Objective: To investigate the antagonistic effect of ML355, the 12-lipoxygenase (12-LOX) inhibitor, on lipopolysaccharide (LPS)-induced inflammatory response in mice and its molecular mechanism.
Methods: (1) In vivo experiment: 24 adult male C57BL/6 mice were randomly divided into control group [intraperitoneal injection of 100 μL dimethyl sulfoxide (DMSO) 1 hour in advance and then intraperitoneal injection of 0.9% normal saline], LPS group (intraperitoneal injection of LPS 20 mg/kg) and ML355 pretreatment groups (15 mg/kg, 30 mg/kg of ML355 intraperitoneal injection 1 hour in advance before LPS administration). The mice were euthanized 12 hours after the model was established, and the peripheral blood, peritoneal lavage fluid and peritoneal macrophages (PMs) were collected. The levels of plasma and peritoneal lavage fluid 12-hydroxyeicosatetraenoic acid (12-HETE), peritoneal lavage fluid interferon-γ (IFN-γ), interleukins (IL-1β, IL-6, IL-10), tumor necrosis factor-α (TNF-α) and serum IFN-γ, IL-1β were measured by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of IFN-γ, IL-1β and arachidonic 15-lipoxygenase (Alox15) in mice PMs were detected by quantitative real time polymerase chain reaction (qRT-PCR). (2) In vitro experiment: the PMs of mice were cultured in vitro and randomly divided into control group (with final concentration of 0.3% DMSO), LPS group (LPS 20 mg/L) and ML355 pretreatment groups (treated with 25 μmol/L, 50 μmol/L of ML355 1 hour before LPS administration). The supernatant and cell mass were collected at different time points stimulated by LPS. The effect of ML355 on the survival rate of PMs were detected by cell counting kit-8 (CCK-8), the levels of 12-HETE, IFN-γ, IL-1β, TNF-α, IL-6 and IL-10 in PMs supernatant were measured by ELISA, the mRNA expressions of IFN-γ, IL-1β and Alox15 were detected by qRT-PCR,and the expressions of 12/15-LOX and mitogen activated protein kinase (MAPK) pathway protein were detected by Western blotting.
Results: (1) In vivo experiment: 12 hours after LPS stimulation,the contents of plasma and peritoneal lavage fluid 12-HETE and inflammatory factors in peritoneal lavage fluid, IFN-γ and IL-1β in serum in LPS model group were significantly higher than those in control group. Compared with LPS group, after 15 mg/kg and 30 mg/kg of ML355 pretreatment, the contents of 12-HETE in plasma and peritoneal lavage fluid and IFN-γ in serum were significantly decreased [12-HETE: plasma (mg/L) was 11.59±1.80, 11.58±1.75 vs. 18.59±1.68, peritoneal lavage fluid (μg/L) was 355.80±59.47, 196.30±88.98 vs. 712.10±44.55; serum IFN-γ (ng/L): 147.60±2.94, 114.20±2.94 vs. 326.40±2.22, all P < 0.05]. After 30 mg/kg of ML355 pretreatment, the level of IFN-γ in peritoneal lavage fluid significantly decreased (ng/L: 228.70±6.94 vs. 309.80±16.37, P < 0.05). The results of qRT-PCR showed that the mRNA expressions of IFN-γ and IL-1β in PMs were significantly increased after LPS administration, and the mRNA expressions of IFN-γ and Alox15 were significantly down-regulated after 15 mg/kg and 30 mg/kg ML355 pretreatment [IFN-γ mRNA (2-ΔΔCt): 1.25±0.25, 0.33±0.07 vs. 2.32±0.13, Alox15 mRNA (2-ΔΔCt): 0.10±0.01, 0.18±0.02 vs. 0.91±0.18, all P < 0.05]. (2) In vitro experiment: compared with the control group, the content of 12-HETE in the supernatant showed an increasing trend after LPS stimulated PMs for 12 hours, but there was no statistical difference. After 25 μmol/L ML355 pretreatment for 12 hours, the content of 12-HETE in PMs supernatant was significantly decreased (μg/L: 39.92±6.22 vs. 79.01±9.82, P < 0.05). Compared with the control group, the levels of IFN-γ, IL-1β, TNF-α and IL-6 in PMs supernatant of LPS group were significantly increased. After 50 μmol/L ML355 pretreatment, the level of IFN-γ in PMs supernatant was significantly decreased (ng/L: 255.30±8.82 vs. 303.10±6.76, P < 0.05). The results of qRT-PCR and Western blotting showed that 12 hours after LPS stimulation, the mRNA expressions of inflammatory factors IFN-γ and IL-1β in PMs were significantly up-regulated compared with the control group, while the mRNA expression of Alox15 had no significant change, and the phosphorylation levels of extracellular signal regulated kinases (ERK), p38MAPK, c-Jun N-terminal kinase (JNK) and the expression of 12/15-LOX protein increased significantly compared with the control group. Compared with LPS group, after 50 μmol/L ML355 pretreatment the mRNA expressions of IFN-γ and IL-1β were significantly down-regulated [IFN-γ mRNA (2-ΔΔCt): 0.16±0.05 vs. 1.25±0.03, IL-1β mRNA (2-ΔΔCt): 4.57±0.19 vs. 7.83±0.56, both P < 0.05], the phosphorylation levels of ERK, JNK and the expression of 12/15-LOX protein were inhibited after ML355 pretreatment, and the decrease of phosphorylation level of JNK was more significant when 50 μmol/L ML355 pretreatment for 12 hours [p-JNK/JNK: 0.96±0.04 vs. 1.20±0.04, P < 0.05]. However, the phosphorylation level of ERK was significantly decreased after 25 μmol/L, 50 μmol/L ML355 pretreatment for 12 hours (p-ERK/ERK: 1.49±0.18, 1.40±0.07 vs. 2.04±0.07, both P < 0.05).
Conclusions: ML355 has antagonistic effect on LPS induced inflammatory response in mice, which may be associated with the suppression on 12/15-LOX, p-ERK and p-JNK proteins.