Purpose: To explore the effects of Triptolide, the principal active diterpenoid from the Chinese Medicinal Herb Tripterygium Wilfordii Hook F that has immunosuppressive and anti-inflammatory properties, on cell proliferation, hyaluronic acid (HA) synthesis, and the expressions of human leucocyte antigen-DR (HLA-DR), intercellular adhesion molecule-1 (ICAM-1) and CD40 on cultured retro-ocular fibroblasts (RFs) from patients with Graves’ ophthalmopathy.
Methods: After two to five passages, cultured RFs were incubated for 48 h within a medium alone or in the presence of recombinant human interferon-γ (IFN-γ) and various concentrations of Triptolide. Cell viability was assessed by MTT (3-[4.5-dimethylahiazol-2-yl]-2,5-diphenyltetrazolium Bromide). RFs proliferation was assessed by [3H]-thymidine incorporation assay. Flow cytometry was used to investigate the amount of HLA-DR, ICAM-1 and CD40. HA synthesis was measured by radioimmunoassay.
Results: Cell viability was not detrimentally affected when incubated with Triptolide from 0.01 µg/L to 10 µg/L for 48 h, and decreased with 20 µg/L Triptolide. The incorporation of [3H]-thymidine of RFs was 55 476 ± 15 842 cpm incubated with medium alone or 18 352 ± 3568 cpm with 10 µg/L Triptolide (t = 5.600, P < 0.01). Initially, the percentage of positive cells of HLA-DR, ICAM-1 and CD40 on RFs were 4.75 ± 2.13%, 17.53 ± 10.12% and 6.38 ± 2.23%, respectively, and the synthesis of HA was 100 ± 12%. Compared with basal values, 48-h incubation with IFN-γ (100 U/mL) significantly enhanced the amount of HLA-DR, ICAM-1 and CD40, and HA synthesis. The values were 60.58 ± 10.12% (t = 13.224, P < 0.01), 62.66 ± 18.17% (t = 5.315, P < 0.01), 57.67 ± 13.61% (t = 9.110, P < 0.01) and 164 ± 22% (t = 9.238, P < 0.01), respectively. Triptolide 0.01 µg/L had little effect on IFN-γ-induced HLA-DR, ICAM-1 and CD40 amounts, as well as HA synthesis. When the concentration ranged from 0.1 µg/L to 10 µg/L, Triptolide inhibited IFN-γ-induced RFs activation in a dose-dependent manner. It was also found that Triptolide had the same inhibiting effects on IFN-γ-induced RFs and skin fibroblasts from patients with normal individual conditions.
Conclusions: Triptolide could inhibit IFN-γ-induced activation of RFs derived from patients with Graves’ ophthalmopathy.