Drp1-mediated mitochondrial fission promotes pulmonary fibrosis progression through the regulation of lipid metabolic reprogramming by ROS/HIF-1α

Abstract

ObjectiveTo confirm the mechanism of dynamic-related protein 1 (Drp1)-mediated mitochondrial fission through ROS/HIF-1α-mediated regulation of lipid metabolic reprogramming in the progression of pulmonary fibrosis (PF). MethodsA mouse model of PF was established by intratracheal instillation of bleomycin (BLM) (2.5 mg/kg). A PF cell model was constructed by stimulating MRC-5 cells with TGF-β (10 ng/mL). Pathological changes in the lung tissue and related protein levels were observed via tissue staining. The indicators related to lipid oxidation were detected by a kit, and lipid production was confirmed through oil red O staining. Inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA). RT–qPCR, Western blotting and immunofluorescence staining were used to detect the expression of genes and proteins related to the disease. We used CCK-8 and EdU staining to confirm cell proliferation, flow cytometry was used to confirm apoptosis and ROS levels, α-SMA expression was detected by immunofluorescence staining, and mitochondria were observed by MitoTracker staining. ResultsThe BLM induced lung tissue structure and alveolar wall thickening in mice. Mitochondrial fission was observed in MRC-5 cells induced by TGF-β, which led to increased cell proliferation; decreased apoptosis; increased expression of collagen, α-SMA and Drp1; and increased lipid oxidation and inflammation. Treatment with the Drp1 inhibitor mdivi-1 or transfection with si-Drp1 attenuated the induction of BLM and TGF-β. For lipid metabolism, lipid droplets were formed in BLM-induced lung tissue and in TGF-β-induced cells, fatty acid oxidation genes and lipogenesis-related genes were upregulated, ROS levels in cells were increased, and the expression of HIF-1α was upregulated. Mdivi-1 treatment reversed TGF-β induction, while H2O2 treatment or OE-HIF-1α transfection reversed the effect of mdivi-1. ConclusionIn PF, inhibition of Drp1 can prevent mitochondrial fission in fibroblasts and regulate lipid metabolism reprogramming through ROS/HIF-1α; thus, fibroblast activation was inhibited, alleviating the progression of PF.