Master's student Affiliated BenQ Hosp., Nanjing Med. Univ., United States
Disclosure(s):
Zexing Lin, M.Med: No financial relationships to disclose
Introduction/Rationale: 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.
