Mechanism of Gzma-Mediated GEF-H1 Activation in Intestinal Epithelial Cells Leading to Intestinal Barrier Dysfunction in Sepsis 2254641

Abstract

Abstract Introduction This study investigates how Granzyme A (GZMA) induces intestinal barrier dysfunction in sepsis, specifically by activating the GEF-H1—RhoA signaling axis. It aims to elucidate the underlying molecular mechanisms and evaluate the therapeutic potential of targeting GZMA and GEF-H1 for sepsis. Methods 1.A cecal ligation and puncture (CLP) mouse model of sepsis was established. qPCR, HE staining, and IF assessed GZMA upregulation, inflammatory cytokine levels, organ damage, and intestinal barrier integrity (via ZO-1/E-cadherin). 2.Intestinal epithelial cells were co-cultured with LPS-stimulated NK92MI cells or treated with recombinant GZMA. Barrier function was evaluated by TEER, permeability assays, and IF for tight junction proteins. 3.Western blot and qPCR analyzed GEF-H1—RhoA pathway components (RhoA, LIMK1, MLC2, cofilin). GEF-H1’s role was confirmed using siRNA knockdown/overexpression in cells and GEF-H1-/- septic mice. Results 1. Sepsis models showed significantly increased GZMA expression and concurrent disruption of the intestinal epithelial barrier. 2.GZMA directly increased intestinal epithelial permeability. Co-culture with LPS-stimulated NK92MI cells or direct GZMA treatment impaired barrier function in vitro. 3.GZMA activated the GEF-H1/RhoA signaling pathway in multiple intestinal epithelial cell lines under septic conditions. 4.Inhibiting GEF-H1 (via KO in mice or siRNA in cells) alleviated intestinal barrier injury, improved survival, and suppressed GEF-H1/RhoA pathway activity. 5.Overexpression or stabilization of GEF-H1 exacerbated epithelial barrier damage in sepsis models. Conclusion GZMA is upregulated in sepsis and disrupts intestinal barrier integrity. The mechanism involves GZMA promoting GEF-H1 phosphorylation, which activates the RhoA—ROCK—MLC2 pathway, leading to dysregulation of tight junction proteins (occludin, claudin-1, ZO-1) and increased permeability. GZMA and GEF-H1 represent promising therapeutic targets for sepsis-induced intestinal barrier dysfunction. Funding Source National Natural Science Foundation of China (82272209) Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)