Effects And Mechanisms Of Artesunate On The Proliferation Of Rabbit Urethral Fibroblasts And Extracellular Matrix Synthesis Induced By TGF-β1

Objective:The treatment of urethral stricture is always a difficult problem in clinic.The effect of clinical application is not good.Therefore,continuing to explore safe and reliable drugs for the treatment of urethral fibrosis is the direction of our research to provide more effective solutions to the problem of urethral fibrosis.TGF-β/Smad is the most important pathway known for the occurrence and development of fibrosis.There is a synergistic effect between Notch pathway and TGF-β1,and Notch1 may be a target for inhibiting urethral fibrosis.The antifibrotic effect of artemisinin and its derivatives in various organs is clear,but the study on the role of artesunate in urethral fibrosis is still insufficient.In this experiment,we observed the effects of Artesunate(ART),a derivative of Artemisinin,on the proliferation,migration and apoptosis of rabbit urethral fibroblasts,which induced by exogenous TGF-β1.Also we observed its effects on the expression of related proteins on TGF-β/Smad and Notch pathways.The experiment clarify the effect of Artesunate on urethral fibroblasts and its possible mechanism,with a view to providing a protocol for the treatment and prevention of urethral strictures.Methods:Primary urethral fibroblasts were isolated from rabbit urethra and cultured.They were identified as fibroblasts by Cy3 fluorescent labeled Vimentin antibodies.The experiment was divided into five groups,including normal group A,control group B(10μg/L TGF-β1),experimental group C(10μg/L TGF-β1+30mg/L ART),experimental group D(10μg/L TGF-β1+60mg/L ART)and experimental group E(10μg/L TGF-β1+120mg/L ART).1.The effects on cell proliferation at different concentrations were detected by enzyme label(CCK-8).2.Cell survival rate under different drug concentrations was detected by Live/Dead method;3.The effect of ART on apoptosis of fibroblasts was detected by flow cytometry(Annexin V-PI double staining).The effect of ART on the cell cycle of fibroblasts was also detected by flow cytometry.4.Transwell was used to detect the effect of ART on the migration ability of fibroblasts.5.The m RNA expressions of TGF-β1,α-SMA,Smad3,Smad7,Col Ⅰ,Col Ⅲ were detected by fluorescence quantitative PCR with ART intervention.6.Western blot was used to detect the expression of α-SMA,Smad3,Col Ⅰ,Col Ⅲ with ART intervention.7.The expression levels and localization of Col Ⅰ,Col Ⅲ after ART intervention were detected by cell immunofluorescence technique.8.Notch1 protein expression was detected by ELISA.To study the inhibitory effect of ART on TGF-β/Smad signaling pathway in rabbit urethral fibroblasts cultured in vitro,we explore a new way to treat fibrosis and make ART have a broader application prospect.Results:1.Transforming growth factor TGF-β1 can promote the proliferation and migration of rabbit urethral fibroblasts.Compared with the control group,the proliferation and migration of rabbit urethral fibroblasts induced by exogenous TGF-β1 decreased with the increase of ART concentration in the experimental groups.2.ART can increase apoptosis rate and slow down proliferation rate by down-regulating the proportion of S-phase cells in the cell cycle;3.TGF-β1 could induce m RNA expressions of α-SMA,Smad3,Col Ⅰ,Col Ⅲ(P < 0.01).With the increase of ART concentration,m RNA expressions of TGF-β1,α-SMA,Smad3,Col Ⅰ,Col Ⅲ decreased while the m RNA expression of Smad7 increased(P < 0.01).4.TGF-β1 could induce the protein expressions of α-SMA,Smad3,Col Ⅰ and Col Ⅲ(P < 0.01),on the contrary,the protein expressions of α-SMA,Smad3,Col Ⅰ and Col Ⅲ in the experimental group decreased with the increase of ART concentration(P < 0.01).5.TGF-β1 increased Notch1 expression(P < 0.01),and Notch1 expression decreased gradually with the increase of ART concentration in the experimental group(P < 0.01).Conclusion:ART can effectively inhibit urethral stricture caused by excessive proliferation of urethral fibroblasts induced by exogenous TGF-β1.The mechanism may be related to the inhibition of ART on the expression of extracellular matrix components type I and III collagen,cytoskeleton protein α-SMA.What’s more,TGF-β/Smad and Notch signaling pathways related proteins TGF-β1,Smad3 and Notch1 m RNA and protein expression decreased.ART has shown great antifibrotic potential function in urethral stricture and deserves further investigation to make significant progress in clinical application in the future.