Celastrol inhibits LPS-induced RA-FLS migration and invasion by suppression of MMP-9 activity.

<p>FLSs were incubated with the indicated concentrations of celastrol for 24 h followed by LPS (1 µg/mL) stimulation. <b>A and B:</b> After 24 h, the mRNA and protein levels of endogenous MMP-1,2,3,9 were measured by qRT-PCR and western blot. GAPDH and β-actin were used as the internal controls, respectively. The histogram shows the mRNA levels from three independent experiments.^**<i>P</i><0.01 versus normal control group, ^⧫<i>P</i><0.05, ^⧫⧫<i>P</i><0.01 versus LPS alone-treated group. <b>C and D:</b> The supernatants were collected and assayed for the amount and activity of secreted MMP-9, by ELISA and gelatin zymography, respectively. The histogram shows the ELISA results from three independent experiments.^**<i>P</i><0.01 versus normal control group, ^⧫<i>P</i><0.05, ^⧫⧫<i>P</i><0.01 versus LPS alone-treated group. <b>E:</b> MMP-9 siRNA was used to study of the role of MMP-9 in LPS-induced migration and invasion of FLSs. After transfection with MMP-9 siRNA or control siRNA for 48 h, and stimulation with LPS, FLSs were allowed to migrate for 24 h. Left, photomicrographs of cells that have passed through membrane (100× magnification). Right, the cell number per microscopic field was plotted (mean ± SD in three independent experiments) in the histogram. ^**<i>P</i><0.01 versus normal control group. ^⧫<i>P</i><0.05 versus LPS and control siRNA treated group.