Effect Of TGF-?- Induced LOXs Overexpression Inhibit By Aptamer Targeting TGF-?? Receptor

Background: Glaucoma is the major cause of irreversible blindness,characterized by irreversible loss of optic neuropathy and visual field loss.Optic nerve lesion performance as the loss of optic nerve ganglion cell,narrow down of rim and excavation of the optic disk.The drug?laser trabeculoplasty?laser peripheral iridoplasty?laser peripheral iridectomy and filtration surgery are the main treatment methods to control intraocular pressure,All of the available treatment,surgery has proven to be the most effective,but after two years of failure rate as high as 15% to 25%,the most common reason is occurred in the surgical site of conjunctival scar.The two most important factors for the formation of scar after filtering bleb were the transdifferentiation of fibroblasts and the over crosslinking of the extracellular matrix,Tenon 's capsule fibroblasts play an important role in the course of the disease.Transforming growth factor beta(TGF-?)has many biological activities,not only in cell growth,embryonic development,the formation of blood vessels,organ formation and immune responses play an important physiological role,is also known as the most effective growth factors involved in wound healing of the whole body.A lot of research showed that cells release of TGF-? can stimulate fibroblast migration,proliferation,differentiation,and promote collagen synthesis and deposition,highly related to their level of activation and scarring.Studies have shown that TGF-?2 plays a key role in the pathological process of filtering bleb fibrosis after glaucoma filtering surgery,and its activation is closely related to the formation of scar.Lysyl oxidases(LOXs)is an important extracellular matrix protease catalyzed cross-linking between collagen and elastin,when there is trauma or inflammation,LOXs over expression,promote the excessive cross-linking of the extracellular matrix,and then woven into a stable network structure,resulting in progressive scarring lesions.At present,it is generally accepted that TGF-?2 is an important upstream factor of LOXs,which can induce the over expression of LOXs in cells or tissues.Therefore,to study the role of LOXs in fibrotic lesions,and to block the LOXs enzyme in order to achieve anti scar,this study can provide a new way for the treatment of anti-scarring.In our previous study,we screened aptamers targeted against the extracellular domain of T?RII called aptamer S58,has been shown to inhibit human Tenon,s fibroblasts transdifferentiation.While it is not clear how S58 suppresses the TGF-?-induced LOX and LOXL Proteins production in the HTFs,this will be further confirmed in the study.Through this study,we can further understand the pathogenesis of filtering bleb scarring after glaucoma filtration surgery,and it is of great significance to improve the efficacy of S58 after the anti-glaucoma surgery.Objective:The main purpose of this paper is to determine whether(1)whether the LOX and LOXL proteins are expressed in the human Tenon's fibroblasts,(2)whether TGF-? induces LOXs proteins expression in the HTFs,and(3)whether aptamer S58 suppresses the TGF-?-induced LOXs production in the HTFs.Methods:1.HTFs(Human Tenon fibroblasts,HTFs)from our previous primary cell culture,Biopsies of Human HTFs were isolated from patients who had undergone strabismus surgery.With adherent method to separate human tenon capsule fibroblasts,cell viability was determined with MTT method.The preliminary study of this subject was approved by the ethics committee of the Institute of field surgery,Daping Hospital,Third Military Medical University.2.The expression and distribution of LOXs protein in HTFs were detected by immunofluorescence assay.3.The cells were exposed to TGF-?2(4.0 ng/m L)for 24 h,The protein expression of LOXs were determined using Western Blot;Detection of LOX protein expression in HTFs by confocal immunofluorescence assay.4.HTFs were treated with increasing concentrations of TGF-?2(0?2?4?8?16ng/ml)for 24 hours,The protein expression of LOXs were determined using Western Blot.5.HTFs were treated with TGF-?2(4ng/m L)for 6,12,24 and 48 hours,The protein expression of LOXs were determined using Western Blot.6.aptamer S58(100n M)prestimulation HTFs 1H,and TGF-?2(4ng/m L)Co stimulatory cells 24 h,The protein expression of LOXs were determined using Western Blot.Results:1.LOXs is expressed in human Tenon 's capsule fibroblasts,and TGF-?2 promotes the expression of LOXs protein.2.TGF-?2 induced expression of secreted LOXs proteins in a concentration-dependent manner.LOX,LOXL2 and the expression of LOXL3 protein increased with the concentration of TGF-?2 has a concentration dependent.LOXL1,LOXL4 increased with the concentration of TGF-?2 have increased,but no obvious concentration dependence.3.TGF-?2 induced secreted LOXs proteins as early as 6 hours and maintained this induction for up to 48 hours.LOX expression increased with the extension of the time,the 24 h reached saturation effect;the expression of LOXL1 protein in 12 h this effect gradually decreased;the expression of LOXL3 protein in 12 h this effect reaches the steady state;the effect of 24 h LOXL4 protein expression gradually decreased,the expression of LOXL2 was increasing trend,with the concentration gradually increased its expression was significantly enhanced.4.TGF-?2 induced LOXs expression is inhibited by treatment with S58 in HTFs.Conclusions:1.It was somewhat surprising to find that LOX or LOXL proteins were expressed in the HTFs,Under the fluorescence microscope,the red fluorescence was arranged in the periphery of the nucleus.2.TGF-?2 Induces LOXs Proteins in HTFs,TGF-?2 Induces LOXs Proteins in a Concentration-and Time-Dependent Fashion.3.S58 treatment significantly attenuated this TGF-?2 induced LOXs Proteins synthesis.