Effect And Mechanisms Of Extracellular Matrix Synthesis In Lens Posterior Capsular Opacification Via Wnt5a Signaling Pathway

Objective Posterior capsular opacification(PCO)is a process of wound-healing and fibrosis of lens tissue,which eventually leads to visual loss.Its pathological mechanism is the residual lens epithelial cells(LECs),under the stimulation of transforming growth factor-β(TGF-β),undergo proliferation,migration and epithelial-mesenchymal transition(EMT)after cataract surgery or lens trauma.The transformed myofibroblasts are the key cell type involved in fibrosis,which induce the deposition and remolding of extracellular matrix(ECM)is an important mechanism of PCO,and overexpressing the cellular cytoskeletal protein α-smooth muscle action(α-SMA).Wnt5 a protein,a member of the non-canonical Wnt protein family,acts as a key regulator in many fibrotic processes,regulating myofibroblasts’ proliferation and transdifferentiation,as well as ECM production and remodeling in fibrotic diseases.This study aims to test the effect and potential mechanism of Wnt5 a protein regulates EMT occurrence,ECM remodeling and cellular morphological change in human lens epithelial cells(HLECs)line SRA 01/04 induced by TGF-β through activating the non-canonical Wnt signaling pathway,which strive to provide effective clinical measures for the prevention and treatment of PCO.Methods Taking HLECs line SRA 01/04 cells as the research object,divided into four groups:TGF-β group,Wnt5 a si RNA group + TGF-β group,Wnt5 a group and control group.SRA01/04 cells were induced with TGF-β to establish a PCO cellular model as TGF-β group,the cells were induced with TGF-β after transfection of Wnt5 a si RNA 24 h as Wnt5 a si RNA group + TGF-β group,only exogenous Wnt5 a protein was added in the SRA 01/04 cells treated as Wnt5 a group,and only untreated SRA 01/04 cells in the control group.Western blot assay was used to detect the expression of Wnt5 a protein,cellular cytoskeletal protein α-SMA and main components of ECM Col-I and fibronectin at the protein level.Immunofluorescence assay was used to show the expression and distribution of cellular cytoskeletal protein α-SMA in HLECs line SRA 01/04 cells,as well as cellular morphological change.Wound healing assay was used to assess the change in the mobility and migrality of HLECs line SRA 01/04 cells after Wnt5 a protein overexpression.Col-I contraction assay was observed the contractility of ECM and cells after overexpression of Wnt5 a protein in HLECs line SRA01/04 cells.Results Western blot assay detection of HLECs line SRA 01/04 cells confirmed that the expressions of each Wnt protein in the TGF-β group was significantly higher than that in the control group,showing a significant difference between the two groups(P < 0.001).The expression of Wnt5 a protein in the TGF-β group was also significantly higher than that in the control group;and its expression at 24 h and 48 h was time-dependent,showing a significant difference between the groups(t=-42.338,P=0.001).Western blot assay showed that in SRA01/04 cells in the TGF-β group,the expression of α-SMA,Col-I and fibronectin were obviously higher than those in the other three groups,showing a significant difference between the groups(F=11161.395,2507.39 and 1971.676,all P < 0.001).Immunofluorescence assay showed that the α-SMA was strongly expressed in SRA 01/04 cells in the TGF-β group compared with the other three groups,and the cellular morphology was significantly changed.Wound healing assay showed that the migration distance of the SRA 01/04 cells in the TGF-β group at 48 h was significantly higher than that in the other three groups,and the migration distance of cells in the TGF-β group within 48 h gradually increased with time,showing a significant difference between the groups(all P < 0.001).Col-I contraction assay showed the gel contraction area ratio of the TGF-β group at 48 h was significantly lower than that in the other three groups,and the gel contraction area ratio of the TGF-β group within 48 h gradually decreased with time,showing a significant difference between the groups(all P<0.001).Conclusion Cell experiments proved that LECs overexpressed Wnt5 a protein under the induction of TGF-β,promoted the expression of cellular cytoskeletal protein α-SMA,main components of ECM Col-I and fibronectin by activating the non-canonical Wnt signaling pathway,and induced cellular morphological changes and ECM contraction,while enabling cells to acquire stronger motility and contractility.In conclusion,Wnt5 a may be a new target for prevention and treatment of fibrotic diseases such as PCO.