Prime-O-glucosylcimifugin attenuates lipopolysaccharide-induced acute lung injury in mice

Abstract

Prime-O-glucosylcimifugin is an active chromone isolated from Saposhnikovia root which has been reported to have various activities, such as anti-convulsant, anticancer, anti-inflammatory properties. The purpose of this study was to evaluate the effect of prime-O-glucosylcimifugin on acute lung injury (ALI) induced by lipopolysaccharide in mice. BALB/c mice received intraperitoneal injection of Prime-O-glucosylcimifugin 1h before intranasal instillation (i.n.) of lipopolysaccharide (LPS). Concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and interleukin (IL)-6 in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). Pulmonary histological changes were evaluated by hematoxylin-eosin, myeloperoxidase (MPO) activity in the lung tissue and lung wet/dry weight ratios were observed. Furthermore, the mitogen-activated protein kinases (MAPK) signaling pathway activation and the phosphorylation of IκBα protein were determined by Western blot analysis. Prime-O-glucosylcimifugin showed promising anti-inflammatory effect by inhibiting the activation of MAPK and NF-κB signaling pathway.

Fig. 1

Effect of Prime-O-glucosylcimifugin on macrophage toxicity. Cells were cultured with Prime-O-glucosylcimifugin (0–100 mg/L) in the absence or presence of 1 mg/L LPS for 18 h. Cell viability was assessed by MTT reduction assays. Data are presented as mean ± SEM of three independent experiments.

Fig. 2

Effects of Prime-O-glucosylcimifugin on LPS-induced cytokine (TNF-α, IL-1β, IL-6 and IL-10) production in vitro. The cells were treated with LPS alone or LPS plus different concentrations (12.5, 25 or 50 mg/L) of Prime-O-glucosylcimifugin for 24 h. The production of TNF-α, IL-1β and IL-6, and IL-10 in the culture supernatant of macrophages was measured by ELISA kits. The values represent mean ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. ^##P < 0.01 indicates significant differences from the unstimulated control group, ^⁎P < 0.05 vs. LPS, ^⁎⁎P < 0.01 vs. LPS.

Fig. 3

Effect of Prime-O-glucosylcimifugin on MAPKs and NF-κB signaling pathways in LPS stimulated RAW cells. RAW 264.7 cells were treated with different concentrations (12.5, 25 or 50 mg/L) of Prime-O-glucosylcimifugin for 1 h before stimulating them with LPS (1 mg/L) for 30 min. Protein samples were analyzed by Western blot with antibodies specific for the phosphorylated forms of ERK, JNK, p38 and IκB. Quantification of protein expression was normalized to β-actin using a densitometer (Imaging System). (A) The phosphorylation of ERK, JNK and p38 after pretreatment with Prime-O-glucosylcimifugin and LPS challenge. (B) The phosphorylation of IκB after pretreatment with Prime-O-glucosylcimifugin and LPS challenge. The data are representative of three independent experiments and expressed as mean ± SEM. ^##P < 0.01 indicates significant differences from the unstimulated control group, ^⁎P < 0.05 vs. LPS, ^⁎⁎P < 0.01 vs. LPS.

Fig. 4

Effect of Prime-O-glucosylcimifugin on the production of TNF-α, IL-1β and IL-6 in BALF of LPS-induced ALI mice. Mice were given an intraperitoneal injection of Prime-O-glucosylcimifugin (2.5, 5 or 10 mg/kg) 1 h prior to administration of LPS. BALF was collected at 7 h following LPS challenge to analyze the inflammatory cytokines TNF-α (Fig. 4A), IL-1β (Fig. 4B) and IL-6 (Fig. 4C). The values presented are the mean ± SEM (n = 6 in each group). ^##P < 0.01 vs. control group, ^⁎P < 0.05, ^⁎⁎P < 0.01 vs. LPS group.

Fig. 5

Effects of Prime-O-glucosylcimifugin on the number of total cells, neutrophils, and macrophages in the BALF of LPS-induced ALI mice. Mice were given an intraperitoneal injection of Prime-O-glucosylcimifugin (2.5, 5 or 10 mg/kg) 1 h prior to an i.n. administration of LPS. BALF was collected at 7 h following LPS challenge to measure the number of total cells (A), neutrophils (B), and macrophages (C). The values presented are the mean ± SEM (n = 6 in each group). ^##P < 0.01 vs. control group, ^⁎P < 0.05, ^⁎⁎P < 0.01 vs. LPS group.

Fig. 6

Effects of Prime-O-glucosylcimifugin on the lung W/D ratio of LPS-induced ALI mice. Mice were given an intraperitoneal injection of Prime-O-glucosylcimifugin (2.5, 5 or 10 mg/kg) 1 h prior to an i.n. administration of LPS. The lung W/D ratio was determined at 7 h after LPS challenge. The values presented are the mean ± SEM (n = 6 in each group). ^##P < 0.01 vs. control group, ^⁎P < 0.05, ^⁎⁎P < 0.01 vs. LPS group.

Fig. 7

Effects of Prime-O-glucosylcimifugin on MPO activity in lungs of LPS-induced mice. Seven hours after LPS instillation, lung homogenates were prepared for determination of MPO activity. MPO activity in the lungs was determined with a kit by measurement of the H₂O₂-dependent oxidation of an o-dianisidine solution. Data are presented as the mean ± SEM (n = 6 in each group). ^##P < 0.01 vs. control group, ^⁎P < 0.05, ^⁎⁎P < 0.01 vs. LPS group.

Fig. 8

Effect of Prime-O-glucosylcimifugin on histopathological changes in lung tissues in LPS-induced ALI mice (A, B, C, D, E: × 100; A1, B1, C1, D1, E1: × 400). Mice were given an intraperitoneal injection of Prime-O-glucosylcimifugin (2.5, 5, 10 mg/kg) 1 h prior to an i.n. administration of LPS. Lungs (n = 3) from each experimental group were processed for histological evaluation at 7 h after LPS challenge. To confirm pathologic changes in lung tissues, we performed hemotoxylin–eosin staining. (A) Normal mice; (B) LPS treated mice; (C, D, E) LPS + Prime-O-glucosylcimifugin-treated mice.