Macrophage accumulations on the injured pulmonary surface accelerate intrathoracic adhesions

We established a novel mouse model of intrathoracic adhesions (IA) to investigate the molecular mechanisms underlying IA formation. RNA sequencing analysis was performed to explore these mechanisms. A 200 μL talc solution was administered into the left thoracic cavity, and the mice were sacrificed at 30 minutes, 3 days, and 9 days post-administration. IA tissues, including the left lung parenchyma and chest wall, were harvested and homogenized using QIAzol lysis reagent. Total RNA was extracted, and the FASTQ file data were trimmed and mapped using the GLC Genomics Workbench. Differential gene expression and enrichment analyses were performed using RNAseq Chef version 1.1.2. According to the bulk RNA analysis, we observed a significant upregulation of genes associated with inflammatory cytokines and macrophage surface markers, including Il6, Ccl2, Il1β, Tgfβ1, Cd163, Cd68, and Cd86, at day 3. Meanwhile, genes related to the extracellular matrix (ECM) and its production processes were significantly upregulated at day 9. Cnet plot showed that the TNF-α and NF-κB signaling pathways were activated during the early phase, while epithelial-mesenchymal transition occurred during the completion phase of IA formation. Ingenuity Pathway Analysis (IPA) on day 3 revealed activation of leukocyte trafficking, maturation, and emigration, along with agranulocyte adhesion and diapedesis.