Targeting TNFR1 by salvianolic acid B alleviates sepsis-induced acute lung injury and pulmonary-intestinal epithelial barrier damage

BACKGROUND: Sepsis-induced acute lung injury (ALI) and intestinal injury were characterized by dysregulated systemic inflammation and organ dysfunction. Host-directed therapy (HDT) that modulates excessive immune responses is a promising complementary strategy. Salvianolic acid B (Sal B) has been reported to modulate various signaling pathways, yet whether it targets TNFR1 to simultaneously inhibit NF-κB, necroptosis, and p-MLCK/p-MLC2 in septic ALI remains unexplored. PURPOSE: This study aimed to investigate the protective effects of Sal B against sepsis-induced lung-intestine injury and to elucidate its underlying molecular mechanisms, with a focus on identifying its potential cellular target. METHODS: A murine model of sepsis-induced ALI was established via lipopolysaccharide (LPS) challenge. H&E staining, ELISA, immunohistochemistry analyses were performed to assess lung, intestinal injury and their barrier damage. In vitro, models of LPS/zVAD-induced macrophage necroptosis and tumor necrosis factor-α (TNF-α) induced epithelial barrier damage were established in J774A.1, THP-1, A549, and HCT116 cells. Cell death, cytokine secretion, the key proteins of necroptosis and signal molecules related to epithelial barrier integrity were evaluated using LDH/PI assays, ELISA, western blotting, and immunofluorescence. By using gene silencing and overexpression techniques, the role of tumor necrosis factor receptor 1 (TNFR1) in regulating the necrotic apoptosis process of macrophages infected with LPS was elucidated. The Sal B-TNFR1 potential interaction was validated using drug target identification methods such as molecular docking and site-directed mutagenesis. RESULTS: In vivo, Sal B alleviated pulmonary edema, histopathological damage in lung and colon, and reduced systemic and local levels of TNF-α and interleukin-1β (IL-1β). It concurrently suppressed the activation of the phospho myosin light-chain kinase/phospho myosin light chain 2 (p-MLCK/p-MLC2) pathway and restored expression of tight junction proteins, while inhibiting the necroptosis pathway. In vitro, Sal B inhibited TNF-α-induced barrier damage in epithelial cells and LPS/zVAD-induced necroptosis in macrophages. Mechanistically, Sal B exhibits a strong binding potential with TNFR1 and may exert protective effects by targeting TNFR1 to modulate necroptosis and pulmonary and intestinal epithelial barrier damage, ultimately attenuating LPS-induced the lung-intestine injury. CONCLUSION: By potentially targeting TNFR1, Sal B exerts protective effects on both lung and intestine. These findings underscore the therapeutic potential of Sal B as a novel HDT strategy.