Investigation Of Therapeutic Molecular Compounds In Duchenne Muscular Dystrophy

Duchenne Muscular Dystropy(DMD)is a lethal X chromosome-linked inhereditary neuromuscular disorder,caused by frame-disrupting mutations in the DMD gene,which result in the absence of functional dystrophin protein.There is no effective treatment available in the clinic.The typical clinical features of DMD patiens are progressive body-wide muscle-wasting with most of patients die of cardiomyopathy and respiratory failure at later stages.In the present study,we aim to screen a library consisting of clinically applicable and food-grade small compounds.Through intramuscular and systemic evaluation of these compounds in DMD mouse models(mdx),we wish to identify candidate compounds which can promote muscle satellite cell proliferation,muscle regeneration and improve muscle pathologies and reduce inflammation.Thus,we wish to provide a candidate therapeutic compound for DMD treatment.To screen compounds for promoting cell proliferation,murine C2C12 myoblast was used as an in vitro model and cell proliferation was measured with cell counting.Mdx mice were used as in vivo models for intramuscular and systemic screening.Immunostaining,collagen staining and histopatholgical staining were used to measure the effect of candidate compounds on muscle regeneration and pathologies in mdx mice.Two different delivery routes including oral and intravenous injection were tested to optimize the administration route.Immunostaining and H&E staining were used to measure the change in muscle regeneration and pathologies.To determine the long-term beneficial effect of candidate G1 on muscle functions,regeneration,we administered G1 into adult mdx mice for 3 months intravenously.Grip strength,running wheels,body-weight change,serum biochemical parameters and immunostaining were used to determine the effect of repeated administrations of G1 on muscle function,regeneration and pathologies.To verify the involvement of actively proliferating satellite cells in G1-treated samples,cardiotoxin-injured C57BL6 mice and neonatal mdx mice were treated with G1 and assayed by immunostaining,histopathological staining and flow cytometry.To elucidate the mechanism underpinning the functionality of G1,Western blotting and quantitative RT-PCR were used to measure the phosphorylation and expression of mTORC1 signal pathway and related genes.Also fluorescence confocal microscopy was used to visualize the localization of mTORC1 in lysosomes.In vitro screening results demonstrated that G1 significantly enhanced cell proliferation compared to other compounds.The number of cells rose to 26000 from 8000 at 24 hr after treatment.Local intramuscular results in mdx mice indicated that the number of embryonic myosin heavy chain(eMyHC)was significantly increased up to 326 and showed significant increases compared to G2,G3 and G5,suggesting that G1 can promote muscle regeneration.5% G1 showed comparable activity to 10% and outperformed 2.5% in promting muscle regeration,suggesting 5% is the dose required for the optimal effect.Systemic evaluation of G1 in adult mdx mice revealed that G1 significantly elicited muscle regeneration in peripheral muscle with gastrocnemius and triceps showing the best effects,compared to other compounds.Also G1 could significantly reduce fibrosis in peripheral muscle with triceps showing significantly reduced fibrotic area(5%),compared to the saline group.Evaluation of different administration routes revealed that intravenous injection induced more beneficial effects than dietary supplementation.Long-term evaluation of G1 in mdx mice induced functional and pathological improvemens demonstrated by significantly increased grip strength and rounds of running wheels and significantly reduced fibrotic areas.Importantly,the number of satellite cells was significantly increased in skeletal muscle from G1-treated mdx mice compared to the saline group with the protein level of Pax7 reaching 1.6-fold higher than the saline group.G1 also promoted the proliferation of satellite cells in skeletal muscles from cardiotoxin-injued C57BL6 mice and neonatal mdx mice.Examination on the phosphorylation of mTORC1 and mTORC1-related downstream signal proteins such as p70S6 k and 4E-BP1 indicated that increased levels of phosphorylation of mTORC1,p70S6 k and 4E-BP1 in G1-treated samples compared to the saline group,with 4E-BP1 showing the most significant effect.Also the mRNA expression of cell cycle-related genes such as cyclin D1 was significantly up-regulated in muscle from G1-treated mdx mice compared the saline group.Muscle regeneration was significantly compromised in muscle from mdx mice treated with mTORC1 inhibitor.The translocation of activated mTORC1 from cytoplasma to lysosome was observed in muscle cells in the presence of G1.Altogether,these data demonstrated that G1 can activate mTORC1 signal pathway and thus trigger the prolifertation of satellite cells and muscle regeneration.In summary,we identified a food-grade small compound showing the capability of promoting satellite cell proliferation and muscle regeneration in vitro and in mdx mice.Long-term evaluation of G1 in mdx mice demonstrated that repeated administration of G1 can significantly improve functions and pathologies of mdx mice without any detectable toxicity.The mechanistic study elucidated that G1 promote satellite cell proliferation and muscle regeneration by triggering mTORC1 signalling pathway.This study provides a candidate compound for the supportive treatment of DMD and also shed light on the role of G1 as a signaling molecule for mTORC1 pathway.