Introduction Intestinal ischemia-reperfusion (I/R) injury, a common and severe clinical condition with high morbidity and mortality, burdens healthcare systems. Our previous investigations established that a nano-delivery system enabled targeted rapamycin delivery to intestinal I/R injury sites with therapeutic efficacy. While calcium/calmodulin-dependent protein kinase IIδ (CaMK2D) has been implicated in myocardial injury and tumorigenesis, its role in intestinal I/R pathophysiology remains unexplored. This study investigates the therapeutic mechanisms of rapamycin in intestinal I/R injury by modulation of CaMK2D signaling. Methods An oxygen-glucose deprivation/reperfusion (OGD/R) model in Caco-2 human colorectal cancer cells and a murine intestinal I/R model were established. Small interfering RNA (siRNA) and hesperadin (HES) were used to inhibit CaMK2D expression. Transcriptomic profiling was performed via RNA sequencing (RNA-Seq) with subsequent bioinformatic analysis including differential gene expression, MCODE-based protein interaction network clustering, and RAPA-CaMK2D molecular docking studies. Cellular assays included qRT - PCR, western blotting (WB), Fluo-3 calcium flux analysis, flow cytometry, and Enzyme-linked immunosorbent assay (ELISA). In animal experiments, HE staining, immunohistochemistry, TUNEL assay, WB, and ELISA were employed. Results Both cellular and murine models demonstrated a significant upregulation of CaMK2D phosphorylation with intestinal epithelial apoptosis, barrier dysfunction, and enhanced inflammatory response during I/R. CaMK2D knockdown using siRNA attenuated these pathological manifestations, vice versa. Bioinformatic analysis revealed a CaMK2D-dominated regulatory module (ranked fifth) enriched in calcium-mediated signaling pathways. Mechanistically, I/R induced CaMK2D activation exacerbated inflammatory cascades, epithelial apoptosis, and tight junction disruption. Rapamycin treatment (1.5 mg/kg, i.p.) ameliorated these effects by decreasing CaMK2D expression and phosphorylation (WB, P 0.01), pro-inflammatory cytokine levels (ELISA, P 0.01), while preserving intestinal integrity as evidenced by histological analysis (IHC, P 0.05). Discussion Our findings establish CaMK2D hyperactivation as a key to intestinal I/R injury. The therapeutic potential of rapamycin derived from its ability to suppress CaMK2D signaling axis, providing a novel pharmacological strategy for intestinal I/R management.
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