statistical differences (P<0.01). The difference between 150 μM ART group and 300 μM ART group had no statistically significance (P>0.05), others showed statistical differences when compared any other two intervention groups (P<0.05). According to the result of MTT assay, the concentration of ART in the followed experiment were 0,300,600,1200 and 2400 μM.3. In the morphology observation, cells were shrinked and turn round, losing cytoplasm after the treatment of ART. Changes were obviously with the increase of ART concentration.4. The expression levels of TGF-β1 and ITGB1 mRNA were decreased after interfered by various concentrations of ART with FQ-RT-PCR, compared with the blank control group showed statistical differences (P＜0.05). Also, the expression level of MMP-1 mRNA was significantly increased in a dose-dependent manner at the same time, with statistical significance (P＜0.05). The difference between any two intervention group also showed statistical differences (P＜0.05).5. The expression of TGF-β1 and ITGB1 protein were decreased after interfered by various concentrations of ART with Western Blot, the differences compared with the blank control group, except the 300 μM ART group had no statistical sense(P>0.05), others showed statistical differences (P＜0.01). The differences between any two intervention groups, except the difference of ITGB1 protein level between 300 μM ART group and 600 μM ART group had no statistically significance (P>0.05), others showed statistical differences (P＜.05).7. ELISA also showed that the concentration of MMP-1 level in the conditioned culture media was significantly increased. The differences compared with the blank control group, except the 300 μM ART group had no statistical sense (P>0.05), others showed statistical differences (P＜0.01). The differences between any two intervention groups also showed statistical differences (P＜0.05).Conclusion:1. Single human skin hypertrophic scar epithelial cells were got with trypsin and were able to grow adhering on cell culture flask, and can purified by digested with trypsin.2. The results suggest that ART inhibits the epithelial cells proliferative activity, and have influence on the shape of cells, the effects are in a concentration-dependent manner.2. ART can effectively down-regulate the expressions of TGF-β1 and ITGB1 on both mRNA and protein level. At the same time, ART can effectively up-regulate the expression of MMP-1, all effects are in a concentration-dependent manner. A further research is still needed to explore whether the expression of TGF-β1, ITGB1 and MMP-1 in epithelial cells can affect the compound of collagen and extracellular matrix, the decomposition of collagen and the tissues fibrosis in corium layer and subcutaneous tissue.