Senkyunolide I protect against lung injury via inhibiting formation of neutrophil extracellular trap in a murine model of cecal ligation and puncture

Background: Senkyunolide I (SEI), a component of a Chinese herb named Ligusticum Chuanxiong hort, which is included in the formulation of Xuebijing Injection, a medication used to treat sepsis in China. Our previous study showed that SEI was protective against sepsis-associated encephalopathy and the present study was performed to investigate the role of SEI in sepsis-induced lung injury in a murine model of cecal ligation and puncture (CLP).

Methods: SEI (36 mg/kg in 200 μl) or vehicle was administered immediately after CLP surgery. The lung injury was assessed 24 h later by histopathological tests, protein concentration in the bronchoalveolar lavage fluid (BALF), neutrophil recruitment in the lung tissue (myeloperoxidase fluorescence, MPO), pro-inflammatory cytokines and oxidative responses. Platelet activation was detected by CD42d/GP5 immunofluorescence and neutrophil extracellular trap (NET) were determined by immunofluorescence assays and Enzyme linked immunosorbent assay (ELISA) of MPO-DNA. In vitro experiments were performed to detect the level of MPO-DNA complex released by SEI-treated neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) or co-cultured with platelets from CLP mice.

Results: SEI administration relieved the injury degree in CLP mice according to the histopathological tests (P < 0.05 compared with DMSO + CLP group). Protein level in the BALF and neutrophil infiltration were remarkably reduced by SEI after CLP surgery (P < 0.05 compared with DMSO + CLP group). TNF-α, IL-1β and IL-6 were decreased in the plasma and lung tissues from CLP mice treated with SEI (P < 0.05 compared with DMSO + CLP group). The phosphorylation of JNK, ERK, p38 and p65 were all inhibited by SEI (P < 0.05 compared with DMSO + CLP group). Immunofluorescence of MPO showed that neutrophil number was significantly lower in SEI treated CLP mice than in vehicle treated CLP mice (P < 0.05). The CD42d/GP5 staining suggested that platelet activation was significantly reduced and the NET level in the lung tissue and plasma was greatly attenuated by SEI treatment (P < 0.05 compared with DMSO + CLP group). In vitro experiments showed that the MPO-DNA level stimulated by PMA was significantly reduced by SEI treatment (P < 0.05 compared with DMSO treatment). Co-culture neutrophils with platelets from CLP mice resulted in higher level of MPO-DNA complex, while SEI partly reversed such effects of platelet on NET formation.

Conclusions: SEI was protective against lung injury induced by CLP in mice. The NET formation was significantly reduced by SEI treatment, which might be involved in the mechanism of the protective effect.