Micro-CT dynamic changes and molecular mechanisms in a pulmonary nodule mouse model induced by Benzo[a]pyrene and lipopolysaccharide

Background: Pulmonary nodules are clinically critical due to their high prevalence and association with early lung cancer. A mouse model is ideal for pulmonary nodule research. However, existing models perform poorly in simulating the slow occurrence and progression of nodules in humans. Establishing a stable, reproducible, and clinically relevant mouse model is essential for elucidating molecular mechanisms and providing a preclinical platform, motivating this study. This study aimed to establish a mouse model of pulmonary nodules induced by Benzo[a]pyrene (B[a]P) and lipopolysaccharide (LPS), and to clarify their dynamic changes using Microcomputed Tomography and molecular mechanisms. Methods: C57BL/6J mice with equal numbers of males and females were instilled intratracheally with B[a]P once a week for 4 times, followed by intratracheal instillation of LPS once every 2 weeks for 5 times. The primary outcomes of this study were the evaluation of the incidence, number, and diameter of nodules by Microcomputed Tomography and the classification of nodule types through hematoxylin and eosin (H&E) staining. Secondary outcomes included the evaluation of nodules on the surface of the lungs. Then, the gene expression in lung nodules was detected using RNA sequencing, and the core genes of inflammatory nodule progression and malignant transformation were validated. Results: At week 20, the incidence, number, and diameter of nodules were 25%, 1.00±0.00, and 0.65±0.09 mm, respectively. Histopathological analyses indicated that these nodules were inflammatory. By week 28, the incidence, number, and diameter of nodules had increased to 41%, 1.45±0.28, and 0.98±0.12 mm, respectively. Histopathological results showed that 16.7% of nodules were classified as adenocarcinoma in situ (AIS), and the remaining were inflammatory. At week 36, the incidence, number, and diameter of nodules further increased to 70%, 1.71±0.36, and 1.07±0.14 mm, respectively. Histopathological results showed the proportion of AIS further increased to 30.8% among nodules. Overall, the incidence, number, and diameter of nodules significantly increased over time, and the proportion of AIS among nodules also increased. The RNA sequencing results showed that nuclear factor-kappa B was persistently expressed throughout nodules progression, promoting inflammatory nodules to AIS, validated by real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). Furthermore, enzyme-linked immunosorbent assay (ELISA) results confirmed that persistent secretion of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) establishes a pro-inflammatory microenvironment, promoting inflammatory nodule formation and malignant transformation to AIS. Conclusions: B[a]P and LPS could induce the formation of inflammatory nodules, and individual nodules gradually progress to AIS. Nuclear factor-kappa B persistently expressed throughout nodules progression may play a role in this process.