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

Abstract

Objective

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.

Methods

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).

Results

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.

Conclusion

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.

Introduction

The diaphragm, functioning as the primary respiratory muscle, is essential for maintaining continuous respiration and pulmonary function throughout life [1]. Current evidence suggests that more than 50% of critically ill patients undergoing controlled mechanical ventilation (CMV) develop ventilator-induced diaphragmatic dysfunction (VIDD) within 24 h of intubation [2]. As recently reviewed, VIDD is characterized by significant diaphragm atrophy and reduced contractile function, which may be associated with impaired diaphragmatic nerve activation [2, 3]. VIDD contributes to weaning failure, extended ICU hospitalization, increased mortality, and greater healthcare expenditures. Current prevention and treatment strategies for VIDD remain limited and include pharmacological approaches, inspiratory muscle training, diaphragmatic nerve stimulation, and minimizing ventilator support. Therefore, there is an urgent need to identify more effective methods to prevent VIDD in patients on mechanical ventilation(MV).

The pathogenesis of VIDD mainly involves the following aspects: changes in diaphragm muscle fiber structure, increased oxidative stress, mitochondrial damage, abnormalities in protein and lipid metabolism, autophagy, remodeling of the sarcoplasmic reticulum RyR1, etc [4, 5]. and may involve the activation of multiple signaling pathways. Multi-omics analyses have demonstrated that long-term CMV-induced diaphragmatic dysfunction is closely associated with CMV-induced systemic cytokine storm and excessive inflammation [6]. However, the specific inflammatory signaling pathways involved in the local diaphragmatic tissue of VIDD have not yet been fully elucidated.

RNA sequencing (RNA-Seq), a branch of next-generation sequencing, provides comprehensive gene expression information at the mRNA level and enhances the understanding of phenotypic variations and gene regulatory mechanisms by identifying differentially expressed genes (DEGs). Functional enrichment analysis can reveal molecular alterations and involved pathways in diseases [7]. To our knowledge, this study is the first to investigate the inflammatory molecular mechanisms of VIDD using the diaphragm mRNA transcriptome, offering a novel perspective for future research on VIDD pathophysiology.

In this study, we utilized RNA-seq combined with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses to investigate inflammation-linked signaling pathways and identify key genes in the diaphragms of a rat model of VIDD.

Materials and methods

Research animal

Adult male Wistar rats (specific-pathogen-free (SPF) grade; body weight, 350–400 g; production license: SCXK (Shanghai) 2018-0006) were obtained from Shanghai Bicaikeyi Biotechnology Co., Ltd. and housed in an SPF-grade animal facility (temperature approximately 25 °C, relative humidity 50%-70%). This study was conducted in strict accordance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH) and was approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (Approval No: IACUC-1905030). The study is reported in compliance with the ARRIVE guidelines.

Main reagents and equipment

Sodium pentobarbital (P3761) was purchased from Sigma, USA. Paraformaldehyde (P0099) was obtained from Shanghai Biotek Biotechnology Co., Ltd. MHCfast antibody (GB112130-100), MHCslow antibody (GB112131-100), HRP-labeled goat anti-rabbit IgG (H + L) (GB23303), and 3,3’-diaminobenzidine (G1212-200T) were procured from Wuhan Sivol Biotechnology Co., Ltd. TRIzol reagent (12183555) was sourced from Invitrogen, USA. High-Capacity cDNA reverse transcription kit (4368814) was purchased from Thermo Fisher Scientific, USA. A small ventilator (Model 665 A) was supplied by Harvard Apparatus, USA.

VIDD model

Nine adult male Wistar rats were randomly divided into three groups: CMV 0 h group, CMV 6 h group, and CMV 12 h group (n = 3). All animals received anaesthesia induction with intraperitoneal pentobarbital sodium (40 mg/kg) and underwent cervical incision to expose the trachea. For all groups, a jugular vein catheter was placed for continuous intravenous anaesthesia infusion (pentobarbital sodium, 10 mg/kg/h), and body temperature was maintained at 37 ± 0.5 °C using a heating pad throughout the experiment. In the CMV 6 h and CMV 12 h groups, tracheostomy was performed, and the animals were connected to a small ventilator (60 breaths/min, tidal volume 5 ml/kg, positive end-expiratory pressure 8 cmH2O) for 6–12 h, respectively. In the CMV 0 h group, tracheostomy and mechanical ventilation were omitted; after tracheal exposure, the cervical incision was sutured. The CMV 0 h group was maintained under continuous anaesthesia and instrumentation for approximately 30 min before euthanasia. At the end of the respective period, all animals were euthanized by intraperitoneal injection of sodium pentobarbital (150 mg/kg), strictly following the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals: 2020 Edition. Left diaphragm specimens were fixed in 4% paraformaldehyde, while right diaphragm specimens were rapidly frozen in liquid nitrogen and stored at − 80 °C.

Hematoxylin-eosin (HE) staining

Diaphragm tissues fixed in paraformaldehyde were subjected to gradient alcohol dehydration, transparency with a fixative, paraffin embedding in 60 °C paraffin, and sectioning. The sections were incubated in a 40 °C water bath, dried in a 60 °C oven for 3 min, then dewaxed, rehydrated, and HE-stained. After air-drying and neutral gum sealing, pathological changes in the diaphragm were observed. For quantitative assessment of myofiber atrophy, cross-sectional area (CSA) was measured using ImageJ software.

Immunohistochemical staining

The diaphragm tissue samples were fixed in paraformaldehyde and embedded in paraffin, followed by incubation with 1% BSA to block non-specific binding sites. The samples were incubated overnight at 4 °C with MHCfast and MHCslow primary antibodies (1:500), followed by incubation at room temperature for 1 h with horseradish peroxidase-labeled secondary antibodies (HRP-labeled goat anti-rabbit IgG (H + L), 1:500). Finally, the sections were counterstained with hematoxylin and incubated for 10 min with 3,3’-diaminobenzidine for color development.The areas positive for MHCfast and MHCslow were quantified by microscopic scanning and measured using ImageJ software.

Total RNA extraction, cDNA library construction, and transcriptome sequencing

Total RNA was isolated and purified from diaphragm tissue samples using TRIzol reagent. The quality of total RNA was assessed using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent, CA, USA). A RIN value > 7.0 was set as the criterion for inclusion in subsequent experiments. cDNA libraries were constructed by Hangzhou Lianchuan Biotechnology Co., Ltd., and sequencing was performed on the Illumina NovaSeq 6000 platform. Raw RNA-Seq data were exported in fastq format.

Differential gene expression screening

The raw sequencing data were processed to remove adapters, low-quality sequences, and duplicates to obtain clean data. The data were then aligned to the reference genome using HISAT2, followed by preliminary gene/transcript assembly with StringTie. Final assembly annotations were generated by comparing transcripts with reference annotations using gffcompare. FPKM quantification was performed with the ballgown package, and significant inter-sample differences were analyzed using the edgeR package in R (thresholds set at FC > 2-fold or FC < 0.5-fold with P-value < 0.05).

Functional enrichment analysis

Functional enrichment analysis of DEGs was performed using the GO database for biological processes, cellular components, and molecular functions, and the KEGG database for pathway annotation. Terms with a P-value < 0.05 and false discovery rate (FDR) < 0.05 were considered statistically significant.

qRT-PCR

Total RNA was extracted from the diaphragm according to the TRIzol protocol, and reverse transcription was performed to generate cDNA using a reverse transcription kit. Real-time PCR was conducted with cDNA as the template. The reaction conditions were: 95 °C pre-denaturation for 2 min, followed by 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 20 s, for a total of 40 cycles. GAPDH was used as the internal reference, and data were calculated using the 2⁻ΔΔCt method.

The sequences of the primers (rat-derived) are as follows:

Il1b, forward:5’-GGGCCTCAAGGGGAAGAATC-3’,reverse:5’-ATGTCCCGACCATTGCTGTT-3’;

Il6,forward:5’-CCAGTTGCCTTCTTGGGACT-3’,reverse:5’-TCTGACAGTGCATCATCGCT-3’;

Myd88,forward:5’-CGCTCAACGTGGGAGTGAG-3’,reverse:5’-GGCATCCAACAAACTGCGAG-3’;

Gadd45a, forward:5’-CCGGACTTACACTGTGTGCT-3’,reverse:5’-ATCACCGTTCGGGGAATCAC-3’;

Irs1,forward:5’-TATCTGCATGGGTGGCAAGG-3’,reverse:5’-GGTAGCACCTGGGATGTAGC-3’;

Camk2a, forward:5’-GCCCGGGAGTATTACAGTGA-3’,reverse:5’-GGGTTGATGGTCAGCATCTT-3’;

Dusp10,forward:5’-TGCTGGGGATCAATGAAGCTG-3’,reverse:5’-TGAGATCCTGAGGTCGGACA-3’;

Ppp3ca, forward:5’-GAAAGCCGTTCCATTTCCGC-3’,reverse:5’-CACGAGGCTTCCCATCGTTA-3’;

Ntrk2,forward:5’-AGATCTCGCTTCCACTGTATAGCAT-3’,reverse:5’-GGCTTGACATCTTAATCAGCTTTTG-3’.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 9.0 software. Data were expressed as mean ± SD. Between-group differences were analyzed using one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. A P-value < 0.05 was considered statistically significant.

Results

CMV-induced diaphragmatic muscle fiber atrophy and inflammatory cell infiltration

H&E staining of rat diaphragmatic sections revealed distinct morphological differences between groups. Control muscle fibers showed normal architecture: regular cross-sectional profiles, clear striations, tight arrangement, and peripheral nuclear positioning. In contrast, the CMV 6 h and 12 h groups exhibited irregular fiber contours, disordered striations, widened intercellular spacing, irregular nuclear distribution, and significant inflammatory cell infiltration (Fig. 1A). Quantitative analysis of myofiber CSA confirmed these observations: the mean CSA was significantly reduced in the CMV 6 h group (P < 0.05) and further decreased in the CMV 12 h group (P < 0.01), demonstrating a time-dependent progression of atrophy (Fig. 1B). Immunohistochemistry was performed to detect MHCslow and MHCfast expression. Quantitative analysis of the positive area percentage per field of view revealed no statistically significant difference in the CMV 6 h group for either isoform (P > 0.05), whereas both were significantly reduced after 12 h of mechanical ventilation (P < 0.05) (Fig. 2).

Fig. 1.

H&E staining of the diaphragm in rats after CMV (Scale bar = 100 μm). (A) H&E staining of diaphragm sections from CMV 0 h, 6 h, and 12 h groups. (B) Quantitative analysis of myofiber CSA. Data are mean ± SD(n = 3). *, P < 0.05; **, P < 0.01 vs. CMV 0 h group

Fig. 2.

Immunohistochemical detection of MHCslow and MHCfast expression in the diaphragm of CMV model rats (Scale bar = 20 μm). (A) Immunohistochemical staining of MHCslow and MHCfast in the CMV 0 h, 6 h, and 12 h groups. (B and C) Quantitative analysis of MHCslow- and MHCfast-positive area percentage per field of view. Data are mean ± SD(n = 3). *, P < 0.05; **, P < 0.01 vs. CMV 0 h group

Experiment and sample reliability

Principal component analysis (PCA) of mRNA expression levels showed clear separation between the control and CMV groups, with samples clustering by experimental condition, suggesting distinct transcriptomic profiles between the cohorts (Fig. 3A and C). In addition, box plots confirmed the comparability of expression distributions between groups, with no significant differences observed, indicating suitability for differential expression analysis (Fig. 3B and D). These findings collectively demonstrate the reliability of the VIDD model and the quality of the sequencing data, providing a solid foundation for further mechanistic studies.

Fig. 3.

PCA and expression level box plots of mRNAs. PCA showed that samples from the same group clustered together, indicating high reproducibility, while samples from different groups were clearly separated, suggesting distinct transcriptomic profiles between the control and CMV groups (A and C). Box plots showed no significant difference in mRNA expression distribution between the control and CMV groups, indicating suitability for gene expression comparison (B and D)

Differential gene heatmap and volcano plot

High-throughput RNA-seq was employed to identify DEGs between groups. Compared with the control group, the CMV 6 h group yielded 2,048 DEGs, including 321 upregulated and 1,727 downregulated (P < 0.05, Fig. 4A); the CMV 12 h group yielded 1,495 DEGs, including 534 upregulated and 961 downregulated (P < 0.05, Fig. 4D). Volcano plots (Fig. 4B and E) and heatmaps (Fig. 4C and F) were generated to visualize the differentially expressed mRNAs.

Fig. 4.

Expression profiles of differential mRNAs. Compared with the control group, the CMV groups showed distinct mRNA expression profiles: (A and D) the total number of up-regulated and down-regulated mRNAs; (B and E) volcano plots of differentially expressed mRNAs; (C and F) heat maps of differentially expressed mRNAs

Functional enrichment analysis

GO analysis revealed that the CMV 6 h group comprised 1,310 differentially expressed genes related to molecular function (n = 262), cellular components (n = 179), and biological processes (n = 869) (P < 0.05, Fig. 5A). The CMV 12 h group comprised 1,017 differentially expressed genes related to molecular function (n = 185), cellular components (n = 149), and biological processes (n = 683) (P < 0.05, Fig. 5C). KEGG pathway analysis showed that among the top 20 significantly enriched pathways, the CMV 6 h and 12 h groups were commonly enriched in pathways associated with inflammatory responses, aldosterone synthesis and secretion, oxytocin signaling pathways, ECM-receptor interactions, and insulin signaling pathways (P < 0.05, Fig. 5B and D).

Fig. 5.

GO and KEGG analysis of differentially expressed mRNAs. GO functional analysis (A and C) and KEGG pathway enrichment analysis (B and D) were performed on differentially expressed mRNAs in the CMV 6 h and CMV 12 h groups compared with the control group

Bubble plot of signaling pathways associated with inflammatory responses in VIDD

To explore the mechanisms underlying VIDD, KEGG pathway analysis was performed. In the CMV 6 h group, 534 DEGs were enriched in 20 signaling pathways associated with inflammatory responses, whereas 341 DEGs were enriched in 15 such pathways in the CMV 12 h group. The MAPK, PI3K-Akt, and Calcium signaling pathways, which are known to play important roles in inflammatory responses, showed significant enrichment at both time points (P < 0.05; Fig. 6).

Fig. 6.

Bubble plot of signaling pathways associated with inflammatory responses. The bubble plot showed that the three pathways that were significantly enriched in both the CMV 6 h (A) and CMV 12 h (B) groups were MAPK, PI3K-Akt, and Calcium signaling

Heatmaps of genes enriched in MAPK, PI3K-Akt, and calcium pathways

Heatmaps were generated to visualize the expression of genes enriched in these three signaling pathways in the CMV 6 h and 12 h groups. The results demonstrated that key genes in the MAPK pathway, including Il1b, Gadd45a, Pgf, Map3k6, and Myd88, were upregulated at both 6 h and 12 h, while 23 key genes, such as Cacnb1, Nr4a1, and Rps6ka2, were downregulated. In the PI3K-Akt pathway, key genes such as Pgf, Csf3r, Il6, and Eif4ebp1 were upregulated, whereas 17 key genes, including Nr4a1 and Ppp2r3a, were downregulated. In the Calcium signaling pathway, Adora2a was upregulated, and 15 key genes, including Phkg1 and Calm2, were downregulated (P < 0.05; Fig. 7).

Fig. 7.

Heatmaps of key genes involved in MAPK, PI3K-Akt, and Calcium signaling pathways. In CMV 6 h (A) and 12 h (B) groups, the key genes enriched in these three pathways were shown by heat maps

RT-PCR validation of key genes

Nine key genes identified by RNA-seq were randomly selected for RT-PCR validation. As shown in Fig. 8, compared with the 0 h group, the mRNA levels of Il1b, Il6, and Myd88 in the diaphragm tissue significantly increased after 6 h of mechanical ventilation (P < 0.05), while the mRNA levels of Dusp10, Irs1, and Ntrk2 significantly decreased (P < 0.01). After 12 h, the mRNA levels of Il1b, Il6, Myd88, and Gadd45a significantly increased (P < 0.01), whereas Dusp10, Irs1, and Ntrk2 remained significantly decreased (P < 0.01). In contrast, Camk2a and Ppp3ca showed no significant changes at either time point (P > 0.05).

Fig. 8.

RT-qPCR validation of key gene mRNA levels in diaphragmatic tissues Compared to 0 h, CMV rats showed significant increases in Il1b, Il6, and Myd88 mRNA levels and decreases in Dusp10, Irs1, and Ntrk2 after 6 h of ventilation. After 12 h, Il1b, Il6, Myd88, and Gadd45a levels increased, while Dusp10, Irs1, and Ntrk2 decreased, consistent with RNA-seq results.*, P < 0.05; **, P < 0.01 vs. CMV 0 h group

Discussion

This investigation yielded two principal findings. First, histological assessment revealed diaphragmatic muscle fiber atrophy and inflammatory cell infiltration in rats at 6 and 12 h post-CMV exposure. Second, RNA-Seq profiling of diaphragmatic tissue, followed by KEGG pathway enrichment analysis, identified significant dysregulation in key signaling pathways known to be critically involved in inflammatory responses, including MAPK, PI3K-Akt, and Calcium signaling. Differential expression of pivotal genes within these pathways was subsequently confirmed by RT-PCR. The findings of this study provide new research insights for a deeper understanding of the inflammatory mechanisms of VIDD.

VIDD is a common complication observed in critically ill patients undergoing MV. Diaphragmatic atrophy reportedly progresses rapidly, with ultrasonographic studies indicating a 9% reduction in diaphragmatic thickness after just one day of MV therapy [8]. It has also been reported that diaphragm disuse atrophy may lead to a progressive reduction in diaphragmatic thickness at an average rate of 6% per day in some patients [9]. Consistent with these clinical observations, a recent study using diaphragm biopsies from brain-dead donors mechanically ventilated for at least 24 h confirmed that MV is associated with proteolysis, decreased protein synthesis, and upregulation of inflammatory pathways in the diaphragm [10]. Furthermore, animal studies have demonstrated that diaphragmatic atrophy occurs even earlier, within 12 h of MV initiation [11–13]. In light of the above, this study selected 6 and 12 h as the time points for investigation.

Based on these time points, the structural and molecular evidence obtained in this study supports the successful establishment of the VIDD model. Our H&E staining results revealed progressive myofiber atrophy and inflammatory cell infiltration at 6 h and 12 h after CMV, and quantitative analysis demonstrated a time-dependent reduction in myofiber CSA. Our immunohistochemical analysis further showed a significant decline in both MHCslow and MHCfast expression at 12 h.Studies directly linking MHC expression to diaphragmatic contractile function support our findings.Petrof et al. demonstrated that a reduction in maximum isometric tension in the mdx mouse diaphragm was accompanied by decreased IIx/b fiber MHC expression [14]. Hooijman et al. further showed in mechanically ventilated ICU patients that reduced MHC expression was directly correlated with decreased specific diaphragm fiber force, providing direct evidence linking MHC decline to contractile dysfunction [15]. Zheng et al. demonstrated that upregulation of MHC expression was accompanied by improved diaphragm contractility in VIDD rats [16]. Collectively, these findings support that diaphragmatic atrophy and contractile dysfunction are closely interrelated, as summarized by Guan et al. [17].Together with these literature findings, our results indicate that the VIDD model was successfully established in this study.

Current research hotspots on the molecular mechanisms of VIDD primarily focus on diaphragmatic autophagy, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and oxidative stress. Yong et al. demonstrated that MV induces significant changes in mitochondrial dynamics, including increased mitochondrial fission, reduced fusion, and elevated mitochondrial autophagy, leading to diaphragmatic atrophy and contractile dysfunction [18]. MV has been shown to induce ER and oxidative stress in the diaphragm, and inhibiting ER stress or treating with antioxidants can alleviate VIDD. Li et al. suggested that ER stress likely promotes oxidative stress by inhibiting PGC-1 expression in the diaphragm, with a positive correlation between the two. The endoplasmic reticulum stress inhibitor 4-PBA alleviates MV-induced oxidative stress in rat diaphragm, whereas antioxidants failed to improve endoplasmic reticulum stress in the diaphragm [19]. In summary, these pathophysiological processes are not isolated but interconnected and mutually influential. Although the mechanisms involved in VIDD are highly complex, they commonly affect the expression of inflammatory mediators. These findings indicate that diaphragmatic inflammatory response is a critical component in the pathogenesis of VIDD. For instance, studies have demonstrated that the TLR4 and NF-κB signaling pathways may trigger sepsis-associated acute inflammatory responses and muscle protein degradation, thereby mediating the pathogenic mechanisms of VIDD [12]. Previous research on the interaction between inflammatory cascades and VIDD has often been based on VIDD models accompanied by sepsis, exploring the connections between sepsis, MV, and these inflammatory cascades. The novelty of this study lies in eliminating sepsis as a potential synergistic factor contributing to VIDD and, more objectively, demonstrating the inflammatory effects of CMV on the diaphragm through pathological examination and sequencing. To date, the key genes and critical signaling pathways associated with inflammatory responses in VIDD models have not been fully elucidated. Accordingly, this study sought to identify these expression changes.

The MAPK and PI3K-Akt signaling pathways are known to play important roles in inflammatory responses. These pathways regulate numerous cellular processes, including cell adhesion, migration, growth, proliferation, differentiation, and cell cycle progression, by activating a series of substrate phosphorylation events [20, 21]. Their role in diaphragm-related diseases remains controversial. Studies have reported abnormal activation of the MAPK, NF-κB, and PI3K/Akt signaling pathways in the diaphragm of Duchenne muscular dystrophy mice during the pre-necrotic and necrotic stages [22]. Acute hypoxic stress reportedly increases HIF-1α protein expression in the diaphragm of mice and activates the MAPK, mTOR, Akt, and FoxO3a signaling pathways, promoting diaphragmatic protein synthesis and cell survival [23]. The activation of these pathways serves as a beneficial compensatory mechanism for hypoxia-induced diaphragmatic weakness. Similarly, Liu et al. demonstrated that Neuregulin-1β can inhibit oxidative stress and inflammation by upregulating the PI3K/Akt signaling pathway, thereby alleviating sepsis-induced diaphragmatic dysfunction [24]. The present study revealed that CMV induces inflammatory responses in the rat diaphragm, with the MAPK and PI3K-Akt signaling pathways being significantly enriched in diaphragmatic tissues. Building on the above findings, a better understanding of the precise roles of these signaling pathways and the identification of key genes will facilitate the discovery of effective therapeutic targets for VIDD.

Calcium signaling events are closely associated with inflammatory processes. The increase of intracellular calcium leads to mitochondrial damage, release of reactive oxygen species (ROS), and ultimately activation of the NLRP3 inflammasome [25]. The intracellular calcium signaling mediated by the hepatitis C virus (HCV) core protein, which is associated with phospholipase C activation, induces NLRP3 inflammasome activation and IL-1β release in macrophages, representing the molecular mechanism by which HCV causes liver inflammation [26]. Network pharmacological analysis of the anti-inflammatory mechanism of Reduning Injection revealed that the Calcium signaling pathway, estrogen signaling pathway, PI3K-Akt signaling pathway, and cGMP-PKG signaling pathway play key roles [27]. Disrupting calcium homeostasis by altering the normal opening of calcium channels in plasma and ER membranes can promote iron accumulation and ER stress response, increase ROS production, and lead to ferroptosis and inflammation in microglia [28]. In recent years, researchers have also recognized the important role of calcium homeostasis in diaphragm muscle fibers during MV in VIDD. Studies have shown that mitochondrial ROS production is a necessary condition for MV-induced RyR1 leakage, which leads to increased cytosolic free Ca²⁺ levels, thereby accelerating proteolysis and the development of VIDD. It remains unclear whether the increase in cytosolic free Ca²⁺ levels in the diaphragm induced by MV is sufficient to cause mitochondrial Ca²⁺ overload and stimulate additional mitochondrial ROS production [29]. The sequencing results revealed significant enrichment of the Calcium signaling pathway, suggesting that this pathway indeed plays a crucial role in the inflammatory response of the diaphragm. A deeper understanding of the regulatory mechanisms underlying this signaling pathway in VIDD is an important direction for future research.

Among the DEGs validated in this study, several have been implicated in inflammatory or atrophic processes in other tissues, but their coordinated expression changes in the diaphragm during CMV have not been previously reported. Mechanical ventilation activates the TLR9-MyD88-NF-κB pathway in alveolar macrophages [30, 31], consistent with our finding of Myd88 upregulation in the diaphragm, suggesting that similar pro-inflammatory signaling may occur in diaphragmatic tissue during CMV. Preclinical evidence has established a functional link between IL-1β and IL-6 in skeletal muscle inflammation. Using human skeletal muscle cells, Cordeiro-Santanach et al. demonstrated that IL-1β directly stimulates IL-6 secretion [32]. In an aged rat model, Tazawa et al. further showed that leptin-induced upregulation of Il1b and Il6 is associated with increased expression of the atrophy-related proteins MuRF1 and Atrogin-1 [33]. Our observation of concurrent Il1b and Il6 upregulation in the diaphragm following CMV is consistent with this cascade, raising the possibility that CMV-induced inflammation may trigger IL-1β-driven IL-6 production and potentially contribute to diaphragmatic atrophy. In cancer cachexia, Irs1 ubiquitination and degradation promote muscle wasting [34], consistent with the downregulation of Irs1 observed in our study, suggesting that suppression of anabolic signaling may likewise contribute to diaphragmatic atrophy. Dusp10 is known to negatively regulate MAPK-mediated inflammatory signals [35], whereas Ntrk2 primarily functions in neurotrophic support [36]. The downregulation of Dusp10 and Ntrk2 in our CMV model suggests that these genes are responsive to mechanical ventilation, although their specific roles in diaphragmatic inflammation or atrophy remain to be elucidated. Taken together, these findings provide the first evidence that coordinated expression changes of these genes may promote diaphragmatic inflammation and atrophy in VIDD.

Indeed, this study has limitations that should be acknowledged. First, the sample size was relatively modest (n = 3 per group), time-matched spontaneously breathing controls were not included, and direct measurements of diaphragmatic contractile function were not performed. These factors may limit the rigor of the experimental design. Second, the activation status of the implicated signaling pathways was derived from mRNA expression data rather than protein-level validation, and the findings presented here are primarily correlative at this stage, as interventional experiments to establish causality were not conducted. However, our team is actively pursuing follow-up studies to address these aspects, including functional assessments, protein validation, and interventional designs. We believe that the current findings provide a valuable foundation for these ongoing investigations.

Conclusion

In conclusion, this study found that critical pathways linked to inflammation, including MAPK, PI3K-Akt, and Calcium signaling, along with their associated genes, are closely associated with diaphragmatic structural damage and inflammatory responses in rat VIDD, and may be involved in the development of dysfunction. These findings provide a basis for future mechanistic studies to further explore the roles of these pathways in VIDD.

Author contributions

The experiments were performed and the manuscript was written by Yang Chen, Rongrong Xu, and Zhicheng Huang. Data analysis and manuscript revision were carried out by Kai Qiu, Nanyuan Gu, Longhuan Zeng and Fu Tian. Qiangqiang Shi and Hui Chen supervised the research and also revised the manuscript. All authors read and approved the final manuscript.

Funding

Zhejiang Provincial Health Commission Science and Technology Program(2025HY0771); Zhejiang Provincial Health Commission Science and Technology Program(2025HY0774); Jinhua Public Welfare Technology Application Research Project (2023-4-217).

Data availability

The clean read data have been deposited in the Genome Sequence Archive (GSA) under the accession number CRA045075 and can be accessed at. https://ngdc.cncb.ac.cn/gsa/browse/CRA045075.Other. datasets are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study was conducted in strict accordance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH) and was approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (Approval No: IACUC-1905030). The study is reported in compliance with the ARRIVE guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.