Autophagy induced by brazilin limits NF-κB activation and inflammatory response in RA FLS.

<p>(A, B) After pretreatment with brazilin for 48 h, RA FLS were treated with 1 μg/ml of LPS (A) or 15 ng/ml of TNF (B) as for various times as indicated. Whole cell lysates were separated by SDS-PAGE and the immunoblotting was performed using indicated antibodies. (C) RA FLS were pretreated with brazilin for 48 h in the absence or presence of NAC (10 mM), and then treated with TNF as indicated times. Whole cell lysates were separated by SDS-PAGE and the immunoblotting was performed, as in (A). (D, E). RA FLS were pretreated with brazilin for 48 h in the absence or presence of NAC (10 mM). Supernatants were harvested at the indicated times after the commencement of LPS stimulation, and the levels of IL-6 and IL-8 were measured by ELISA. (F) RA FLS transfected with either a lentivirus expressing control shRNA (shCont) or shRNA specific for Atg5 (shAtg5) were cultured for 48 h. Consequently, cells were infected with recombinant adeno-virus expressing NF-κB-Luc at 200 PFU/cells for 2h, and then replaced with DMEM containing 10% FBS. After 24 h of infection. cells were treated for 6 h with LPS (1 μg/ml), and luciferase assay was performed as described in Materials and Methods. Knock down efficiency of Atg5 in RA FLS were assessed by immunoblotting (inset). (G) After RA FLS were transduced with shCont or shAtg5, the cells were further treated with LPS (1 μg/ml) for 24 h. The levels of IL-6 and IL-8 in the supernatant were measured by ELISA. Each columns shows mean ± S.E. from the three independent experiments. *<i>P</i><0.05, compared with shCont-transfected cells.</p

ROS production plays an important role in autophagy activation and early event of cell death in response to brazilin.

<p>(A) RA FLS were treated with 25 μg/ml of brazilin for various times, as indicated. CM-H₂-DCF-DA (1 μM) was added 30 min before end of treatment. ROS were measured with a flow cytometer (left panel) as described in materials and methods. Data were processed and quantified with the FlowJo software (right panel). (B) RA FLS were treated with brazilin (25 μg/ml) in the absence or presence of NAC (10 mM), Mito-TEMPO (100 μM) and DPI (10 μM) for 9 h. Generation of ROS were measured, as in (A) (C) RA FLS were pretreated with NAC (10 mM), Mito-TEMPO (100 μM) and DPI (10 μM), and then followed by brazilin (25 μg/ml) for 24 h. Whole cell lysates were separated by SDS-PAGE and the immunoblotting was performed with anti-LC3 and actin antibodies. (D, E) RA FLS were treated with brazilin (25 μg/ml) in the absence or presence of NAC (10 mM) or Mito-TEMPO (100 μM) for indicated times. Cell were visualized with a normal inverted microscope (D) and the cell viability was determined (E), as describe in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136122#pone.0136122.g001" target="_blank">Fig 1A</a>. (F) HDF were treated with brazilin (25 μg/ml) for various times, as indicated and the levels of ROS were determined, as in (A).</p

Failure to restoration from brazilin-induced cytotoxicity by blockade of autophagy in RA FLS.

<p>(A) RA FLS were treated with brazilin (25 μg/ml), Fas ligand (FasL, 1 μg/ml) plus cycloheximide (CHX, 10 μg/ml), MNNG (0.5 mM) and <i>t</i>-BHP (100 μM) in the absence or presence of pancaspase inhibitor (z-VAD-FMK, 20 μM) or necrosis inhibitor (IM54, 10 μM). The cells were collected and cell viability was then determined by trypan blue exclusion assay. The results are presented as the mean ± S.E. from the three independent experiments. (B) HDF were treated with brazilin (25 μg/ml) in the absence or presence of z-VAD-FMK (20 μM) or IM54 (10 μM) as indicated times, and cell viability was determined, as in (A). *<i>P</i><0.05, compared with brazilin only treated group. (C, D) RA FLS were pretreated with autophagy inhibitor, NH₄Cl (5 mM) and chloroquine (CQ, 5 mM) for 30 min, and then followed by 25 μg/ml of brazilin as indicated times. (E) HDF were pretreated with rapamycin (100 nM) for 30 min, and then followed by 25 μg/ml of brazilin as indicated times. The cell viability was determined, as describe in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136122#pone.0136122.g001" target="_blank">Fig 1A</a>.</p

Restoration of cellular survival against from brazilin-induced cytotoxicity in RA FLS, but not in normal fibroblasts.

<p>FLS from RA patients (A) and several normal fibroblasts (dermal fibroblast from human foreskin tissues, NIH3T3, COS-7 and MEF) (B) were treated with indicated concentrations of brazilin. The cells were collected and cell viability was then determined by trypan blue exclusion assay. The results are presented as the mean ± S.E. from the three independent experiments.</p