Assessment of the role of inflammation-linked signaling pathways in ventilator-induced diaphragmatic dysfunction in...

To investigate the key genes and inflammatory signaling pathways involved in the pathogenesis of ventilator-induced diaphragmatic dysfunction (VIDD) in rats, with the aim of identifying potential therapeutic targets.

Adult male Wistar rats were randomly assigned to a control (0 h) group, a 6-hour controlled mechanical ventilation (CMV 6 h) group, and a 12-hour controlled mechanical ventilation (CMV 12 h) group, with 3 rats in each group. After model establishment, diaphragmatic tissues were collected for hematoxylin-eosin (HE) staining, immunohistochemical staining, and RNA extraction. HE staining was used to assess pathological changes and quantify myofiber cross-sectional area (CSA); immunohistochemistry was employed to detect the expression of slow (MHCslow) and fast (MHCfast) myosin heavy chain isoforms and quantify the percentage of positive area per field of view; and transcriptome sequencing (RNA-Seq) was utilized to analyze mRNA expression changes across groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to determine the biological functions and pathways associated with significant differentially expressed genes (DEGs).

HE staining revealed diaphragmatic muscle fiber atrophy in both the CMV 6 h and 12 h groups, accompanied by varying degrees of inflammatory cell infiltration. Quantitative analysis showed that myofiber CSA was significantly reduced in the CMV 6 h group (P < 0.05) and further reduced in the CMV 12 h group (P < 0.01) compared with the control group.Immunohistochemical analysis showed no statistically significant difference in MHCslow and MHCfast expression in the CMV 6 h group compared to the control group (P > 0.05), whereas the percentage of positive area for both MHCslow and MHCfast was significantly reduced in the CMV 12 h group (P < 0.05). RNA-Seq identified 2,048 DEGs in the CMV 6 h group (321 upregulated and 1,727 downregulated) (P < 0.05) and 1,495 DEGs in the CMV 12 h group (534 upregulated and 961 downregulated) (P < 0.05). GO analysis revealed that the CMV 6 h group comprised 1,310 DEGs related to molecular functions (n = 262), cellular components (n = 179), and biological processes (n = 869) (P < 0.05). The CMV 12 h group comprised 1,017 DEGs related to molecular functions (n = 185), cellular components (n = 149), and biological processes (n = 683) (P < 0.05). KEGG pathway analysis showed that the top 20 significantly enriched pathways in the CMV 6 h and 12 h groups included inflammatory responses, aldosterone synthesis and secretion, oxytocin signaling pathways, ECM-receptor interaction, and insulin signaling pathways (P < 0.05). The most significantly enriched pathways known to play important roles in inflammatory responses included MAPK, PI3K-Akt, and Calcium signaling pathways, with key genes in these pathways screened and validated using RT-qPCR.

MAPK, PI3K-Akt, and Calcium signaling pathways, along with their associated genes, are associated with diaphragmatic structural damage and inflammatory responses in VIDD in rats, warranting further investigation into their potential roles in dysfunction.