Molecular Study from the Signaling Pathways of Four Potential asthma triggers: AKT1, MAPK13, STAT1, and TLR4

Abstract

Asthma is a chronic and heterogeneous inflammatory airway disease with diverse clinical endotypes and limited curative treatment options. Recent systems biology analyses identified four potential molecular triggers-AKT1, MAPK13, STAT1, and TLR4-as candidate regulators of asthma-associated signaling pathways. This study aimed to validate the expression of these four proteins and their downstream signaling elements in peripheral blood mononuclear cells (PBMCs) from patients with allergic asthma (AA), nonallergic asthma (NA), and healthy controls (HC), to explore their potential as biomarkers or therapeutic targets. For that, PBMC samples were collected from 45 AA patients, 17 NA patients, and 15 HC subjects. Gene and protein expression of AKT1, MAPK13, STAT1, and TLR4 were quantified using RT-qPCR and Western blotting. Expression patterns were compared across groups and stratified by asthma severity. Correlations with clinical parameters (FEV1, FVC, FeNO, IgE, eosinophil counts) and treatment regimens were also assessed. All four target genes showed significantly reduced expression in asthma patients compared to controls (p < 0.001), with the most marked downregulation in NA patients. At the protein level, MAPK13 and TLR4 showed significant differential expression. Stratification by severity revealed a stepwise reduction in gene expression in AA patients, correlating with disease severity, whereas NA patients showed uniformly low expression regardless of severity. Multiple pathway-related genes, including RELA, SMAD3, NFATC1, and ALOX5, were also downregulated, particularly in NA patients. Notably, differential correlations were observed between gene expression and lung function parameters in AA vs. NA groups. In conclusion, this study supports the potential involvement of AKT1, MAPK13, STAT1, and TLR4 in asthma pathogenesis and highlights differences between allergic and nonallergic asthma at the molecular level. These proteins and their associated pathways may serve as future targets for biomarker development or endotype-specific therapies. Further studies in larger and more diverse cohorts, including functional validation, are warranted.

Keywords: AKT1; MAPK13; STAT1; TLR4; allergic asthma (AA); asthma; biomarkers; gene expression; nonallergic asthma (NA); peripheral blood mononuclear cells (PBMCs); signaling pathways.

Figure 1

Differential gene and protein expression results of AKT1, MAPK13, STAT1, and TLR4 in PBMCs samples. (A) The relative expression of each gene is represented as the mean relative quantification (RQ) in each asthma group compared to healthy controls. Error bars represent the standard error. (B) (i) Protein expression results are displayed in a box-and-whisker plot, which shows the relative intensity (RI) of each band detected by Western blot and the β-Actin band in each sample. (ii) A representative example of a Western blot including two control subjects (HC), five allergic asthmatic patients (AA), and three nonallergic asthmatic patients (NA) is shown. Statistically significant differences between groups, according to the nonparametric Mann–Whitney test, are indicated: HC* and HC^## (p < 0.05 and p < 0.001, respectively) for statistically significant differences between the control group and the indicated clinical group; * and ** (p < 0.05 and p < 0.01, respectively) for statistically significant differences between the two clinical groups indicated by the horizontal bar.

Figure 2

Differential gene expression of potential asthma triggers according to asthma severity in (A) allergic asthmatic (AA) patients and (B) nonallergic asthmatic (NA) patients. The relative expression of each gene is represented as the mean relative quantification (RQ) in each asthma group compared to healthy controls. Error bars represent the standard error. The gene expression was compared among severities and with healthy control (HC) subjects by non-parametric Mann–Whitney test, using the R program, and differences were considered significant with adjusted p-values under 0.05. Statistically significant differences between HC and the indicated severity subgroup are shown as HC**, HC^#, and HC^## (p ≤ 0.01, p ≤ 0.005, and p ≤ 0.001, respectively). Statistically significant differences among indicated subgroups are shown as *, **, and ^# (p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively). AA_S, AA_Mo, AA_M: severe, moderate, and mild allergic asthmatic patients, respectively; and NA_S, NA_Mo, NA_M: severe, moderate, and mild nonallergic asthmatic patients, respectively.