Niclosamide Inhibits TGF-β1-induced Fibrosis Of Human Tenon’s Fibroblasts By Regulating The MAPK-ERK1/2 Signaling Pathway

ObjectiveThe prevention of postoperative bleb scarring is an effective way to improve the long-term curative effect of glaucoma filtration surgery.Use of more effective antifibrotic drugs with fewer adverse effects may be a good way to address the problem.In the present study,we explored if niclosamide inhibits human Tenon’s fibroblasts(HTFs)induced by transforming growth factor-β1(TGF-β1)and the underlying mechanisms.MethodsPrimary HTFs were stimulated with TGF-β1 to induce fibrotic phenotype,and the cells were treated with niclosamide.Cell viability and proliferation was detected using CCK-8 assay.Wound healing assay was used to assess cell migration.Flow cytometry was used to detect the effect of niclosamide on cell apoptosis.Immunofluorescence assay was used to show cytoskeletal changes.Real-time quantitative PCR and western blotting assay were used to detect the mRNA and protein expression of alpha-smooth muscle actin,type Ⅰ collagen,and type Ⅲ collagen in HTFs treated with niclosamide,respectively.In addition,transcriptome sequencing assay was used to screen the potential signaling pathway of niclosamide inhibiting TGF-β1-induced HTFs fibrosis.ResultsThe results showed that the proliferation capacity of HTFs was decreased in concentration and time dependence after treatment with different concentrations of niclosamide for 12,24,and 48 h.Niclosamide attenuates HTFs migration and counteracts the pro-migration effect of TGF-β1.It also increases HTFs apoptosis rate.Niclosamide not only reduced the mRNA and protein levels of TGF-β1-induced alpha-smooth muscle actin,type I collagen,and type III collagen in HTFs,but also counteracted TGF-β1-induced cytoskeletal changes and reduced vimentin expression.Moreover,KEGG pathway enrichment analysis of transcriptome data showed that niclosamide was involved in the mitogen-activated protein kinase-extracellular signal-regulated kinase 1/2(MAPK-ERK1/2)signaling pathway,and the validated expression levels of some significantly different differential genes in this pathway were consistent with the trend of transcriptome sequencing.Niclosamide and ERK inhibitor downregulated the protein level of TGF-β1-induced phosphorylated ERK1/2 in HTFs.Activators of MAPK-ERK1/2 signaling pathway up-regulated the protein level of phosphorylated ERK1/2 in HTFs.However,niclosamide reversed this change.ConclusionsThe results showed that niclosamide not only inhibited TGF-β1-induced HTFs proliferation,migration and fibrosis gene expression,but also reversed TGF-β1-induced cytoskeleton changes and extracellular matrix component deposition.In addition,niclosamide may inhibit TGF-β1-induced HTFs fibrosis by regulating the MAPK-ERK1/2 signaling pathway.In conclusion,niclosamide is a potential antifibrotic candidate,which is promising for clinical application in the future to improve the success rate and long-term efficacy of glaucoma filtration surgery.