PG490-88, a derivative of triptolide, blocks bleomycin-induced lung fibrosis

Abstract

In this study we evaluate the antifibrotic properties of PG-490-88, a water-soluble derivative of triptolide. Triptolide is an oxygenated diterpene that is derived from a traditional Chinese herb that has potent immunosuppressive and antitumor activity. We used the intratracheal bleomycin mouse model and found that PG490-88 inhibits fibrosis in the bleomycin group when given the same day or 5 days after bleomycin. PG490-88 also markedly reduced the number of myofibroblasts in the bleomycin treatment group. An enzyme-linked immunosorbent assay of transforming growth factor (TGF)-beta in the bronchoalveolar lavage fluid showed a significant decrease in TGF-beta in the PG490-88-treated groups compared to the bleomycin-treated group. Additionally, triptolide blocked bleomycin-induced increase in TGF-beta mRNA in cultured normal human lung fibroblasts. The efficacy of PG490-88 when administered late after bleomycin installation suggests a potential role in the treatment of idiopathic pulmonary fibrosis.

Figure 1.

A: H&E stain: ×100. a: Saline control, normal lung architecture. b: Saline plus PG490-88, normal lung architecture. c: Bleomycin alone, extensive inflammation and fibrosis. d: Bleomycin plus PG490-88 (same day), patchy areas of inflammation with minimal fibrosis. e: Bleomycin plus PG490-88 (5 days later), patchy areas of inflammation with minimal fibrosis. B: Comparable sections of mouse lung to Figure 1 ▶ stained with trichrome: saline control: normal lung architecture (a); saline plus PG490-88, normal lung architecture (b); bleomycin alone, extensive areas of collagen (stained blue) (c); bleomycin plus PG490-88 (same day), minimal collagen (d); bleomycin plus PG490-88 (5 days later), minimal collagen (e).

Figure 2.

Alpha smooth-muscle actin stain of mouse lung sections (original magnification, ×100) that detects myofibroblasts (blue arrow) and airway smooth muscle cells that are a positive control (yellow arrow). Left: Bleomycin alone, extensive accumulation of α-smooth muscle actin-positive myofibroblasts. Center: Bleomycin plus PG490-88 (same day), small number of myofibroblasts. Right: Bleomycin plus PG490-88 (5 days later), scattered areas of myofibroblasts. Consecutive sections of mouse lung tissue stained with H&E are shown for comparison.

Figure 3.

Total hydroxyproline content ± SE in μg/lung. In the bleomycin plus PG490-88 (5 days) group, PG490-88 was administered 5 days after bleomycin. Four independent experiments were used for the statistical analysis that is outlined in Materials and Methods. *, P < 0.01 compared to bleomycin alone.

Figure 4.

BALF levels of active TGF-β (pg/ml) ± SE measured by ELISA. *, P < 0.01 compared to bleomycin alone.

Figure 5.

A: RT-PCR of TGF-β mRNA in NHLFs stimulated for 24 hours with 0.1 μg/ml or 10 μg/ml of bleomycin in the presence or absence of PG490 (20 ng/ml). GAPDH is used as a loading control. B: The ratio of TGF-β/GAPDH mRNA is shown.

Figure 6.

Molecular structure of PG-490-88 and triptolide. PG490-88 is a succinate salt-water soluble derivative of triptolide that is converted to triptolide in the serum.