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Antioxidant effects of bis-indole alkaloid indigo and related signaling pathways in the experimental model of Duchenne muscular dystrophy

Abstract

Indigo is a bis-indolic alkaloid that has antioxidant and anti-inflammatory effects reported in literature and is a promissory compound for treating chronic inflammatory diseases. This fact prompted to investigate the effects of this alkaloid in the experimental model of Duchenne muscular dystrophy. The main aim of this study was to evaluate the potential role of the indigo on oxidative stress and related signaling pathways in primary skeletal muscle cell cultures and in the diaphragm muscle from mdx mice. The MTT and Neutral Red assays showed no indigo dose-dependent toxicities in mdx muscle cells at concentrations analyzed (3.12, 6.25, 12.50, and 25.00 μg/mL). Antioxidant effect of indigo, in mdx muscle cells and diaphragm muscle, was demonstrated by reduction in 4-HNE content, H2O2 levels, DHE reaction, and lipofuscin granules. A significant decrease in the inflammatory process was identified by a reduction on TNF and NF-κB levels, on inflammatory area, and on macrophage infiltration in the dystrophic sample, after indigo treatment. Upregulation of PGC-1α and SIRT1 in dystrophic muscle cells treated with indigo was also observed. These results suggest the potential of indigo as a therapeutic agent for muscular dystrophy, through their action anti-inflammatory, antioxidant, and modulator of SIRT1/PGC-1α pathway.

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Funding

The authors received financial support from the Coordenação de Pessoal de Nivel Superior Brasil (CAPES) – Finance Code 001, the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process: 15/03726-8; 13/17732-4; 20/09733-4; 21/09693-5), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; process #311166/2016-4; 427859/2018-2) and the FAEPEX. D.S.M, G.L.R. and C.C.L were the recipients of a CAPES fellowship. C.C. was the recipient of a CNPq fellowship. H.N.M.S. and M.J.S. are the recipient of a CNPq fellowship.

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Contributions

E.M. and M.J.S. conceived and designed the experiments. E.M. coordinated the research. D.S.M. performed the experiments and G.L.R., H.N.M.S., C.C., C.C.L., and E.C.L.P. assisted with the experiments. E.M., D.S.M, and M.J.S. participated in drafting the article. All authors read and approved of the version to be submitted.

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Correspondence to Elaine Minatel.

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The authors declare no competing interests.

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Supplementary information

Fig. 1S

Targets of indigo treatment in mdx muscle cells and diaphragm muscle. Indigo up-regulated; Indigo down-regulated. (PNG 51 kb)

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Fig. 2S

Indigo chemical structure and experimental design of indigo treatment in vitro, groups and analyses. (PNG 29 kb)

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Fig. 3S

Experimental design of indigo treatment in vivo, groups and analyses. (PNG 17 kb)

High resolution image (TIF 51 kb)

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Mizobuti, D.S., da Rocha, G.L., da Silva, H.N.M. et al. Antioxidant effects of bis-indole alkaloid indigo and related signaling pathways in the experimental model of Duchenne muscular dystrophy. Cell Stress and Chaperones 27, 417–429 (2022). https://doi.org/10.1007/s12192-022-01282-0

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  • DOI: https://doi.org/10.1007/s12192-022-01282-0

Keywords

  • Indigo
  • Oxidative stress
  • Inflammatory process
  • Activators mitochondrial biogenesis
  • mdx mice