Enhanced muscle regeneration in MDX Duchenne muscular dystrophy-mice, proven by CD44 & MYH3, on consumption of A.Pullulans produced Neu REFIX ß-Glucan

Topic:

Pre-Clinical Research

Poster Number: Virtual

Author(s):

Samuel JK Abraham, MD, PhD, FRCP, CACR, University of Yamanashi, Chuo, Yamanashi, Japan & Sophy Inc., Nyodogawa, Kochi, Japan, Shuji Sakamoto, PhD, Laboratory of Molecular Biology, Science Research Center, Kochi University, Nankoku, Japan, Takuma Higuchi, PhD, Laboratory of Molecular Biology, Science Research Center, Kochi University, Nankoku, Japan, Subramaniam Srinivasan, MD, FACP, Nichi-In Centre for Regenerative Medicine (NCRM), Chennai, India, Vidyasagar Devaprasad Dedeepiya, MD, DNB, Nichi-In Centre for Regenerative Medicine (NCRM), Chennai, India, Nobunao Ikewaki, PhD, Dept. of Medical Life Science, Kyushu University of Health and Welfare, Nobeoka, Japan, Masaru Iwasaki, MD, PhD, CACR, University of Yamanashi, Chuo, Yamanashi, Japan, Mathaiyan Rajmohan, MSc, Nichi-In Centre for Regenerative Medicine (NCRM), Chennai, India, Rajappa Senthilkumar, PhD, GN Corporation Co Ltd, Kofu, Yamanashi, Japan, Senthilkumar Preethy, BDS, MSc, Nichi-In Centre for Regenerative Medicine (NCRM), Chennai, India

Background: Duchenne muscular dystrophy (DMD), an X-linked fatal myogenic disorder is characterized by continuous muscle degeneration and regeneration cycles resulting in extensive fibrosis and progressive muscle mass loss. Numerous innovative and experimental therapies focus on increasing dystrophin levels have been proposed, but clinical results have been unsatisfactory. This could be attributed to insufficient amelioration of inflammation, fibrosis, apart from limited muscle regeneration and maturation. After proving the anti-inflammatory, and anti-fibrotic effects of N-163 strain of Aureobasidium pullulans produced 1,3-1,6 ß-glucan (Neu REFIX) in clinical and pre-clinical studies, we have now evaluated its effects on muscle regeneration and maturation, through the expression of CD44 and Myosin heavy chain (MYH3) in a mdx mice model of DMD.
Methods: 45 mice were studied in the three groups, 15 per group; Gr.1 normal mice (control), Gr.2 mdx mice as vehicle, and Gr.3 mdx mice, which were orally administered the Neu-REFIX for 45 days (3 mg/kg dose). Immunohistochemistry analysis (IHC) for CD44 expression and immunofluorescence analysis of MYH3 expression were performed in the muscles obtained by biopsy post-intervention and the positive areas were evaluated using H-score and sketchandcalc software, respectively.
Results: Intensity of IHC staining of CD44 had a higher H-score of > 2.0 in the Neu-REFIX group compared to a score of 1.0 in Gr.2 and 0.1 in Gr.1. Immunofluorescence staining showed larger MYH3-staining positive area in N-163 group compared to that in the Gr.2, amounting to 20% higher muscle regeneration in Neu-REFIX group.
Conclusion: With Neu-REFIX ß-glucan, increase in MYH3 expression in skeletal muscle of MDX mice representing foetal myosin /early muscle development, is indicative of successful muscle regeneration. Increase in CD44, a marker for Hyaluronic acid (HA) with Neu-REFIX indicates maturation of the regenerated muscle. HA interacts with extra-cellular matrix constituents playing a crucial role in muscle development and maintenance which is hampered in DMD, has now been proven to be tackled by Neu-REFIX. The anti-inflammatory and anti-fibrotic potentials along with increased plasma dystrophin, having been proven earlier, it is worth considering this safe food supplement for validation in larger clinical studies as a disease modifying adjunct, in slowing down the progress of DMD.