Peritonitis caused by LPS is a severe clinical challenge, which causes organ damage and death. However, the mechanism of LPS-induced peritonitis has not been fully revealed yet. Here, we investigated the transcriptome profile of the peritoneal tissue of LPS-induced peritonitis in mice. A model of LPS-induced peritonitis in mice was established (LPS 10 mg/kg, i.p.), and the influence of TAK 242 (TLR4 inhibitor) on the level of inflammatory cytokines in mouse peritoneal lavage fluid was investigated by using an ELISA test. Next, the peritoneal tissues of the three groups of mice (Control, LPS, and LPS+TAK 242) (n = 6) were isolated and subjected to RNA-seq, followed by a series of bioinformatics analyses, including differentially expressed genes (DEGs), enrichment pathway, protein-protein interaction, and transcription factor pathway. Then, qPCR verified-hub genes that may interact with TAK 242 were obtained. Subsequently, the three-dimensional structure of hub proteins was obtained by using homology modeling and molecular dynamics optimization (300 ns). Finally, the virtual docking between TAK 242 and hub proteins was analyzed. Our results showed that TAK 242 significantly inhibited the production of inflammatory cytokines in the peritoneal lavage fluid of mice with peritonitis, including IL-6, IFN-γ, IL-1β, NO, and TNF-α. Compared with the Control group, LPS treatment induced 4201 DEGs (2442 down-regulated DEGs and 1759 up-regulated DEGs). Compared with the LPS group, 30 DEGs were affected by TAK 242 (8 down-regulated DEGs and 22 up-regulated DEGs). A total of 10 TAK 242-triggered hub genes were obtained, and the possible docking modes between TAK 242 and hub proteins were acquired. Overall, our data demonstrated that a large number of DEGs were affected in LPS-triggered peritonitis mice. Moreover, the TLR4 inhibitor TAK 242 is capable of suppressing the inflammatory response of LPS-induced peritonitis. Our work provides clues for understanding the pathogenesis of LPS-induced peritonitis in mice.

Purpose: The critical role played by miRNAs in sepsis-induced acute peritonitis has been repeatedly validated. This research focuses on elucidating the influence of endothelial progenitor cells (EPC)-derived exosomal miRNA-31 (exo-miR-31) on septic peritonitis (SP) and the role of E-selectin (SELE) in this process. Methods: Differences in serum miRNA-31 and soluble SELE (sSELE) expression between SP cases and healthy controls (HCs) were identified. Additionally, we built a mouse colon ascendens stent peritonitis (CASP) model and an in vitro sepsis model via lipopolysaccharide (LPS) stimulation of mouse venous endothelial cells to verify the specific mechanism of EPC-derived exo-miR-31 on SP in vitro and in vivo. Results: As indicated by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) results, SP cases showed markedly reduced serum miR-31 levels and evidently elevated sSELE expression than HCs, with the expression of both genes altered more significantly as the condition worsened. Correlation analysis suggested a strong association between serum miRNA-31 and sSELE levels and sepsis severity. miR-31 in the peritoneal lavage fluid (PLF) of CASP model mice was down-regulated, while sSELE and inflammatory factors (IFs) TNF-α, IL-6 and IL-10 were notably increased. Gimsa staining revealed the massive recruitment and aggregation of nucleated cells in the abdominal cavity of CASP mice, and HE staining showed extensive neutrophilic infiltration in lung, liver and colon tissues, with obvious inflammatory tissue destruction. Besides, large numbers of bacteria were detected in the peripheral blood and PLF of CASP mice, with many CASP mice dying within 48 hours, as indicated by the bacterial smears and culture tests. EPC-derived exo-miR-31 was effective in elevating PLF miR-31 expression in CASP mice while suppressing sSELE, TNF-α, IL-6 and IL-10 levels. In EPCs-Exos^miR-31 group, significant reductions in nucleated cell counts in the mouse PLF, neutrophil infiltration in lung, liver and colon tissues, and peripheral blood and PLF bacterial loads were observed, as well as an obvious extension in survival. In addition, in vitro experimental results of cell sepsis showed that miR-31 could obviously block the inhibitory effect of LPS stimulation on the proliferation level of mouse venous endothelial cells and inhibit the level of apoptosis. SELE, Caspase-3 and Bax protein levels were markedly lower in LPS+miR-31 group as compared to LPS and LPS-miR-NC groups, while Bcl-2 was increased (P<0.05). Dual luciferase reporter gene assay identified the role of miR-31 as the direct target of SELE. Conclusions: EPC-derived exo-miR-31 can effectively alleviate SP, possibly through targeted inhibition of SELE, providing a potential new direction for future prevention and treatment of SP.