As a common inhalable hazardous particle, silica is also a versatile industrial material with broad applications ranging from food and cosmetics to scientific research (Xie et al., 2024, Shoul et al., 2022). The ubiquitous use of silica in industries has led to pervasive exposure to silica particles across multiple environments. Silicosis results from chronic inhalation of respirable crystalline silica dust, pathologically characterized by diffuse pulmonary fibrosis and silicotic nodule formation. This progressive condition ultimately leads to respiratory dysfunction and may culminate in fatal respiratory failure (Li et al., 2018, Sun et al., 2019). Currently, the molecular mechanisms underlying silicosis have not been fully elucidated, and require further investigation.

Inhalation of silica particles affects a variety of cell types, including macrophages, dendritic cells, neutrophils, fibroblasts, and epithelial cells (Gilberti et al., 2008, Kang and Lim, 2012, Ou et al., 2023, Refsnes et al., 2021, Satpathy et al., 2015). As one of the main effector cells of silicosis, macrophages play important roles in the development of pulmonary inflammation and fibrosis. Upon inhalation of silica particles, alveolar macrophages engulf the silica to form phagocytic vesicles. These vesicles subsequently merge with lysosomes, leading to lysosomal damage. This process activates the NLRP3 inflammasome and triggers IL - 1 - dependent inflammation, ultimately resulting in fibrosis (Hamilton et al., 2008, Barnes et al., 2019).

Cholesterol 25-hydroxylase (CH25H) is an endoplasmic reticulum-resident, multi-pass membrane enzyme that catalyzes the hydroxylation of cholesterol to produce 25-hydroxycholesterol (25-HC), thereby contributing to cellular cholesterol homeostasis. Ch25h is reported to involve in periodontal inflammation (Tran et al., 2023), brain inflammation (Jang et al., 2016), vascular inflammation (Canfran-Duque et al., 2023) and adipose tissue inflammation (Russo et al., 2020). Madenspacher, et al (Madenspacher et al., 2023). showed that Ch25h knockout significantly decreases inflammatory level in mice with lipopolysaccharide (LPS) -induced acute lung injury. Ch25h deficiency alleviates TLR3 agonist-induced macrophage inflammation and LPS-induced microglial inflammation (Gold et al., 2014, Wong et al., 2020). Besides, both Ch25h and its downstream product 25-hydroxycholesterol have been reported to promote the activation of NLRP3 inflammasome (Jang et al., 2016, Wong et al., 2020). In sodium dextran sulfate-induced colitis models, deficiency of Ch25h mitigates the extent of intestinal fibrosis (Raselli et al., 2019). However, its impact on pulmonary fibrosis in silicosis models remains unexplored, highlighting the need for further investigation in this area.

In this study, we performed mRNA sequencing to characterize the pulmonary transcriptomic alterations in a murine silicosis model. To investigate the functional role of Ch25h, lentivirus-mediated gene silencing was implemented in vivo. Systematic histopathological analyses were conducted using hematoxylin-eosin (H&E) staining for tissue morphology, Masson's trichrome for collagen deposition, coupled with immunohistochemistry (IHC) and immunofluorescence for protein localization. Inflammatory cytokine profiles were quantitatively assessed by enzyme-linked immunosorbent assay (ELISA). Our findings establish CH25H as a critical regulator of silicosis pathogenesis and elucidate its mechanistic contributions to disease progression.