Peucedanol ameliorates LPS-induced inflammation in RAW264.7 cells and CLP-induced sepsis in mice by inhibiting TLR4/myD88/NF-κB pathway

Abstract

AbstractBackgroundPreviously, it has reported that Peucedanol (PEU) possesses anti-bacterial activity. However, its effect and mechanism against inflammation remains unclear.MethodsIsothermal titration calorimetry (ITC) was used to assess binding affinities of PEU to pathogen associated molecular patterns (PAMPs) Kdo2-Lipid A (KLA), oligodeoxynucleotide 1826 (ODN 1826), and peptidoglycan (PGN). A lipopolysaccharide (LPS)-induced RAW264.7 cell inflammation model and a cecum ligation and a puncture (CLP)-induced mouse sepsis model were used to assess efficacy and mechanism of PEUin vitroandin vivo. 16S ribosomal RNA (16S rRNA) sequencing was used to assay characteristics of intestinal flora of the sepsis mice.ResultsPEU had a moderate binding to KLA and ODN 1826. PEU significantly reduced supernatant tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), and downregulated protein expressions of toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 8 (MyD88), and nuclear factor kappa-B (NF-κB) in the LPS-treated cells. PEU remarkably increased the survival rate, reduced the serum TNF-α and IL-6 levels, attenuated the CLP-induced pathological damage of intestine, increased proliferation-related proteins Bmi1 and Lgr5. Further, the anti-inflammatory effects of PEU were not significantly abolished in the present of chloroquine (CQ). Meanwhile, PEU significantly increased Chao1 index of the intestinal flora at the early stage of sepsis. In addition, PEU significantly changed composition of the flora at both phylum and genus levels. Moreover, PEU significantly affected metabolism-related pathways such as tricarboxylic acid (TCA) cycle, fatty acid degradation, secondary bile acid biosynthesis, and others.ConclusionsTaken together, PEU significantly inhibits LPS-induced inflammationin vitroand CLP-induced sepsisin vivo. Further, its anti-inflammatory effect is independent of the TLR4/myD88/NF-κB pathway. In addition, PEU improves the intestinal flora imbalance at the early stage of sepsis.