Andrographolide reduces skeletal muscle damage in mdx mice. To augment the extent of muscle fibrosis, three-month-old mdx mice were subjected to an exercise protocol for three months [6, 25]. During this period, mice were treated with 1 mg/kg andrographolide or vehicle (intraperitoneal (ip) injections three times per week, six animals per group). (A) H&E staining of tibialis anterior (TA) muscles showed a striking reduction in the damaged areas of muscle in andrographolide-treated mdx mice compared with untreated mdx mice. Upper panel shows 100X magnification pictures (scale bars = 200 μm), while the bottom panel shows 400X magnification pictures (scale bars = 50 μm). (B) Evans blue dye uptake in TA muscle fibers from wild type (WT), vehicle-treated mdx mice, and andrographolide-treated mdx mice. Nuclei were labeled with Hoechst. Mice were injected ip with 1% Evans blue dye 24 hours before muscle fixation (scale bar = 200 μm). (C) Serum creatine kinase (CK) was measured to evaluate skeletal muscle damage. The bar graph shows a significant reduction in serum CK activity in andrographolide-treated mdx mice compared with vehicle-treated mdx mice. Values are expressed as mean ± SD of three independent experiments, using five mice for each experimental condition. (*P < 0.05 relative to WT mice; #P < 0.05 relative to vehicle-treated mdx mice). The recovery score was 49.5%.