Distinct Src, STAT3, and MAPK Immunoreactivity in Peripheral Blood-Derived Cell-Block Preparations and Qualitative Protein Patterns in Isolated PBMCs from Patients with Type 1 and Type 2 Diabetes Mellitus

Background: Type 1 Diabetes Mellitus (T1DM) and Type 2 Diabetes Mellitus (T2DM) have distinct pathogenic mechanisms but share inflammatory and immune-related alterations. Src, signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase (MAPK) signaling pathways play important roles in immune regulation and cellular stress responses. However, patterns of these signaling-related proteins in peripheral blood-derived cellular preparations and isolated Peripheral blood mononuclear cells (PBMCs) from patients with T1DM and T2DM remain incompletely characterized. This study aimed to compare STAT3, Src, Extracellular signal-regulated kinases ½ (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) immunoreactivity in peripheral blood-derived cell-block preparations and to descriptively examine total and phosphorylated protein patterns in isolated PBMCs from patients with T1DM and T2DM. Methods: Peripheral blood samples were obtained from 15 patients with T1DM, 15 patients with T2DM, and 15 healthy controls. For Western blot analysis, PBMCs were isolated from heparinized blood using Ficoll–Paque density-gradient centrifugation. For immunohistochemistry, separate Ethylenediaminetetraacetic acid (EDTA)-anticoagulated peripheral blood samples were processed by direct centrifugation to obtain cellular pellets, which were fixed and paraffin-embedded. STAT3, Src, ERK1/2, and p38 MAPK immunoreactivity was evaluated semi-quantitatively in these peripheral blood-derived cell-block preparations, whereas total and phosphorylated protein patterns were examined qualitatively by Western blot analysis of isolated PBMCs. Results: Immunohistochemical analysis revealed significant differences in STAT3, Src, ERK1/2, and p38 MAPK immunoreactivity among the three groups (all p < 0.001). STAT3 and ERK1/2 immunoreactivity was strongest in T1DM, whereas Src and p38 MAPK immunoreactivity was most prominent in T2DM. Qualitative Western blot analysis showed visually different total and phosphorylated protein band patterns among the displayed representative samples, including more prominent total STAT3 bands in the Control and T1DM samples and greater relative p-STAT3 band intensity in the T2DM samples. However, the groups differed significantly in age (p < 0.001), and detailed clinical covariates were unavailable; therefore, the observed between-cohort differences cannot be attributed specifically to diabetes type. Conclusions: In this exploratory cohort, differences in STAT3, Src, ERK1/2, and p38 MAPK immunoreactivity were observed among the three study groups, together with qualitative Western blot differences among the displayed representative PBMC samples. Because of substantial age imbalance, unavailable clinical covariates, the semi-quantitative nature of immunohistochemical scoring, and the absence of participant-level quantitative Western blot data, these observations cannot be interpreted as diabetes-type-specific signaling differences or evidence of pathway activation. Further studies using age-matched, clinically characterized cohorts, quantitative protein analyses, and cell-subset-specific approaches are required.