NAD + /NADH homeostasis, a key regulator of redox metabolism, is implicated in Achilles tendinopathy (AT) pathogenesis. Methylene blue (MB), a mitochondria-targeting antioxidant, holds potential for restoring NAD metabolism. The study aimed to investigate the role of NAD dysmetabolism in AT, evaluate the effect of MB in restoring NAD homeostasis and alleviating tendinopathy. Published single-cell RNA sequencing data were analysed, and human Achilles tendons were harvested for histological evaluation and ex vivo explant cultures. Rat experimental tendinopathy models and primary rat tenocytes were utilized. Key outcomes included quantification of NAD + /NADH, ATP, and reactive oxygen species (ROS); JC-1 assays for mitochondrial membrane potential. Immunohistochemical staining and western blot analysis used to evaluate expression of proteins (Col1, Col3, NDUFB8, AIF etc …). Mitochondrial respiratory chain complex I (MC1) activity and AIF expression were modulated using rotenone (a selective MC1 inhibitor) and siRNA-mediated knockdown, respectively. NAD homeostasis orchestrated metabolic and biosynthetic programming to counteract oxidative stress in tendinopathy. MB restored NAD homeostasis, enhanced ATP production, and reduced ROS accumulation, while improving collagen fiber organization. MC1 and AIF synergistically regulated NAD metabolism in AT. Rotenone (Rot) inhibited the vitality of MC1, as MB restored NAD homeostasis even under the treatment of Rot but failed to reverse oxidative stress, matrix degeneration, and reduced cell viability. In contrast, AIF deficiency induced MC1 dysfunction in AT, yet MB rescued NAD dysregulation, oxidative stress, and matrix degeneration under AIF knockdown conditions. We demonstrated that MB targets NAD metabolism to regulate AIF-driven mitochondrial repair, promoting MC1 biogenesis in both human and rat AT, thereby maintaining energy metabolism and redox homeostasis. Collectively, our work highlights the central role of NAD metabolism in AT pathogenesis and supports the potential of developing a therapeutic strategy targeting MB, AIF, and MC1. Importantly, the characterization of emergent functional and mechanistic properties of MB establishes a translational paradigm for old drug repurposing, with highlighting potential for convenient clinical translation.
Jiang et al. (Mon,) studied this question.