The Role And Mechanisms Of TGF-β/Smad3 Signaling Pathway In The Development Of Posterior Capsule Opacification

ObjectivePosterior capsule opacification(PCO)is the most common complication of modern phacoemulsification combined with intraocular lens implantation and is the main reason for postoperative visual decline.It is currently believed that the proliferation,migration,epithelial-mesenchymal transition(EMT)of residual lens epithelial cells(LECs)and extracellular matrix deposition after cataract surgery are the main reasons for the development of PCO.At present,the most effective treatment is Nd:YAG laser posterior capsulotomy,but this method may cause complications such as macular edema and retinal detachment.This study is to explore the pathogenesis of PCO and provide new ideas for the diagnosis and treatment strategy of PCO.Transforming growth factor-beta(TGF-β)is considered to be the most potent cytokine in the induction of LECs transdifferentiation,and inhibition of TGF-β signaling pathway may provide a new treatment for posterior capsule opacification.Smad3 is a key molecule in the TGF-β/Smad signaling pathway.To elucidate the precise regulation of the biological effects of TGF-β/Smad3 signaling pathway on the pathogenesis of PCO,this study intends to identify the regulatory role of TGF-β/Smad3 signaling pathway in the formation of PCO by cell experiments and animal models.Methods Part I: In vitro cell experiments,we used the recombinant TGF-β2 to induce the PCO model of the lens epithelial cells(HLE-B3).The Smad3 inhibitor SIS3 was added or not in the medium,and the cell viability of the LECs was detected by CCK-8 assay.The inhibitory effect of SIS3 on Smad3 was examined by Western blot analysis.Experimental group: A: normal control group;B: negative control group:DMSO;C: TGF-β2 group;D: TGF-β2+SIS3 group.The relative mRNA expression of α-smooth muscle actin(α-SMA),transcription factor Snail1 and Slug,epithelial marker E-cadherin,fibronectin(FN)and collagen type I during EMT was detected by RT-PCR.Immunofluorescence was used to detect the expression of fibronectin and E-cadherin.Part II: In the animal model experiment,a 26 G hypodermic needle was used to pierce the lens anterior capsule of C57BL/6J mice to induce LECs epithelial-mesenchymal transition,providing a model basis for the next experiment.1.Histological observation of the healing of lens anterior capsule injury in mice.2.Real-time quantitative PCR was used to detect the relative expression of α-SMA,CDH1(E-cadherin),lumican,osteopontin(OPN),FN and collagen type I after lens anterior capsule injury in mice.3.Immunofluorescence staining was used to detect the expression of α-SMA,lumican and FN in situ proteins after lens anterior capsule injury in mice.Part III: To further investigate the role of Smad3 deletion on lens epithelial-mesenchymal transformation,Smad3 knockout mice(Smad3 knocout,Smad3 KO/Smad3-/-)and wild type mice(wide type,WT/Smad3+/+)were used to establish a lens anterior capsular injury model.1.Histological observation of the healing of lens injury in Smad3 KO and WT mice.2.Real-time quantitative PCR was used to detect α-SMA,Snail1,Slug,Twist1,SIP1,E-cadherin,lumican,OPN,hyaluronidases(HASs),FN,collagen type I mRNA relative expression in Smad3 KO and WT mice after surgery.3.Western blot analysis was used to detect α-SMA,lumican,OPN and FN protein expression in Smad3 KO and WT mice after surgery.4.Immunofluorescence staining was used to detect α-SMA,E-cadherin,lumican,OPN,hyaluronic acid(HA),FN,collagen type I in situ protein expression in Smad3 KO and WT mice after surgery 5.TUNEL staining was used to detect the number of apoptosis in Smad3 KO and WT mice after surgery.Results 1.In vitro experiments,SIS3 can effectively inhibit the expression of p-Smad3,and when the final concentration of SIS3 is 3μM,the inhibition of Smad3 is the strongest.2.SIS3 effectively decreased the expression of transcription factors Snail1 and Slug,down-regulated the expression of α-SMA,up-regulated E-cadherin,and inhibited the expression of extracellular matrix components fibronectin and collagen type I.3.Using the 26 G needle to pierce the lens anterior capsule experiment,the results showed that the expression of α-SMA,lumican,OPN,FN,collagen type I increased,the expression of E-cadherin decreased,and the EMT of LECs was successfully induced.The method is simple,safe,repeatable and low cost,and provides a basis for further experiments in vivo.4.In vivo animal experiments showed that Smad3 deletion can inhibit the expression of α-SMA,transcription factor Snaill,Slug,Twist1 and SIP1,promote the expression of E-cadherin,and inhibit the expression of extracellular matrix such as lumican,OPN,HA,FN and collagen type I.ConclusionsIn this study,TGF-β/Smad3 is involved in the regulation of lens epithelial-mesenchymal transition by cell experiments and animal model experiments.Smad3 is a key target of TGF-β/Smad3 signaling pathway;blocking Smad3 can inhibit the effect of TGF-β/Smad3 signal pathway on the EMT of lens epithelial cells;Smad3 may be a potential new ideas for fibrotic diseases such as posterior cataract and anterior subcapsular cataract.