The Effect And Mechanism Of Plumgagin On Synoviocytes In Rheumatoid Arthritis

ObjectiveRheumatoid arthritis（RA）is an autoimmune disease that can be gradually developed worse,with high morbidity and disability rates^[1],the pathogenic mechanisms of RA have not been fully elucidated,and there is still no safe and effective drug treatment.In recent years,Traditional Chinese medicine monomers have received more and more attention in the treatment of RA,which provides a new method for the treatment of RA.At present,several studies have demonstrated that Plumbagin（PLB）has strong anti-inflammatory,anti-tumor and anti-liver fibrosis effects,but its mechanism is not fully understood.This study performed to explore effects of Plumbagin（PLB）on human Rheumatoid arthritis fibroblast-like synoviocytes（RA FLSs）and its possible mechanism.Methods1.Human RA FLSs were isolated from RA patients,Enzyme digestion method isolated synovial fibroblasts,using optical microscope to observe cell morphology.Using immunocytochemistry method for detecting FLSs marker Vascular cell adhesion molecular-1（VCAM-1）.2.Cell viability and proliferation were measured with MTT assay.4-7generation cells were used to following experiments,the cells were divided into8 groups:blank control group（Con group）,Lipopolysaccharide（LPS）group（cells were treated with1μg/m L LPS）,LPS+PLB group（cells were treated with1μg/mL LPS+0.5,1.5,3,5,7.5,10μmol/L PLB）,treated for 24 h.The pro-inflamm atory cytokines were assessed using ELISA assay,in this assay,RA FLS were divided into four groups:Con group（blank control group）,LPS group（cells were treated with1μg/mL LPS）,and LPS+PLB group（cells were treated with 1μg/mL LPS+0.5,3μmol/L PLB）,In following tests,RA FLSs were divided as this method.3.The cell apoptosis was assessed using Hoechst33342 staining assay and flowcytometry assay（FCM）.Expression levels of Bcl-2,Bax,cleaved-Caspase3proteins were detected with Western Blot,after intervented 24 hours by diferent concentrations of PLB in RA FLSs.4.Transwell assay was used to test invasion ability of RA FLSs.Wound healing assay and Transwell assay were used to detect metastasis ability in RA FLSs.The expression of MMP-2,MMP-9 levels were assassed using Western Blot.5.Expression levels of p-JAK2 and p-STAT3 proteins were detected with Western blotting.Results1.The results showed that the cell was fibroblast-like ynoviocytes（FLSs）and had uniform morphology and appearance when it was in the 3^rdd passage,and the cells had long spindle-shape.FLSs expressed VCAM-1.These suggested that harvested from Enzyme digestion method were FLSs.2.The result of MTT assay found that PLB could dose-dependently inhibited LPS-induced cell viability and proliferation ability（P=0.035,P=0.002,P=0.005,P=0.000,P=0.0 00,P=0.000）,the IC50 for LPS-stimulated RA FLSs of PLB was3.149μmol/L,choose the concentration of 0.5 and 3.0μmol/L PLB for subsequent assays.ELISA assays results showed that LPS increased the production of TNF-αand IL-6（TNF-α:P=0.004 vs Con group,IL-6:P=0.007 vs Con group）.PLB decreased LPS-stimulated production of TNF-αand IL-6（TNF-α:P=0.009 vs LPS group,IL-6:P=0.001 vs LPS group）in the cells.3.FCM results showed that PLB dose-dependently increased apoptosis rate of human RA FLSs in vitro（P=0.002,P=0.000 vs LPS group）.Further more,Western Blot（WB）results showed that,PLB could upregulate the expre ssions of Bax and cleaved–Caspase3（P=0.0004,P=0.000 vs LPS group）,and decreased the Bcl-2 protein expression（P=0.000 vs LPS group）.4.Transwell assay discoveries were that PLB could inhibit invasion and migration of RA FLSs（P=0.005,P=0.000 vs LPS group）.The Western Blot results showed that PLB could inhibit MMP-2,MMP-9 expression（MMP-2:P=0.005,P=0.000 vs LPS group;MMP-9:P=0.001,P=0.000 vs LPS group）.5.The result presented that PLB could down regulate the JAK2 and STAT3pho sphorylation level（p-JAK2:P=0.015、P=0.000 vs LPS group,p-STAT3:P=0.028、P=0.003 vs LPS group）.Conclusions1.Plumbagin inhibited proliferation and LPS-stimulated production of TNF-αand IL-6 in the RA FLSs.2.Plumbagin induced apoptosis in RA FLSs is mediated through upregulation of protein Bax and downregulation of Bcl-2 protein.3.Plumbagin inhibited invasion and migration of RA FLSs were mediated through decreasing the expression of MMP-2 and MMP-9.4.Plumbagin could inhibit JAK2/STAT3 signaling pathway.