THU0024\u2005INHIBITION OF CELL PROLIFERATION AND PROMOTION OF INTERLEUKIN-8 PRODUCTION BY T-614 IN CULTURED HUMAN AORTIC ADVENTITIAL FIBROBLASTS

Abstract

Background Takayasu’s arteritis (TA) is an inflammatory fibrosing arteritis that affects predominantly the aorta and its main branches. Recently, growing evidence supports that adventitial fibroblasts play an essential role in vascular inflammation [1].Tumor necrosis factor-α (TNF-α) can reportedly induce inflammation of vascular adventitial fibroblasts[2]. T-614(iguratimod), a novel disease-modifying antirheumatic drug, has been widely used in rheumatoid arthritis in China and Japan [3]. However, the effect and mechanism of T-614 on TA have received little attention. Objectives We report the effects of T-614 on cell proliferation and interleukin-8 (IL-8) production in cultured human aortic adventitial fibroblasts (HAAF), and explored its possible effect on the treatment of TA. Methods HAAF were cultured with 0, 5, 50, 100, or 250 μg/ml T-614 in the absence or presence of 10 ng/ml TNF-α in vitro. Cell viability of HAAF was determined by a modified MTT assay. Supernatant IL-8 level was measured by enzyme linked immunosorbent assay. Results (1)After subculture, HAAF were polygonal or spindle-shaped under the microscope (Figure 1A, B).(2)In the presence of TNF-α, compared with the contrast group, cell viability of HAAF significantly decreased in 50, 100, and 250 μg/ml T-614 treatment groups (OD value: P<0.01, P<0.001, P<0.001, respectively; survival fraction (SF): P<0.05, P<0.001, P<0.001, respectively) (Table I, Figure 2). However, there was no significant difference in cell viability between TNF-α stimulated and unstimulated groups at the same concentration of T-614. In the absence and presence of TNF-α, T-614 suppressed HAAF cell viability dose-dependently (OD value: r = -0.915, P =0.000 and r = -0.926, P =0.000, respectively; SF: r = -0.897, P =0.000; r = -0.885, P =0.000, respectively). (3)In the absence of TNF-α, compared with the contrast group, IL-8 level in 5 and 100 μg/ml T-614 treated groups were significantly higher(P<0.05); in the presence of TNF-α, IL-8 level in 5, 50, and 100 μg/ml T-614 treated groups were significantly higher (P<0.001, P<0.001, P<0.01, respectively); (Figure 3). There was a negative correlation between supernatant IL-8 level and the concentration of T-614 in groups stimulated with TNF-α (r = -0.670, P =0.000). TNF-α increased IL-8 level in the control group and various concentrations of T-614 treated groups (all P<0.001). Table I. Effects of T-614 on TNF-α stimulated cell viability of HAAF (OD value) T-614(μg/ml) OD value 0 ng/ml TNF-α 10 ng/ml TNF-α 0 0.424±0.038 0.414±0.042 5 0.404±0.050 0.440±0.054 50 0.347±0.061*** 0.358±0.040** 100 0.267±0.034*** 0.302±0.039*** 250 0.096±0.019*** 0.092±0.009*** TNF-α, tumor necrosis factor-α; HAAF, human aortic adventitial fibroblasts. Data are shown as mean ± SD of 3 independent experiments, each in triplicate. *** P < 0.001, ** P < 0.01 vs. control group(0μg/ml T-614) within each group alone. Conclusion T-614 can inhibit the proliferation of HAAF and promote IL-8 production; therefore, it may provide a new immunotherapeutic intervention for TA. References [1] Maiellaro, K. and W.R. Taylor, The role of the adventitia in vascular inflammation. Cardiovasc Res, 2007. 75(4): p. 640-8. [2] He, Y., et al., SIRT6 inhibits TNF-alpha-induced inflammation of vascular adventitial fibroblasts through ROS and Akt signaling pathway. Exp Cell Res, 2017. 357(1): p. 88-97. [3] Wei, Y., et al., Inhibitory Effect of a Novel Antirheumatic Drug T-614 on the IL-6-Induced RANKL/OPG, IL-17, and MMP-3 Expression in Synovial Fibroblasts from Rheumatoid Arthritis Patients. Biomed Res Int, 2015. 2015: p. 214683.Abstract THU0024 Figure 1Abstract THU0024 Figure 2Abstract THU0025 Figure 2 Acknowledgement Funding: This project was supported by grants from China International Medical Foundation(Z-2014-06-2-1636).The sponsors had no role in the study design, data collection and analysis, decision to publish, or preparation. Disclosure of Interests None declared