Bromfenac Protects Against TGF-?1-mediated Fibrotic Effects On Human Pterygium Fibroblasts And Human Conjunctival Fibroblasts

Background and ObjectivePterygium is a common ocular surface disease,is an invasive and benign overgrowth of fibrovascular conjunctiva over the cornea with unknown cause,which is believed to be related to chronic inflammatory stimulus,including ultraviolet(UV)irradiation and sawdust exposure.The prominent pathogenesis of pterygium is a combination of the processes of proliferation,migration,epithelial-mesenchymal transition(EMT)of pterygial progenitor cells,and the activation of myofibroblasts triggered by many cytokines under the stimulation of chronic inflammation.Among these,studies have reported that transforming growth factor beta 1(TGF-?1),an important inflammatory mediator,has a higher expression level in pterygium tissues.TGF-?1 is considered to be related to the development and progression of pterygium.Excessive wound healing after pterygium excision surgery often leads to fibrosis,which indicates the recurrence of pterygium.It has been reported that non-steroidal anti-inflammatory drugs(NSAIDs)could relive inflammatory response,reverse EMT progression and inhibit the activation of myofibroblasts,which has also been proved to reduce the recurrence rates of fibrotic diseases such as breast cancer effectively.Besides,cyclooxygenase-2(COX-2)was found to have a significantly higher expression levels in pterygium tissues than their paired conjunctival tissues.Treatment with COX-2 inhibitors appeared to prevent pterygium cells proliferation effectively,but the anti-fibrotic effects of COX-2 inhibitors,and the underlying pharmacological mechanisms are still unclear.The aims of the present study were to determine the effects of TGF-?1 in human pterygium fibroblasts(HPFs)and human conjunctival fibroblasts(HConFs),such as inducing the production of extracellular matrix(ECM),activation of myofibroblasts,cellular migration and proliferation.The present study also aimed to investigate the role of NSAIDs bromfenac in TGF-?1-induced production of ECM,activation of myofibroblasts,cellular migration and proliferation in HPFs and HConFs,as well as to explore the potential mechanisms beneath.MethodsFirstly,the HPFs and HConFs were divided into five groups:group exposed to 20 ng/ml TGF-?1(4 groups)and control group(1 group).The protein expression levels of mesenchymal markers fibronectin(FN),collagen 3(COL3),and a-smooth muscle actin(a-SMA)in HPFs and HConFs exposed to TGF-?1 for 6h,12h,24h,and 48h were detected by western blotting assay,respectively.Besides,after exposed to TGF-?1 for 15min,30min,1h,2h,and 6h,the phosphorylation and total levels of relevant signal molecules were measured by western blotting assay,which including AKT,ERK1/2,GSK-3?-S9,smad2/3 and p38 MAPK.In order to investigate the potential inhibitive effects of bromfenac on the related markers of the production of ECM,activation of myofibroblasts,cellular migration and proliferation in the TGF-?1-induced HPFs and HConFs,cells were divided into four groups,respectively:control group;20 ng/ml TGF-?1 group;20 ng/ml TGF-?1+90ug/ml bromfenac group;90ug/ml bromfenac group.Each group of HPFs and HConFs were treated simultaneously for 48h,and the protein expression levels and distribution of FN,COL3,and ?-SMA were detected by western blotting assay and immunofluorescence,the changes of relevant signal molecules were measured by western blotting assay,while the migration capability of the cells was determined by the wound healing assay,and the proliferation ability of the cells was determined by CCK-8 assay.To further confirm the potential inhibitive effects of bromfenac on the production of ECM,activation of myofibroblasts,cellular migration and proliferation,HPFs and HConFs were treated with three different concentrations(i.e.30ug/ml,60ug/ml,and 90ug/ml)of bromfenac for 48h,the protein expression levels of FN,COL3,matrix metalloproteinase 3(MMP3),?-SMA,and survivin were detected by western blotting assay and quantitative real-time PCR,the changes of relevant signal molecules were measured by western blotting assay.To further explore the mechanisms underlying the inhibitive effects of bromfenac on TGF-?1 induced production of ECM and activation of myofibroblasts,the PI3K inhibitor wortmannin,MEK inhibitor U0126,GSK-3? inhibitor LiCl or SB216763 were used separately on the TGF-?1 induced HPFs and HConFs,so as to investigate the effects of AKT,ERK1/2,and GSK-3? signaling molecules on the production of ECM and activation of myofibroblasts in HPFs and HConFs.The expression levels of FN,COL3,and ?-SMA were detected at protein levels by western blotting assay,besides,western blotting assay was also used to measure the changes of relevant signal moleculesResultsThe protein expression levels of FN,COL3,and ?-SMA were elevated significantly after exposed to TGF-?1 for 48 h in HPFs and HConFs,the cellular migration and proliferation ability also increased.Treatment with bromfenac inhibited the expression of FN,COL3,and a-SMA induced by TGF-?1,the migration and proliferation ability of the cells were also attenuated.TGF-?1 rapidly phosphorylated AKT,ERK1/2,GSK-3p-S9,smad2/3,and p38 MAPK,which in turns regulating the fibrosis of HPFs and HConFs.The phosphorylation of AKT,ERK1/2,and GSK-3?-S9 induced by TGF-?1 could be suppressed by bromfenac.Treatment with bromfenac alone on HPFs and HConFs inhibited the expression of FN,COL3,a-SMA,and survivin at both protein and mRNA levels,as well as inhibited the phosphorylation levels of AKT,ERK1/2,and GSK-3?-S9.Blocking PI3K/AKT(wortmannin),MEK/ERK(U0126)or GSK-3?(LiCl or SB216763)pathway all could suppress the expression of FN,COL3,and a-SMA induced by TGF-?1.Using LiCl to inactivate GSK-3? could increase the level of p-GSK-3?-S9,but couldn't reverse the inhibitory effect of bromfenac on the expression of FN,COL3,and a-SMA,on the contrary,using LiCl to elevate the level of p-GSK-3?-S9 is accompanied by a decrease in FN,COL3,and a-SMA protein levels.ConclusionBromfenac inhibited the TGF-?1-induced production of ECM,activation of myofibroblasts,cellular migration and proliferation in HPFs and HConFs.Bromfenac suppresses the production of ECM and activation of myofibroblasts induced by TGF-?1 through influencing AKT and ERK1/2 pathways.Increasing the level of p-GSK-3?-S9 inhibited the TGF-?1-induced production of ECM and activation of myofibroblasts in HPFs and HConFs.