Vitamin D attenuates the progression of pulmonary fibrosis via inhibiting thymidine kinase 1/PFKFB3-driven glycolysis

Pulmonary fibrosis is characterized by the excessive deposition of extracellular matrix produced from myofibroblasts in the lungs. Enhanced glycolysis has been linked to fibroblast-myofibroblast transition (FMT) during pulmonary fibrogenesis, however, there is still a lack of pharmaceutical agents to target the metabolic dysregulation. Emerging evidence highlights Vitamin D’s anti-fibrotic potential, yet its role in modulating fibroblast glycolysis and pulmonary fibrosis remains unclear. MRC-5 cells and mouse primary lung fibroblasts treated with TGF-β1 combined with Vitamin D were used to explore the role of vitamin D on fibroblast activation and glycolysis in vitro. Silica and bleomycin-induced pulmonary fibrosis mouse model was established to evaluate the antifibrotic effects of Vitamin D and the glycolysis inhibitor 3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one (3PO) in vivo. Cell metabolism assays were performed to determine the glycolytic rate. RNA sequencing was utilized to analyse the underlying molecular mechanisms by which Vitamin D affects fibroblast activation and glycolysis. Transcriptomic analysis and subsequent experiments demonstrated that TK1 acts as a functional downstream effector of Vitamin D, and its expression was negatively regulated by Vitamin D. Knockdown of TK1 inhibited the profibrotic effects of TGF-β1 by reducing fibroblast glycolysis. Furthermore, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key driver of glycolysis, was identified as a target of TK1. Mechanistically, Vitamin D could reduce the expression of TK1, thereby partly blocking fibroblast glycolysis and activation by inhibiting PFKFB3. In a murine model of silica or bleomycin-induced fibrosis, both Vitamin D and 3PO treatment alleviated pulmonary fibrosis. Our findings revealed that Vitamin D could attenuate pulmonary fibrosis by blocking fibroblast glycolysis and FMT through the TK1-PFKFB3 pathway. Vitamin D supplementation or targeting fibroblast glycolysis could be promising treatment strategies for pulmonary fibrosis.