The Mechanism Of Ceruloplasmin In Regulating Fibrosis In Orbital Fibroblasts

Background: Thyroid-associated ophthalmopathy(TAO)is an autoimmune disease with complex pathogenesis.As a common pathological change,fibrosis occurred with the change of phenotypic and functional changes of orbital fibroblasts(OFs),however,the mechanism has not been fully cleared.Ceruloplasmin(CP)regulates the metabolic process and oxidative stress.The level of CP induces oxidative stress injury of cells and fibrosis.However,whether CP participated in the occurrence of TAO is not clear.Aim: Determine the expression of CP in orbital tissues from TAO,and detecting the effect of CP in the mechanism of TAO.Methods: The differentially expressed genes were analyzed through bioinformatics methods;After being sectioned and stained by HE and Masson,orbital tissues were observed under the microscope;Quantitative Real-time PCR(QPCR)was used to verify the expression level of CP and cathepsin C;Primary OFs were isolated through enzyme digestion and the cell types were identified by immunofluorescence staining;Different concentration of TGF-β was incubated with OFs for different times,then MTT was used to test the cell viability and QPCR was used to test the expressed level of CP,cathepsin and the biomarkers of phenotypic and functional changes of OFs;The level of CP was overexpressed or knock-down through liposome transfection and verified by QPCR;immunofluorescence staining,Western blot,QPCR,and ELISA were used to test the phenotypic and functional changes of OFs after liposome transfection.The phosphorylation level of key proteins was tested by Western blot to detect the downstream pathways of CP.Results: Through analyzing GSE58331 and GSE105149 from GEO datasets with bioinformatics methods,five differential expressed genes were identified,and the level of CP and cathepsin C were significantly downregulated(P<0.05).The clinical information and orbital tissues were collected from five TAO patients and four healthy controls.Significant fibrosis was observed in orbital tissues from TAO after staining the sections.The cells isolated through enzyme digestion from orbital tissues were identified to be OFs since vimentin and fibronectin were expressed on cells.After incubated with TGF-β,cell viability and the level of vimentin and fibronectin were positively related with the concentration of TGF-β and incubating time(P<0.05).Through this process,the level of CP and cathepsin were downregulated(P<0.05).CP was successfully overexpressed or knockdown in OFs through liposome transfection.After overexpression of CP in OFs,the cell viability was elevated while the level of vimentin,fibronectin,and collagen Ⅰ was downregulated(P<0.05).After the knockdown of CP in OFs,the cell viability decreased while the level of vimentin,fibronectin,and collagen Ⅰ was upregulated(P<0.05).In addition,the phosphorylation level of key proteins in TGF-β/Smad,MAPK,and AKT pathways were negatively correlated with the level of CP(P<0.05).Conclusion: CP was a protective factor in TAO,and the level of CP was negatively correlated with cell viability and the expression of fibrosis-related proteins of OFs.Upregulated expression of CP may have a therapeutic effect on TAO.