| BackgroundGlaucoma filtration surgery(GFS)is the conventional surgical treatment for glaucoma.The key to success is the formation of a long-lasting functional filtering bleb under the conjunctiva.However,excessive scarring in bleb will lead to surgical failure by hindering the outflow and absorption of aqueous humor.The scar formation is the result of fibrosis in conjunctiva and subconjunctiva tissues of the surgical area,which is characterized by activation of myofibroblasts and massive deposition of extracellular matrix(ECM).Transforming growth factor β(TGF-β)is a key growth factor involved in myofibroblasts activation,promoting target cells proliferation,migration,transdifferentiation and ECM production.Activated myofibroblasts,which characteristically express α-smooth muscle actin(α-SMA),have stronger contractile and secretory abilities to promote wound closure and ECM deposition.What’s more,myofibroblasts have the potential of dedifferentiation,which can be induced to lose the characteristic phenotype and revert to inactive precursor cells.Myofibroblast dedifferentiation can promote fibrosis reversion.Therefore,inhibiting myofibroblasts activation and promoting myofibroblasts dedifferentiation are two directions for antifibrosis therapy,which can alleviate the progression of fibrosis from inhibiting and reversing respectively.Cyclooxygenase 2(COX-2)is a key enzyme for prostaglandins synthesis in inflammatory and proliferation-related diseases and can be involved in the regulation of several fibrotic diseases.The non-steroidal anti-inflammatory drugs can affect the development of fibrosis by inhibiting the function of COX-2.Bromfenac sodium(BS),a commonly used non-steroidal anti-inflammatory drugs in ophthalmology,has the advantage of efficient COX-2 inhibition and low corneal toxicity.However,the role of BS in bleb scarring after GFS has not been reported.PurposeTo investigate the effect and mechanism of BS on fibrosis from inhibiting and reversing by using the TGF-β1-induced human conjunctival fibroblast(Hcon F)fibrosis model.Then to further study the effect of BS on filtering bleb scarring after GFS by using the mouse GFS model.Methods1.Building fibrosis model in Hcon F by TGF-β1 stimulation.The proliferation of cells was detected by CCK-8.Immunofluorescence assay was used to detect the expression of α-SMA and F-actin.Western blot assay was used to detect the expression of α-SMA,Collagen-1,Fibronectin,COX-2,p-Smad2/3,Smad2/3,p-ERK1/2,ERK1/2,p-AKT and AKT.2.To investigate the effect and mechanism of BS pretreatment on fibrosis in Hcon F induced by TGF-β1.CCK-8 assay was used to detect the cell proliferation.Immunofluorescence assay was used to detect the expression of COX-2.Western blot assay was used to detect the expression of α-SMA,Collagen-1,Fibronectin,COX-2,pSmad2/3,Smad2/3,p-ERK1/2,ERK1/2,p-AKT and AKT.The expression of COX-2was altered by small interfering RNA(si RNA)transfection and lentiviral infection respectively to explore the effect of COX-2 on fibrosis and the mechanism of BS pretreatment inhibitive effect on fibrosis.CCK-8 assay was used to detect the cell proliferation.Western blot assay was used to detect the expression of Bax,Bcl2,cleavePARP,α-SMA,Collagen-1,Fibronectin,COX-2,p-AKT and AKT.3.Hcon F was induced by TGF-β1 for 48 h,and then TGF-β1 was removed.The cells were cultured continuously with or without BS for another 72 h.The expression ofα-SMA,Collagen-1 and Fibronectin were detected by immunofluorescence,q PCR and Western blot assays to explore the effect of BS on myofibroblasts dedifferentiation and fibrosis reversion4.C57BL/6J mice were used to construct GFS surgical model,the IOP was measured at 0,3 days after surgery,the morphology of the filtering blebs was observed and photographed at 3 days after surgery.Then the mice were randomly divided into 5groups including Normal,GFS,MMC,BS subconjunctival injection and BS eyedrop.The morphology of the filtering blebs was observed and photographed at 3,7,14,21,28 days after GFS.All the mice were killed at 28 days after surgery,and the paraffin sections of eyes were labeled with HE and Masson staining to detect the morphology and local collagen deposition,frozen tissue sections were used to detect the expression of α-SMA,Collagen-1 and Fibronectin in the bleb.Results1.The treatment of 5ng/ml TGF-β1 promoted the proliferation of Hcon F at 24,48 and 72h.TGF-β1 promoted the expression of α-SMA,Collagen-1,Fibronectin in Hcon F,and the effect was most obvious at 48 h.After TGF-β1 treatment for 48 h,the morphology of Hcon F widened from a slender spindle to a hypertrophic star or triangle shape with the thickening of skeleton and neo-expression of α-SMA.The expression of COX-2,p-Smad2/3,p-ERK1/2 and p-AKT were induced by TGF-β1,which increased in the early stage and gradually decreased in the later stage.2.100μg/ml BS was safe and effective according to the results of CCK-8.Pretreatment of 100μg/ml BS inhibited the proliferation and the expression of α-SMA,Fibronectin,COX-2,p-AKT induced by TGF-β1 in Hcon F.In order to further study the mechanism of BS inhibiting fibrosis,the expression of COX-2 was changed by the si RNA transfection and overexpression lentivirus infection.The silence of COX-2inhibited the proliferation and the expression of α-SMA,Collagen-1,Fibronectin,COX-2,p-AKT induced by TGF-β1 in Hcon F,and also promoted the expression of apoptosis-associated proteins Bax/Bcl2 and cleave-PARP in myofibroblasts.The overexpression of COX-2 promoted the proliferation and the expression of α-SMA,Collagen-1,Fibronectin,COX-2,p-AKT in Hcon F.The use of PI3 K inhibitor LY294002 reversed the increasing of α-SMA,Collagen-1,Fibronectin caused by COX-2 overexpression.3.After the treatment of 5ng/ml TGF-β1 for 48 h,the morphology of Hcon F widened from a slender spindle to a hypertrophic star or triangle shape.And the morphology could be maintained for 72 h after removal of TGF-β1,with positive expression of α-SMA,Collagen-1 and Fibronectin.However,the treatment of BS for72 h after TGF-β1 removing induced dedifferentiation of myofibroblasts,with the cell morphology returning to the fibroblast-like shape,and the down-expression of α-SMA,Collagen-1 and Fibronectin,which is independent of the inhibitive effect of BS on COX-2.4.The mouse GFS model was successfully constructed.A raised and slightly white bleb was observed at the surgical site 3 days after GFS.Analysis of the IOP ratio showed that the IOP decline significantly at 3 days after GFS.5.The blebs in different groups at different time after surgery were evaluated according to Kronfeld’s classification.The raised filtering blebs could be seen in each group at 3 days after surgery.The filtering blebs of GFS group began to flatten and disappear at 14 days after surgery,accompanied by neovascularization.However,the blebs in MMC,BS subconjunctival injection and BS eyedrop groups become flat and absent from 21 days after surgery.Analysis of blebs survival showed that the survival of filtering blebs was significantly different among the four groups(P=0.0056).Compared with the GFS,the survival rates of the MMC(P=0.0033),BS subconjunctival injection(P=0.0168),and BS eyedrop(P=0.0259)groups were higher at 28 days after surgery.But there was no difference among MMC,BS subconjunctival injection and BS eyedrop groups.6.The results of HE and Masson staining revealed that the subconjunctival tissue in normal eyes was loose.The bleb tissue of GFS group comprised of thick strands of fibrous material overlying the sclera,with increasing of fibroblasts and collagen fiber.The subconjunctival tissue of MMC and BS eyedrop groups were also loose containing fewer collagen fiber.The staining of bleb tissue in BS subconjunctival injection group revealed a thinner but tightly packed bleb,with an obvious lacunae between conjunctiva and sclera,and fewer collagen deposition overlying the sclera.7.Results of immunofluorescence showed that a small amount expression of α-SMA,Collagen-1 and Fibronectin were observed in subconjunctiva of normal mice.And the expression of α-SMA,Collagen-1 and Fibronectin were upregulated in the blebs of GFS group.While the expression of these proteins all decreased after the treatment of MMC and BS.Conclusion1.TGF-β1 treatment induced Hcon F fibrosis model successfully.TGF-β1promoted the expression of COX-2,p-Smad2/3,p-ERK1/2,p-AKT time-dependently.2.Pretreatment of BS inhibited the fibrosis by inhibiting the proliferation,myofibroblast transdifferentiation and Fibronectin synthesis induced by TGF-β1 in Hcon F.The inhibitive effect of BS may be achieved by destroying the signaling pathway of TGF-β1/COX-2/AKT.3.The myofibroblasts induced by TGF-β1 persisted for at least 72 h.BS promoted the dedifferentiation of myofibroblasts independent of COX-2,with the cell morphology returning,down-expression of myofibroblast marker α-SMA and reduction of ECM synthesis capability.BS reversed TGF-β1-induced fibrosis in Hcon F.4.Both treatments of BS including BS subconjunctival injection and BS eyedrop prolonged the survival of filtering blebs by inhibiting the formation of scar in blebs after GFS via suppressing the accumulation of myofibroblasts and the deposition of ECM... |
PDF Full Text Request