S58 Prevents Fibrosis Via Activation Of PI3k/Akt/Nrf2 Signaling Axis In Rabbits Of Glaucoma Filtration Surgery

Background: Glaucoma filtration surgery(GFS)is currently an effective method for the treatment of poorly controlled intraocular pressure(IOP)in glaucoma.However,postoperative scarring of the conjunctival sac often results in surgical failure.Although the clinical application of 5-fluorouracil(5-FU)and mitomycin C(MMC)have improved the success rate of filtration,there are still some serious complications,such as leakage of filtrating blebs,shallow anterior chamber after operation,and corneal toxicity.Therefore,the search for a more effective and safe anti-scarring drug remains the focus of research.Some literatures indicate that oxidative and anti-oxidant balance regulate TGF-?s-induced fibrosis through different pathways,and show that the increase of oxidative stress is essential for the development of fibrotic diseases.Therefore,targeting TGF-?-induced and ROS-induced oxidative stress signals may provide new approaches for the treatment of fibrotic diseases.In the previous research,we used the Systematic Evolution of Ligands by Exponential Enrichment(SELEX)technology to screen and optimize those with targeted T?R II,non-toxic and easily chemically modified nucleic acid aptamer S58.Whether S58 inhibits the binding of TGF-?2 to its receptors can improve the intracellular stress level,regulate the oxidation-antioxidation imbalance to inhibit the formation of scars after glaucoma filtering surgery,and it needs to be further elaborated,which provides experimental basis for the research of anti-scar treatment.Objective: In this study,in vivo/vitro experiments was conducted to verify the interference effect of S58 targeting T?R II on the biological effect of ?GF-?2,and to explore the mechanism of S58 in improving the oxidationantioxidation homeostasis of injured HConFs and inhibiting scars after GFS.Methods:a)In vitro experiments: Human Conjunctival blasts(HConFs)are used for cell experiments.1.The three-dimensional model of S58 and extracellular receptor proteins T?R II period of crystal structure were established,confirmed that was most likely in solution with the aptamer combined with 3D crystal structure,and half the DNA and protein molecules flexible molecular docking experiment,statistic clustering analysis of the combination of DNA aptamer,and accurate analysis of the binding site.2.Flow cytometry technology and laser confocal microscopy were used to observe the binding of fluorescent-labeled S58 to fibroblasts,and siRNAT?R II knockdown experiment was used to verify the anti-TGF-?2-induced fibrosis of S58 targeted T?R II.3.HConFs was induced by different concentrations of TGF-?2(0,1,2,4,10,20 ng/m1)for 24 h,and TGF-?2(4 ng/m1)for HConFs at different times(0,24,48,72 h).Western blotting or RT-PCR was used to detect the expression of proteins or genes related to cell fibrosis;The expression level of ?-SMA was detected by confocal cell immunofluorescence.4.TGF-?2(4 ng/m1)-induced HConFs were treated with NAC for 24 h,and the expression of fibrosis and related antioxidant proteins or genes were detected by western blotting or RT-PCR.Moreover,the expression levels of collagen-1 and ?-SMA were detected by confocal cell immunofluorescence assay.5.HConFs induced by TGF-?2(4 ng/m1)was treated with S58 for 24 h,and the expression of related antioxidant proteins or genes was detected by western blotting or RT-PCR;Intracellular ROS levels was measured by flow cytometry.6.HConFs induced by TGF-?2(4 ng/m1)was treated with S58 at 24 h,and the expression of proteins or genes related to cell fibrosis was detected by Western blotting or RT-PCR;Confocal cell immunofluorescence assay was used to detect the expression levels of collagen,?-SMA,collagen-1 and fibronectin,and the scratch experiment and CCK-8 assay were used to evaluate the effect of S58 on the proliferation and migration of TGF-?2-induced HConFs.7.Gene knockout of siRNA-Nrf2 or LY294002(an inhibition of PI3k/Akt)were used to verify the anti-fibrosis effects of S58 on TGF-?2-induced HConFs by western blotting and confocal cell immunofluorescence to detect the expression of fibrotic proteins.b)Animal experiment: New Zealand rabbits after GFS were used as models to study the therapeutic effect of S58 on postoperative filtrating bleb fibrosis.1.The experimental groups were: Normal group;Sham group;S58 group: S58 20nM/drop(4 times/day);MMC group: MMC(0.2 mg/m1).2.The morphology of rabbit eye filtrating blebs 3 days,1 w,2 w and 4 w after surgery were observed,the changes of IOP under slit lamp were taken photos and measured.3.Histological examinations of pathological tissues and staining analysis of H&E,Masson,s trichrome staining and Sirius red staining.Results: 1.S58 inhibited TGF-?2-induced HConFs fibrosis by increasing TGF-?2 and T?R II in combination with steric resistance;2.TGF-?2 induced intracellular oxidative stress response and increased fibrosis levels in HConFs cells;3.TGF-?2 increased HConFs oxidative stress and induced fibrosis;4.NAC reduced TGF-?2-induced HConFs fibrosis by inhibiting ROS production,suggesting that ROS played an important role in TGF-?2-induced HConFs fibrosis;5.S58 promoted the antioxidant defense of TGF-TGF 2-induced HConFs;6.S58 reduced TGF-?2-induced HConFs fibrosis;7.S58 reversed TGF-?2-induced HConFs fibrosis by activating the PI3k/Akt/Nrf2 signaling pathway;8.S58 could effectively reduce the degree of fibrosis of the filtrating blebs after the surgery and prolonged the existence time of the filtrating blebs.Conclusion: This study suggests that S58 can improve postoperative results of GFS by activating the intracellular antioxidant defense PI3k/Akt/Nrf2 signaling pathway.