Antagonistic Effect Of ML355 On Lipopolysaccharide Induced Inflammatory Response In Mice And Its Molecular Mechanism

Objective:To investigate the antagonistic effect of ML355,a 12-Lipoxygenase(12-LOX)inhibitor,on lipopolysaccharide(LPS)-induced inflammatory response in mice and its molecular mechanism.Methods:1.24 adult male C57BL/6 mice were randomly divided into 4 groups,control group,LPS group(intraperitoneal injection of LPS 20mg/kg),ML355 pretreatment group(30 mg/kg?15 mg/kg of ML355 intraperitoneal injection 1h in advance+LPS).The mice were killed 12 hours after the model was established,and the peripheral blood,peritoneal lavage fluid and peritoneal macrophages were collected.The levels of plasma and peritoneal lavage fluid 12-Hydroxyeicosatetraenoic acid(12-HETE),peritoneal lavage fluid interferon-y(IFN-?)?interleukin(IL-1??IL-6?IL-10)?tumor necrosis factor-?(TNF-?)and serum IFN-y?IL-1? were measured by enzyme linked immunosorbent assay(ELISA).The mRNA expressions of IFN-? IL-1? and Alox-15 were detected by Real time quantitative polymerase chain reaction(qRT-PCR).2.The primary peritoneal macrophages(PMs)of mice were cultured in vitro and randomly divided into four groups:control group,LPS group(LPS 20mg/L),ML355 pretreatment group(25umol/L?50umol/L of ML355 interfere 1 hour in advance+LPS).The supernatant and cell mass were collected at different times stimulated by LPS.The mRNA expressions of IFN-?,IL-1? and Alox-15 were detected by qRT-PCR,the levels of 12-HETE?IFN-??IL-1??IL-6?TNF-? and IL-10 in the supernatant were measured by ELISA,and the expressions of 12/15-LOX and MAPK pathway protein were detected by Western blot.Results:1.The contents of plasma and peritoneal lavage fluid 12-HETE in LPS model group was significantly higher than that in control group.Compared with LPS model group,ML355 pretreatment can significantly reduce the contents of 12-HETE in plasma and peritoneal lavage fluid,and the levels of IFN-y in serum and peritoneal lavage fluid.ML355 can significantly down regulate the levels of IFN-?,IL-1? and Alox-15 expression.2.In vitro experiment,compared with the control group,the content of 12-HETE in the supernatant showed an increasing trend after LPS stimulated peritoneal macrophages,but there was no statistical difference.After ML355 administration,the content of 12-HETE was significantly decreased.Compared with LPS group,the level of IFN-y in cell supernatant was significantly decreased,the gene expressions of IFN-y and IL-1? was down-regulated,the phosphorylation level of p38 MAPK has no significant difference,while the phosphorylation levels of ERK?JNK and the expression of 12/15-LOX protein decreased after ML355 pretreatment.Conclusion:(1)ML355 inhibited LPS induced inflammatory response in mice.(2)ML355 can significantly reduce the production of IFN-y in peritoneal macrophages stimulated by LPS,which may be associated with the suppression on 12/15-LOX,p-ERK and p-JNK proteins.