Transcriptome analysis
RNA sequencing (RNA-seq) was performed using murine lungs from PBS group (n=5), Si group (n=6), High PFD group (n=7), High BIBF group (n=4) and Low COM group (n=5). The varying sample sizes in the RNA-seq among groups were attributed to underperforming sample extraction or sequencing library construction. mRNA library was generated by generating paired 150 bp long end reads using the Illumina Hiseq platform. HISAT2 software was used with default parameters (v.2.1. 0) to align reads to the reference genome (mouse: GRCm38) (Pang et al., 2021). Then samtools transformed the sam files to bam format. Read counts were calculated using featureCounts (version) with the annotation file (GTF file). Normalization and gene differential expression analysis were conducted by the R package DEseq2 (v.3.10). Differentially expressed genes (DEGs) were identified withP -values < 0.05 in PBS group compared to Si group, Si group compared to PFD group, BIBF group, and COM group, respectively. Heatmap of log-transformed gene counts was plotted for DEGs in each comparison using the R package pheatmap (v1.0.12). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using the R package clusterProfiler (v4.4.4) (Yu et al., 2012) withP -values < 0.05 as the significance threshold in up-regulated and down-regulated DEGs separately. Volcano plot, bar plot, venn diagram and multi-dot plot were plotted by bioinformatics (https://www.bioinformatics.com.cn, last accessed on 10 June 2023), an online platform for data analysis and visualization.