Andrographolide attenuates skeletal muscle dystrophy in mdx mice and increases efficiency of cell therapy by reducing fibrosis

Abstract

Background: Duchenne muscular dystrophy (DMD) is characterized by the absence of the cytoskeletal protein dystrophin, muscle wasting, increased transforming growth factor type beta (TGF-β) signaling, and fibrosis. At the present time, the only clinically validated treatments for DMD are glucocorticoids. These drugs prolong muscle strength and ambulation of patients for a short term only and have severe adverse effects. Andrographolide, a bicyclic diterpenoid lactone, has traditionally been used for the treatment of colds, fever, laryngitis, and other infections with no or minimal side effects. We determined whether andrographolide treatment of mdx mice, an animal model for DMD, affects muscle damage, physiology, fibrosis, and efficiency of cell therapy.

Methods: mdx mice were treated with andrographolide for three months and skeletal muscle histology, creatine kinase activity, and permeability of muscle fibers were evaluated. Fibrosis and TGF-β signaling were evaluated by indirect immunofluorescence and Western blot analyses. Muscle strength was determined in isolated skeletal muscles and by a running test. Efficiency of cell therapy was determined by grafting isolated skeletal muscle satellite cells onto the tibialis anterior of mdx mice.

Results: mdx mice treated with andrographolide exhibited less severe muscular dystrophy than untreated dystrophic mice. They performed better in an exercise endurance test and had improved muscle strength in isolated muscles, reduced skeletal muscle impairment, diminished fibrosis and a significant reduction in TGF-β signaling. Moreover, andrographolide treatment of mdx mice improved grafting efficiency upon intramuscular injection of dystrophin-positive satellite cells.

Conclusions: These results suggest that andrographolide could be used to improve quality of life in individuals with DMD.

Keywords: Andrographolide; Cell therapy; DMD; Fibrosis; Skeletal muscle; mdx.

Figures

Figure 1

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%.

Figure 2

Andrographolide reduces skeletal muscle fibrosis in mdx mice. Fibrosis was augmented as explained in the legend of Figure 1. During this period, mice were treated with 1 mg/kg andrographolide or vehicle (intraperitoneal (ip) injections three times per week, six animals per group). Indirect immunofluorescence analysis of (A) collagen I (green) and (B) fibronectin (red) in cryosections of tibialis anterior (TA) muscles from wild type (WT), vehicle-treated mdx mice, and andrographolide-treated mdx mice. Nuclei are stained in blue (Hoechst). Bar corresponds to 200 and 50 μm for 100X and 400X magnification pictures respectively.

Figure 3

Andrographolide reduces the amount of fibrotic proteins in mdx skeletal muscle. Experiments were performed as explained in the legend of Figure 2. Muscle extracts were obtained under each experimental condition and fibrotic proteins were analyzed by western blotting. (A) Fibronectin (FN) and (B) collagen type III (Col III) from tibialis anterior (TA) muscles of wild type (WT), vehicle-treated mdx mice, and andrographolide-treated mdx mice. GAPDH protein levels are shown as loading control; molecular weight markers are shown in kDa. Values are expressed as mean ± SD of three independent experiments, using three mice for each experimental condition. (*P < 0.05 relative to WT mice; #P < 0.05 relative to vehicle-treated mdx mice).

Figure 4

Andrographolide decreases NF-κB activity in vivo. Fibrosis was augmented as explained in the legend of Figure 1. During this period, mice were treated with 1 mg/kg andrographolide or vehicle (intraperitoneal (ip) injections three times per week, six animals per group). The activity of NF-κB was evaluated in formalin-fixed muscle samples by Southwestern blot analysis. A specific DNA was used to detect active NF-κB by immunofluorescence (red). The nuclei were stained with Hoechst (blue). (A) The upper panel shows merged red (active NF-κB) and blue (total nuclei) signals. Bar corresponds to 50 μm. (B) Quantification of total nuclei per field (in 400X magnification pictures).

Figure 5

Andrographolide reduces expression of pro-fibrotic factors and collagen type I in mdx mice. Fibrosis was augmented as explained in the legend of Figure 1. During this period, mice were treated with 1 mg/kg andrographolide or vehicle (intraperitoneal (ip) injections three times per week, four animals per group). (A) Transforming growth factor type beta (TGF-β1), (B) Connective tissue growth factor (CTGF) and (C) collagen type I mRNA levels were determined in tibialis anterior (TA) muscle from wild type (WT), vehicle-treated mdx mice, and andrographolide-treated mdx mice by qPCR using GAPDH as a reference gene. Values correspond to the mean dCT value ± SD of three independent experiments, normalized to WT levels (*P < 0.05 relative to WT mice; #P < 0.05 relative to vehicle-treated mdx mice). The recovery scores for TGF-β1 and CTGF were 56.3% and 63.2 respectively.

Figure 6

Andrographolide reduces transforming growth factor type beta (TGF-β) signaling pathway activity in mdx mice. Indirect immunofluorescence analysis of (A) p-Smad-2 and (D) p-Smad-3 (intracellular TGF-β1 mediators) in cryosections of tibialis anterior (TA) muscle from vehicle-treated and andrographolide-treated mdx mice. Bar corresponds to 200 μm. (B,E) Ratio of p-Smad-2 and p-Smad-3 respectively versus total nuclei. (C,F) Quantification of total nuclei per field (in 400X magnification pictures). Values are expressed as mean ± SD of three independent experiments, using three mice for each experimental condition.

Figure 7

Andrographolide increases skeletal muscle strength in mdx mice. Fibrosis was augmented as explained in the legend of Figure 1. 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) Tibialis anterior (TA) muscles were isolated from wild type (WT), vehicle-treated mdx mice, and andrographolide-treated mdx mice to evaluate isometric specific force (mN/mm2) at different stimulation frequencies (pulses per second, pps). (B) Bar graph shows tetanic specific force. Values are represented as percentage of specific isometric force generated by WT muscle (*P < 0.05 relative to WT mice; #P < 0.05 relative to vehicle-treated mdx mice). The recovery score for the twitch force measurement was 54.2%. (C) Bar graph showing twitch force (*P < 0.05 relative to WT mice; #P < 0.05 relative to vehicle-treated mdx mice). The recovery score for the tetanus measurement was 50.3%. (D) Mice were subjected to an exercise challenge on the treadmill at 15 meters/minute for five minutes and the number of set-backs was counted (n = 7, *P < 0.05 relative to WT mice; #P < 0.05 relative to vehicle-treated mdx mice). The recovery score for this measurement was 45.5%.

Figure 8

Muscle stem cell therapy with satellite cells (SC) is improved by the reduction in muscle fibrosis following treatment with andrographolide. (A) Fibrosis was augmented as explained in the legend of Figure 1. During this period, mice were treated with andrographolide or vehicle (intraperitoneal (ip) injections three times per week, six animals per group). One week after the last drug administration (seven-month-old mice), 500 freshly isolated satellite cells purified from wild type (WT) mice were transplanted into both tibialis anterior (TA) muscles of each mdx mouse. At four weeks after engraftment, the number of fibers expressing dystrophin and collagen I was determined by immunofluorescence analysis of cryosections. The images are representative of two experimental groups with six mice per group. Bar corresponds to 200 μm. (B) Quantification of the data obtained in (A) showing the number of myofibers expressing dystrophin per TA muscle in each case. (C) The number of satellite cells (PAX-7-positive nuclei) was determined for isolated single muscle fibers from extensor digitorum longus muscle (EDL) of each case as an indicator of endogenous SC survival. (D) 500 WT SC freshly isolated as in (A), from the C57-EGFP mice were engrafted in mice treated as in (A) (six animals per group). Both TA muscles were dissected immediately after engraftment or after 2 or 15 days from two animals in each case. Total genomic DNA was purified. EGFP transgene present in the engrafted muscle was detected by qPCR. Values are expressed as mean ± SD of three independent experiments. (*P<0.05 relative to vehicle-treated mice, **P<0.01 relative to vehicle-treated mice at day 15).