| Glaucoma is the world's first irreversible blinding eye disease and has become a serious threat to people's visual function and quality of life.Filtration surgery is the most common treatment to reduce intraocular pressure, but excessive scar tissue reactions reduce the postoperative survival rate of filtering bleb. Currently, 5- fluorouracil( 5- Fu),Mitomycin C( MMC), and other anti-metabolic drugs has been applied to inhibit subconjunctival scarring in high-risk patients, but severe side effects related to the using of these agents(such as filtering bleb leaking,endophthalmitis, low intraocular pressure, shallow anterior chamber,corneal decompensation, corneal endothelial injury, cataracts,scleromalacia and scleritis disease) limit their further application in resisting scarring. Therefore, looking for more effective and with less side effects methods against scarring makes sense to treatment for glaucoma.Filtration surgery can stimulate conjunctival fibroblasts proliferation,migration, differentiation from fibroblasts to myofibroblasts, and promote the secretion of extracellular matrix(ECM); these are important processes of wound healing, but also lead to scarring. Transforming growth factor ?(TGF-?) is a kind of bioactivity peptides secreted by macrophages,lymphocytes, fibroblasts(FB), vascular endothelial cells and stromal cells.TGF-? produced through autocrine or paracrine promotes cell proliferation, differentiation to myofibroblast(MF) and ECM synthesis.These features make TGF-? become an important growth factor topromote fibrosis, which can promote the happening of the organ fibrosis,this is the case in the process of glaucom.On account of this, TGF-?1 was applied to human conjunctival fibroblast(Hcon F) to simulate the wound healing process. Cell proliferation ability was detected by the Cell Counting Kit-8(CCK-8);flow cytometry was used to study cell cycle process; Real-time polymerase chain reactions(RT-PCR) and western blot assays were performed to detect the expression of ?-smooth muscle actin(?-SMA),collagen- I and fibronectin levels, respectively; cell scratch wound and Transwell experiment were performed to test the migration ability of Hcon F. The results showed that TGF-?1 time dependently promoted Hcon F cell proliferation by accelerate cell cycle period. TGF-?1 also accelerated the transformation of human conjunctiva FB to MF. TGF-?1promoted Hcon F cell migration and the ECM synthesis reflected as increased collagen- I and fibronectin expression. The above results show that TGF-?1 promoted Hcon F fibrosis and can be used to simulate the wound healing process after glaucoma filtration surgery in vitro.The chloride ion is the most abundant organisms content anionic,which participates in a variety of biological activity via the process of transmembrane transport by ion channels. Previous findings have demonstrated that chloride channels are widely distributed in mammalian tissues, organs and various kinds of cells, and play important roles in cell volume regulation, transmembrane transport, stabilization of membrane potential, acidification of organelles, regulation of intracellular ph, cell proliferation, differentiation, apoptosis, migration and glandular secretion.As a subtype of the chloride channels family, CLC-2 chloride channel is widely and clearly researched in recent years.Based on this, we examined the effects of 5-Nitro-2-(3-phenylpropylamino) benzoic acid(NPPB) in proliferation, transition of FB to MF, ECM synthesis and migration of Hcon F. Following NPPB treatment in the presence of TGF-?1, Hcon F exhibited reduced proliferation and migration, along with increased transition of FB to MF.NPPB also inhibited cell cycle progression by arresting cells in the G1 phases and reducing collagen I and fibronectin expression. To further investigate the mechanism of chloride channel on Hcon F fibrosis,phosphorylation level phosphoinositide 3-kinase(PI3K) and protein kinase B(Akt) were detected by Western blot. The result showed that TGF-?1 increased phosphorylation of PI3 K and Akt, which were reversed by NPPB.Considering the non-specifically block effect of NPPB to chloride channels, RNA interference technique was applied to investigate the potential role of CLC-2 chloride channel on Hcon F biology. CLC-2si RNA concentration-dependently reduced the gene and protein expression of CLC-2. TGF-?1 treatment increased the expression of CLC-2 which was inhibited by CLC-2 si RNA. TGF-?1 induced Hcon F cell proliferation, migration and transition of FB to MF were also suppressed by CLC-2 si RNA. CLC-2 si RNA transfection inhibited TGF-?1 induced collagen- I and fibronectin synthesis as well as the phosphorylation of PI3 K and Akt in Hcon F.This study discussed the effect of CLC-2 chloride channels in TGF-?1 simulated process of wound healing and the possible mechanism. The result demonstrated that CLC-2 chloride channel regulated HCon F cell proliferation, transformation, migration and ECM synthesis process of Hcon F via PI3K/Akt signaling pathways, which are important molecularmechanisms for scar formation after filtration surgery. Our study provides a new direction for resistance to scarring after filtration surgery. |
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