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Protostemonine (PSN) is the main anti-inflammatory alkaloid extracted from the roots of Stemona sessilifolia (known as “Baibu” in traditional Chinese medicine). Here, we reported the inhibitory effects of PSN on lipopolysaccharide (LPS)-induced macrophage activation in vitro and LPS-induced acute lung injury in mice. Macrophage cell line RAW264.7 cells and mouse bone marrow-derived macrophages (BMDMs) were treated with PSN (1, 3, 10, 30 and 100 μmol/L) for 0.5 h and then challenged with LPS (0.1 μg/mL) for 24 h. Pretreatment with PSN significantly inhibited LPS-induced phosphorylation of MAPKs and AKT, iNOS expression and NO production in the macrophages. C57BL/6 mice were intratracheally injected with LPS (5 mg/kg) to induce acute lung injury (ALI). The mice were subsequently treated with PSN (10 mg/kg, ip) at 4 and 24 h after LPS challenge. PSN administration significantly attenuated LPS-induced inflammatory cell infiltration, reduced pro-inflammatory cytokine (TNF-α, IL-1β and IL-6) production and eliminated LPS-mediated lung edema. Furthermore, PSN administration significantly inhibited LPS-induced pulmonary MPO activity. Meanwhile, LPS-induced phosphorylation of p38 MAPK, iNOS expression and NO production in the lungs were also suppressed. The results demonstrate that PSN effectively attenuates LPS-induced inflammatory responses in vitro and in vivo; the beneficial effects are associated with the decreased phosphorylation of MAPK and AKT and the reduced expression of pro-inflammatory mediators, such as iNOS, NO and cytokines. These data suggest that PSN may be a potential therapeutic agent in the treatment of ALI.

The evolutionary relationship and functional correlation between human formyl peptide receptors (FPRs) and their mouse counterparts remain incompletely understood. We examined three members of the mouse formyl peptide receptor subfamily (mFprs) and found that they differ in agonist preference and cellular distributions. When stably expressed in transfected rat basophilic leukemia (RBL-2H3) cells, mFpr1 was readily activated by N-formylated peptides derived from Listeria monocytogenes(fMIVTLF), Staphylococcus aureus(fMIFL), and mitochondria (fMMYALF). In contrast, the Escherichia coli–derived fMLF was 1000-fold less potent. The aforementioned peptides were much less efficacious at mFpr2, which responded better to the synthetic hexapeptide WKYMVm, the synthetic agonists Quin-C1 (a substituted quinazolinone), and compound 43 (a nitrosylated pyrazolone derivative). Saturation binding assays showed that mFpr1 and mFpr2 were expressed at similar levels on the cell surface, although their affinity for N-formyl-Met-Leu-Phe-Ile-Ile-Lys-fluorescein isothiocyanate varied by more than 1000-fold [dissociation constant (Kd) values of 2.8 nM for mFpr1 and 4.8 μM for mFpr2]). Contrary to these receptors, mFpr-rs1 responded poorly to all the previously mentioned peptides that were tested. Fluorescent microscopy revealed an intracellular distribution pattern of mFpr-rs1. On the basis of these results, we conclude that mFpr1 is an ortholog of human FPR1 with certain pharmacologic properties of human FPR2/ALX, whereas mFpr2 has much lower affinity for formyl peptides. The intracellular distribution of mFpr-rs1 suggests an evolutionary correlation with human FPR3.