Lung decellularized matrix-derived 3D spheroids: Exploring silicosis through the impact of the Nrf2/Bax pathway on myofibroblast dynamics.

Silicosis is an occupational respiratory disease caused by long-term inhalation of high concentrations of free silica particles. Studies suggest that oxidative stress is a crucial initiator of silicosis fibrosis, and previous studies have linked the antioxidative stress transcription factor known as Nrf2 to fibrosis antagonism. Myofibroblasts play a pivotal role in tissue damage repair due to oxidative stress. Unlike physiological repair, myofibroblasts in fibrosis exhibit an apoptosis-resistant phenotype, continuously synthesising and secreting significant amounts of collagen and other extracellular matrices, which could be a direct cause of silicosis fibrosis. However, the relationship and mechanism of action between oxidative stress and myofibroblast apoptosis resistance remain unclear. In this study, a new 3D cell culture model using mice lung decellularised matrix particles and fibroblasts was developed, simulating the changes in myofibroblasts during the development of silicotic nodules. Western Blot results indicate that silica stimulation leads to increased collagen deposition and decreased apoptosis-related protein Bax and oxidative stress-related protein Nrf2 in the 3D spheroid model. Immunofluorescence experiments reveal co-localisation in their expression. In Nrf2 overexpressing spheroids, Bax exhibits significant upregulation. In the Nrf2 knockout spheroids, Bax is also significantly downregulated; after intervention with Bax inhibitors, a significant downregulation of Bax-induced apoptosis was also detected in the Nrf2-overexpressed spheroids. In contrast, Bax-induced apoptosis showed a significant upregulation trend in Nrf2-overexpressed spheroids after intervention with Bax agonists. The results demonstrate that the spheroid model can mimic the development process of silicotic nodules, and silica stimulation leads to an apoptosis-resistant phenotype in myofibroblasts in the model, acting through the Nrf2/Bax pathway. This research establishes a new methodology for silicosis study, identifies therapeutic targets for silicosis, and opens new avenues for studying the mechanisms of silicosis fibrosis.