Effect Of Gypenosides On Inflammation And Fibrosis Of Orbital Fibroblasts In Graves’ Ophthalmopathy

Objective:This study aimed to evaluate the therapeutic effects of gypenosides(Gyps)on inflammation and fibrosis of orbital fibroblasts(OFs)in Graves’ ophthalmopathy(GO)using a combination of bioinformatics and in vitro cell experiments.Methods:1.Investigation of the potential mechanism of Gyps for GO based on bioinformaticsData were extracted by searching Pub Med and Web of Science databases and screening articles describing the targets of Gyps’ role in treating inflammation and fibrosis.Gyps-related targets for inflammation and fibrosis were summarized,and gene ontology analysis and pathway analysis were performed to build a protein-protein and gene-gene interaction network.This process could describe the potential molecular mechanism of Gyps in the treatment of GO from the perspective of network pharmacology.2.OFs extraction,cultivation and identificationThe OFs used in this study were from the orbital connective tissue of the orthopedics surgery of the First Affiliated Hospital of Guangxi Medical University.Primary cells were extracted and cultured by enzyme digestion.Morphological observation and immunohistochemistry(IHC)detecting vimentin(VIM),S100 calcium binding protein B(S100 B),myoglobin(MB),desmin(DES)keratin 17(KRT17)were used to identify the cell type.3.Investigation of the effects of Gyps in protecting OFs in GO through cell experiments in vitroThe CCK-8 kit was used to detect the effect of different concentrations of Gyps on the proliferation of OFs,and the Gyps concentration with the strongest cell activity was selected for subsequent experiments.After pretreatment of OFs with specific concentration of Gyps for 24 hours,IL-1β and TGF-β1 were used to stimulate OFs for 24 hours to build a model of cellular inflammation and fibrosis.Real-time quantitative PCR was used to detect IL-6,IL-8,TNF-α and CCL2,and HAS2,COL1A2,α-SMA and FN1 m RNA expression levels;ELISA was used to detect IL-6,TNF-α and CCL2,and HA,Col I,α-SMA,and FN protein expression levels;WB was used to detect TLR4 and NF-κB,and SMAD2,p-SMAD2,and SMAD4 protein expression levels.Results:1.Bioinformatics analysis proved that Gyps could treat GO with antiinflammatory and anti-fibrotic effectsGyps were proved to against inflammation(working on TNF,IL-1β,IL-5,MAPK14,NFKB1,STAT1,PPARG,and VCAM1)and fibrosis(working on MMP2,MMP9,TIMP1,TIMP2,TGFB1,SMAD7,CCN2,ALDH1B1,and PPARA)through literature data extraction.The gene ontology analysis and pathway analysis of the above targets proved that Gyps could affect the inflammation and fibrosis process through several related pathways.2.Successfully isolated and cultured high-purity OFs and identified the cellsOFs was extracted from the orbital connective tissue by enzymatic digestion.The cell morphology was spindle-shaped,and the cells were adherent,and could form a swirling shape after fusion.IHC results showed that OFs expressed VIM,but not S100 B,MB,DES,and KRT17.3.Cell experiments confirmed the protective effect of Gyps on OFs in GO patients in vitroModels of inflammation and fibrosis of OFs could be successfully constructed by IL-1β and TGF-β1 stimulation,while Gyps could reduce the expression levels of inflammation-and fibrosis-related m RNA and protein.Conclusions:1.Bioinformatics analysis confirmed that Gyps can protect OFs in GO through anti-inflammatory and anti-fibrotic effects.The results can provide a theoretical basis for subsequent cell experiment verification in vitro.2.High-purity OFs can be extracted by enzymatic digestion and can be identified by IHC detection with positive expression of VIM and negative expression of S100 B,MB,DES and KRT17.3.Gyps may have a therapeutic effect on GO by regulating NF-κB signaling pathway for anti-inflammation and regulating TGF / SMADs signaling pathway for anti-fibrosis.