Experimental Study On The Biological Effects Of Artesunate On Rabbit Urethral Fibroblasts

Objective:To observe the effects of artesunate(Artesunate,Art)on the proliferation and apoptosis of urethral fibroblasts cultured in vitro,as well as the transforming growth factor β1(TGF-β1)and matrix metalloproteinases 1(MMP-1)of the cells.To provide experimental basis for clinical application of artesunate in the treatment of urethral scar and prevention of recurrence of postoperative urethral stricture,and to provide ideas for urethral tissue engineering anti-scar therapy.Methods:The urethral fibroblasts of young rabbits were cultured and identified.The experiment was divided into two groups,including the blank control group(Artesunate 0mg/L)and the experimental group(Artesunate 15,30,60,120,240mg/L).1.Using enzyme labeling instrument to detect its effect on cell proliferation at different concentrations(cck8 method);2.Flow cytometry was used to detect the effect of artesunate on apoptosis(Annexin V-PI double staining).3.The expression of TGF-β1 and MMP-1 genes in the cells treated with artesunate was detected by fluorescence quantitative PCR.4.The expression of TGF-β1 and MMP-1 protein in each group was detected by Western blotting.Results:1.With the increase of the concentration of artesunate,the morphological changes of cells became more and more obvious,such as less cytoplasm,shrinking cells and some round cells.2.CCK8 experiment showed that after 24 hours of drug intervention,compared with the control group,the OD value of the experimental group decreased with the increase of drug concentration,and the difference was statistically significant(P < 0.01).After 48 hours of intervention,there were significant differences among the other experimental groups(P < 0.01).While there was no significant difference between 60mg/L and 120mg/L,120mg/L and 240mg/L.3.The results of flow cytometry showed that the apoptosis rate of cells treated with artesunate increased gradually(P < 0.01)in a dose-dependent manner.4.Fluorescence quantitative RT-q PCR detection showed that the expression of TGF-β 1 mRNA in experimental group decreased with the increase of artesunate concentration.Except that there was no significant difference between 40mg/L concentration group and 80mg/L concentration group,there were significant differences among other concentration groups.The expression of MMP-1mRNA in the experimental fibroblasts increased with the increase of drug concentration in the culture medium,and there was significant difference among different concentration groups(P < 0.01).5.With the increase of artesunate concentration,the expression level of TGF-β 1 protein decreased gradually.There were significant differences between each experimental group and the control group(P < 0.01).There were significant differences among other concentration groups(P < 0.05).In addition,artesunate can also induce MMP-1 expression in a dose-dependent manner.There was significant difference between the 80mg/L concentration group and the control group,and there were significant differences among the experimental groups(P < 0.01).Conclusion:Artesunate can inhibit the proliferation of young rabbit urethral fibroblasts,the expression of transforming growth factor-β1,and induce the expression of matrix metalloproteinase-1.At a certain concentration,artesunate can promote the apoptosis of fibroblasts.Its role in the treatment and prevention of urethral fibrosis is worthy of further study.