Study On Effect Of All-trans-retinoic Acid On TGF-β-induced Fibrotic Processes Of Human Conjunctival Fibroblasts

Glaucoma is the most common cause of irreversible blindness worldwide.It is characterized by the development of a specific pattern of visual field loss and optic neuropathy.Pathologic high intra ocular pressure is the main risk factor of glaucoma.Although the mechnism of glaucoma damage to the optic nerve is not clear by far,the higher the eye pressure,the more severe damage to the optic nerve.Nowadays Patients are usually treated using filtration surgery which serves to reduce intraocular pressure in the eye.The five-year survival rate of filtering bleb is less than 50%after operation,since excessive scarring after surgery can lead to the failure of the filtering bleb which is a major problem in glaucoma treatment.Recntly Mitomicin C(MMC)or 5-fluorouracil(5-FU)was used during or post operation in order to inhibit the excessive scarring.But these antimetabolites have the cytotoxic effects which would induce some complications such as filtring bleb leaking,corneal epithelial dysfunction,delayed endophthalmitis and macular edema.So it is of great significance to explore the much more safe and effective anti-fibrosis medications.Human conjunctival fibroblasts(HconFs)are a kind of mesenchyme located at the conjunctival connective tissue having the differentiation function.HconFs are at the state of rest in the normal condition,while they will be activated by the stimulating factors such as operation and inflammation and transform to myofibroblasts(MFs)initiating the wounld healing process.The phenotypic transformation of HconFs to MFs is an important cytological basis of cell fibrosis and drainage channel scarring.Transforming Growth Factor-β(TGF-β)is the most important cytokine for the HconFs activation and gathering.Mastocytes,fibroblasts and inflammatory cells will secret TGF-βunder emergency situation.The binding of TGF-βand receptor activate phosphorylation of P38,Smad2 and Smad3 expression and transfering.TGF-βcould stimulate the extracellular matrix(ECM)synthesis as well as induce transformation of HconFs to MFs who have the much more activation and contractility.TGF-βexpresses in the normal aqueous fluid and the expression will be much higher in the wound healing process.In vitro TGF-βcould induce HconFs and MFs proliferation,migration and collagen contraction.In vitro and vivo inhibition TGF-βcould effectively inhibit the HconFs fibrosis and scar formation.In our previous study,we showed that TGF-βcould stimulate HconFs proliferation,promote HconFs from G1 stage to S and G2/M stage,improve theα-smooth muscle actin(α-SMA),the marker protein of MFs,expression,stimulate HconFs migration and ECM synthesis.In vitro TGF-βcould stimulate HconFs fibrosis and could simulate the wound healing process.Retinoic acid(RA)is the active metabolite of vitamin A and all-trans retinoic acid(ATRA)is one of its subtype.ATRA plays an important role in the process of embryonic development,reproduction,visual sense,cell proliferation,differentiation and apoptosis as well as inflammation.Some researches showed that RA played an important role in the fibrotic diseases such as liver fibrosis,lung fibrosis though multiple mechnisms.RA could inhibit ECM formation and regulate TGF-βsignal pathway.Objective:In our present research we studied the role of ATRA on the TGF-β-induced HconFs proliferation,migration,apoptosis and ECM expression and the possible mechnism.Methods:(1)We found that ATRA could inhibit HconFs proliferation using CCK-8method.Using flow cytometry we found that ATRA could promote HconFs apoptosis.Though cell wound scrach assay we showed that ATRA could inhibit TGF-β-induced HconFs migration.By western blot method we found that ATRA could inhibit collagen-Ⅰand fibronectin expression in ECM.We concluded that ATRA could inhibit TGF-β-induced HconFs fibrosis.(2)In order to study the possible mechnism by which ATRA inhibited TGF-β-induced HconFs fibrosis,we used Western blot method to detect the level of phosphatidylinositide 3-kinase(p-PI3K)/protein kinase B(AKT).We found that ATRA could down-regulate expression of p-PI3K/AKT of TGF-β-induced HconFs.We concluded that ATRA was likely to regulate TGF-β-induced HconFs fibrosis though down-regulating p-PI3K/AKT expression.(3)To further study the role of ATRA on TGF-β-induced HconFs fibrosis,we conducted the vivo test.We detected the effect of ATRA on drainge channel scarring after glaucomafiltration surgery though establishing the rabbit eyes glaucomafiltration surgery model.We checked and recorded the rabbits eye pressures before and after operation.After operation we took the anterior segment photography weekly to observe the filtering bleb forms and areas.The animals were sacrificed at 4 weeks after operation.The specimens were evaluated histologically.We evaluated the expressions ofα-SMA,FN,collagen-Ⅰ,Fibrocectin,Smad2/Smad3 and p-PI3K/AKT in the filtering bleb using immunohistochemistry method and analysed the effect and possible mechnism of ATRA on the drainge channel scarring after glaucomafiltration surgery.Results:The results showed that ATRA could prolong filtering bleb survival time and reduce the expressions ofα-SMA,FN,collagen-Ⅰ,Fibrocectin,Smad2/Smad3 and p-PI3K/AKT in the filtering bleb.This was consistent with the results of experiments in vitro.All the results indicated that ATRA could inhibit filtering bleb fibrosis after glaucomafiltration surgery and the possible mechnism probably was regulating and controling the PI3K/AKT signal path.Conclusions:We concluded that ATRA could regulate TGF-β-induced HconFs proliferation,transformation,migration and ECM synthesis.ATRA could inhibit filtering bleb scarring which provided a new direction for the anti-fibrosis after glaucomafiltration surgery in the future.