RNA Sequencing Reveals Inflammatory Pathways Behind Ventilator-Induced Diaphragm Damage in Rats

Controlled mechanical ventilation can silently injure the very muscle that keeps patients breathing, and a new rat study now maps, at the level of thousands of genes, which inflammatory signaling pathways switch on inside the diaphragm as that damage develops. Using transcriptome RNA sequencing on diaphragm tissue from ventilated rats, researchers identified widespread gene dysregulation and found that three inflammation-linked pathways—MAPK, PI3K-Akt, and Calcium signaling—were significantly enriched at both six and twelve hours of mechanical ventilation, alongside measurable muscle fiber atrophy and inflammatory cell infiltration.

The study, published in BMC Molecular and Cell Biology, set out to identify the key genes and inflammatory signaling pathways involved in the pathogenesis of ventilator-induced diaphragmatic dysfunction, or VIDD, in rats, with the aim of flagging potential therapeutic targets. VIDD is a recognized complication in critically ill patients: current evidence suggests that more than half of critically ill patients undergoing controlled mechanical ventilation develop the condition within twenty-four hours of intubation. It is characterized by significant diaphragm atrophy and reduced contractile function, and it contributes to weaning failure, extended intensive care unit stays, increased mortality, and greater healthcare expenditures.

To build the model, the team used nine adult male Wistar rats, specific-pathogen-free grade and weighing 350 to 400 grams, randomly divided into three groups of three: a control group receiving no mechanical ventilation, a six-hour controlled mechanical ventilation group, and a twelve-hour group. All animals were anesthetized with intraperitoneal pentobarbital followed by continuous intravenous infusion, with body temperature held at 37 plus or minus 0.5 degrees Celsius. In the ventilated groups, tracheostomy was performed and the animals were connected to a small ventilator set at sixty breaths per minute, a tidal volume of five milliliters per kilogram, and positive end-expiratory pressure of eight centimeters of water. Control animals underwent tracheal exposure without ventilation and were maintained under anesthesia for roughly thirty minutes before euthanasia.

After the model was established, diaphragmatic tissues were collected for hematoxylin-eosin staining, immunohistochemical staining, and RNA extraction. The staining revealed clear morphological differences between groups. Control muscle fibers showed regular cross-sectional profiles, clear striations, tight arrangement, and peripheral nuclear positioning, while the ventilated groups exhibited irregular fiber contours, disordered striations, widened intercellular spacing, irregular nuclear distribution, and significant inflammatory cell infiltration. Quantitative analysis of myofiber cross-sectional area confirmed a time-dependent progression of atrophy: the mean cross-sectional area was significantly reduced in the six-hour group and further decreased in the twelve-hour group compared with controls.

Immunohistochemistry added a molecular dimension to the structural findings. The researchers detected expression of the slow and fast myosin heavy chain isoforms, MHCslow and MHCfast, and quantified the percentage of positive area per field of view. At six hours of ventilation there was no statistically significant difference in either isoform compared with controls, but after twelve hours the positive area percentage for both MHCslow and MHCfast was significantly reduced. The authors note that prior work links MHC expression to contractile performance: reduced MHC expression has been correlated with decreased specific diaphragm fiber force in mechanically ventilated intensive care patients, and upregulation of MHC expression has been accompanied by improved diaphragm contractility in rat VIDD models.

The transcriptomic core of the study produced striking numbers. Compared with the control group, the six-hour ventilation group yielded 2,048 differentially expressed genes, with 321 upregulated and 1,727 downregulated, while the twelve-hour group yielded 1,495 differentially expressed genes, with 534 upregulated and 961 downregulated, all at a significance threshold of P less than 0.05. Principal component analysis showed clear separation between control and ventilated samples, with samples clustering by experimental condition, and box plots confirmed comparable expression distributions between groups, supporting the suitability of the data for differential expression analysis.

Functional enrichment analysis then pointed the way toward inflammation. Gene Ontology analysis attributed 1,310 differentially expressed genes in the six-hour group to molecular functions, cellular components, and biological processes, and 1,017 in the twelve-hour group. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that among the top twenty significantly enriched pathways, both ventilated groups were commonly enriched in pathways associated with inflammatory responses, aldosterone synthesis and secretion, oxytocin signaling, extracellular matrix-receptor interactions, and insulin signaling. When the analysis focused specifically on inflammatory signaling, 534 differentially expressed genes in the six-hour group mapped to twenty inflammation-associated pathways and 341 genes in the twelve-hour group mapped to fifteen, with MAPK, PI3K-Akt, and Calcium signaling significantly enriched at both time points.

Within those pathways, the heatmaps revealed coordinated gene shifts. In the MAPK pathway, key genes including Il1b, Gadd45a, Pgf, Map3k6, and Myd88 were upregulated at both time points, while twenty-three key genes such as Cacnb1, Nr4a1, and Rps6ka2 were downregulated. In the PI3K-Akt pathway, Pgf, Csf3r, Il6, and Eif4ebp1 were upregulated while seventeen genes, including Nr4a1 and Ppp2r3a, were downregulated. In the Calcium signaling pathway, Adora2a was upregulated and fifteen genes, including Phkg1 and Calm2, were downregulated. The team then validated nine randomly selected key genes by quantitative reverse transcription PCR. Compared with controls, Il1b, Il6, and Myd88 mRNA levels significantly increased after six hours of ventilation, while Dusp10, Irs1, and Ntrk2 significantly decreased; after twelve hours, Il1b, Il6, Myd88, and Gadd45a were significantly increased, Dusp10, Irs1, and Ntrk2 remained significantly decreased, and Camk2a and Ppp3ca showed no significant changes at either time point, consistent with the sequencing results.

The discussion places these gene changes in a wider biological context. Mechanical ventilation is known to activate the TLR9-MyD88-NF-kappaB pathway in alveolar macrophages, and the observed Myd88 upregulation in the diaphragm suggests similar pro-inflammatory signaling may occur in diaphragmatic tissue. Preclinical evidence links IL-1beta and IL-6 in skeletal muscle inflammation: in human skeletal muscle cells, IL-1beta directly stimulates IL-6 secretion, and in an aged rat model, leptin-induced upregulation of Il1b and Il6 was associated with increased expression of the atrophy-related proteins MuRF1 and Atrogin-1. The concurrent Il1b and Il6 upregulation seen here raises the possibility that ventilation-induced inflammation may trigger IL-1beta-driven IL-6 production and potentially contribute to diaphragmatic atrophy. The downregulation of Irs1 is consistent with cancer cachexia research in which Irs1 ubiquitination and degradation promote muscle wasting, suggesting suppression of anabolic signaling may likewise contribute to diaphragmatic atrophy, while Dusp10, a known negative regulator of MAPK-mediated inflammatory signals, and Ntrk2, primarily involved in neurotrophic support, appear responsive to mechanical ventilation although their specific diaphragmatic roles remain to be elucidated.

The authors are explicit about the limits of the work. The sample size was modest at three rats per group, time-matched spontaneously breathing controls were not included, and direct measurements of diaphragmatic contractile function were not performed. The pathway activation status was derived from mRNA expression data rather than protein-level validation, and the findings are primarily correlative at this stage, since interventional experiments to establish causality were not conducted. Follow-up studies involving functional assessments, protein validation, and interventional designs are planned. Even with those caveats, the study offers a gene-level map of how controlled mechanical ventilation reshapes inflammatory signaling in the diaphragm within hours, and it singles out MAPK, PI3K-Akt, and Calcium signaling, together with genes such as Il1b, Il6, and Myd88, as candidates that warrant further investigation into their potential roles in ventilator-induced diaphragmatic dysfunction.

Subject of Research: Inflammation-linked gene expression changes in the diaphragm of mechanically ventilated rats

Article Title: Assessment of the role of inflammation-linked signaling pathways in ventilator-induced diaphragmatic dysfunction in rats by transcriptome RNA-seq

Article References: Chen, Y., Xu, R., Huang, Z., Zeng, L., Qiu, K., Gu, N., Tian, F., Shi, Q., & Chen, H. (2026). Assessment of the role of inflammation-linked signaling pathways in ventilator-induced diaphragmatic dysfunction in rats by transcriptome RNA-seq. BMC Molecular and Cell Biology, 27(1), Article 37. https://doi.org/10.1186/s12860-026-00599-3

Image Credits: AI Generated

DOI: 10.1186/s12860-026-00599-3

Keywords: ventilator-induced diaphragmatic dysfunction, mechanical ventilation, RNA-seq, diaphragm atrophy, MAPK signaling, PI3K-Akt signaling, calcium signaling, inflammation, rat model, differentially expressed genes, RT-qPCR, critical care