The Mechanisms Of Tanshinone ?A On Promoting Apoptosis Of Fibroblast-like Synoviocytes In Rheumatoid Arthritis

Background:Rheumatoid arthritis(RA)is a chronic autoimmune joint disease characterized by proliferation and infiltration of fibroblast-like synoviocytes(FLS),and poses a non-negligible health and economic burden to patients.One key strategy for rheumatoid arthritis treatment is to induce apoptosis of fibroblast-like synoviocytes.And it has been reported in the literature that Tanshinone ?A(Tan ?A)can attenuate the proliferation of RA-FLS,thereby impeding the progression of RA,but the specific mechanism are not clear.Objective:We aimed to explore the mechanisms of the Tanshinone ?A(Tan ?A)-induced apoptosis of FLS from patients with RA(termed RA-FLS).Methods:1.Preparation of human synovial tissues and FLS:Synovial tissue samples were obtained from 16 patients with RA during joint replacement or synovectomy or at Northern Jiangsu People's Hospital.Healthy synovial tissues from seven traumatic knee patients were used as normal controls.Synovial tissue samples were got during the surgery.FLS were isolated by digestion with 2.5g/l trypsin for 4h at 37?C with gentle agitation.2.RA-FLS was treated with Tan ?A:RA-FLS was seeded in a plate and cultured in medium containing various concentrations of Tan ?A for various hours,respectively.3.Analysis of the apoptosis rate of RA-FLS cells treated with Tanshinone ?A:3.1 The Cell Counting Kit-8(CCK-8)was used to evaluate RA-FLS viability in accordance with the manufacturer's recommendations.3.2 Annexin V-FITC Apoptosis Detection Kit I(BD Pharmingen,Heidelberg,Germany)was used to analyze RA-FLS apoptosis.Apoptosis and apoptosis rate was calculated using a FACSC alibur flow cytometer and Cell Quest software.4.Analysis the possible site of Tanshinone ?A acting on a RA-FLS:4.1 To quantify the Long non-coding RNA(lnc RNA)GAS5 expression in the Tan ?A-treated RA-FLS(RA-FLS + Tan ?A),untreated RA-FLS(RA-FLS-Tan ?A),normal cells(controls),real-time quantitative PCR was performed.4.2Tanshinone ?A-treated RA-FLS cells(RA-FLS+Tan ?A)were transfected with si RNA-GAS5,lnc RNA GAS5 knockdown,and RA-FLS activity was determined using CCK-8.5: Western blot: Western blot was used to determine the expression of caspase-3,caspase-9,Bax,Bcl-2,p-PI3 K,p-AKT and p-m TOR in RA-FLS and compare them to confirm the signaling pathways of Tanshinone ?A pro-apoptosis effects on RA-FLS.6: Statistical analysis:Student's t test was used to analyze the differences between the two groups.One-way ANOVA was used to compare the results of more than two groups.Differences with a P-value of less than 0.05 were considered statistically significant.All statistical analyses were performed using the SAS 6.12 software package.Result:1.The activity of RA-FLS was declined and the apoptosis rate of RA-FLS was increased after the treatment of Tan ?A.2.Long non-coding RNA(lnc RNA)GAS5 expression was significantly decreased in the synovial tissues and RA-FLS,while Tan ?A treatment resulted in anup-regulation of GAS5.Consistently,knockdown of GAS5 using si RNA inhibited RA-FLS apoptosis.3.Lnc RNA GAS5 knockdown down-regulated the expression of cleaved caspase-3 and caspase-9 in the RA-FLS cells and activated the phosphoinositide 3-kinase(PI3K)/AKT signaling pathway.Conclusion:1.Tan ?A promotes RA-FLS apoptosis by up-regulating lnc RNA GAS5.2.Tan ?A promotes RA-FLS apoptosis by enhanced expression of cleaved caspase-3/caspase-9 and inhibited PI3K/AKT signaling.