The Effect Of Artesunate On Human Skin Hypertrophic Scar Fibroblasts Proliferation And Collagen Deposition

Purpose:1. To evaluate the effects of artesunate (ART) on the cellproliferation and collagen deposition by human skin hypertrophic scarfibroblasts (HSFBs).2. To evaluate the mechanism of ART on the collagen deposition of HSFBs.Methods:1. Fibriblasts were derived from human skin hypertrophic scarspecimens and then purified through primary monolayer cell culture andidentified by anti-vimentin and anti-cytokeratin immunocytochemistry staining.2. HSFBs were divided into five treatment groups: control group,75μMART group,150μM ART group,300μM ART group and600μM ART group.The treatment on HSFBs last for24h resecptively in each group.3.Cell proliferation effects was evaluated by MTT assay and morphologicalchanges of HSFBs were observed under inverted microscope respectively.4. The mRNA levels of α2chain of procollagen Ⅰ (proCOL1A2) and α1chain of procollagen Ⅲ (proCOL3A1), mothers against DPP homolog3(Smad3), connective tissue growth factor (CTGF) and type1matrixmetalloproteinases (MMP-1) were detected by fluorescent quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) assessment.5. The protein expression of collagen I (COL-Ⅰ), collagen III (COL-Ⅲ),Smad3and CTGF were detected with Western blot.6. MMP-1protein levels in the conditioned culture media of HSFBs weremeasured using a commercially available enzyme linked-immuno-sorbent assay(ELISA) kit.Results:1. The human skin scar derived frbroblast were in long spindleshape, and were positive stained in Vimentin and negative in cytokeratinimmunocytochemistry staining which confirmed that the cells used in followingexperiment was HSFBs.2. The proliferation of HSFBs can be inhibited by various concentrations ofART detected by MTT assay. compared with the blank control group showedstatistical differences (P<0.01).3. Over the morphology observation, the number and shape of HSFBswere shrink and the original spindles shape cells were turn out to be shorter,round or even disappeared after the treated of ART.4. The expression of proCOL1A2and proCOL3A1mRNA were decreasedby various concentrations of ART with FQ-RT-PCR, compared with the blankcontrol group showed statistical differences (P<0.01). FQ-RT-PCR also showedthat the expression of the expression of Smad3and CTGF mRNA weredecreased by various concentrations of ART. There were statistical differencescompared with the blank control group (P<0.01). While the mRNA level ofMMP-1was significantly increased in a dose-dependent manner at the sametime.5. Compared with the control group, the expression of COL-Ⅰand COL-Ⅲ protein were decreased by various concentrations of ART, and therewere statistical differences (P<0.01). And the comparison between lowconcentration group and high concentration group showed significant statisticaldifferences (P<0.01). Western blot also showed that the expression of Smad3and CTGF proteins in HSFBs were decreased. There were statistical differencescompared with the blank control group (P<0.01).6. ELISA also showed that the MMP-1protein levels in the conditionedculture media were significantly increased. And the comparison between lowconcentration group and high concentration group showed significant statisticaldifferences (P<0.01).Conclusion:1. These results suggest that ART inhibits cell proliferativeactivity of HSFBs, and can lead to reduction of type I and III collagenproduction and deposition in a concentration-dependent manner in a certainconcentration range.2. ART can effectively down-regulate the expressions of Smad3andCTGF, leading to the inhibition of collagen genetic transcription and production.At the same time, ART can effectively up-regulate the expression of MMP-1,which can accelerate the degradation of extracellular matrix. This research canprovide theoretical support for the application of ART to control hypertrophicscar.