12-脂氧合酶抑制剂ML355对小鼠经脂多糖诱导炎症反应的拮抗效应

Antagonistic effect of 12-lipoxygenase inhibitor ML355 on lipopolysaccharide induced inflammatory response in mice

目的探讨12-脂氧合酶(12-LOX)抑制剂ML355对小鼠经脂多糖(LPS)诱导的炎症反应的拮抗效应及其分子机制。方法①体内实验:将24只成年雄性C57BL/6小鼠按随机数字表法分为对照组〔腹腔注射100μL二甲亚砜(DMSO)后1 h再给予0.9%的生理盐水〕、LPS组(腹腔注射LPS 20 mg/kg)、ML355预处理组(给予LPS前1 h腹腔注射ML35515 mg/kg、30 mg/kg进行预处理)。制模后12 h处死小鼠,收集外周血、腹腔灌洗液和腹腔巨噬细胞(PMs),采用酶联免疫吸附试验(ELISA)检测各组血浆和腹腔灌洗液中12-羟基二十碳四烯酸(12-HETE)含量、腹腔灌洗液中γ-干扰素(IFN-γ)、白细胞介素(IL-1β、IL-6、IL-10)、肿瘤坏死因子-α(TNF-α)及血清中IFN-γ、IL-1β水平,采用实时定量聚合酶链反应(qRT-PCR)检测各组小鼠PMs中IFN-γ、IL-1β及花生四烯酸-15-脂氧合酶(Alox15)的mRNA表达。②体外实验:体外培养小鼠原代PMs,按随机数字表法分为对照组(加入终浓度为0.3%的DMSO)、LPS组(给予LPS 20 mg/L)、ML355预处理组(给予LPS前1 h加入25μmol/L、50μmol/L的ML355预处理)。于LPS作用不同时间点收集各组细胞上清液和细胞团块,用细胞计数试剂盒-8(CCK-8)检测ML355对PMs细胞存活率的影响,采用ELISA法检测各组PMs中12-HETE、IFN-γ、IL-1β、TNF-α、IL-6、IL-10的含量,用qRT-PCR检测各组细胞中IFN-γ、IL-1β及Alox15的mRNA表达,用蛋白质免疫印迹试验(Western blotting)检测12/15-脂氧合酶(12/15-LOX)及丝裂素活化蛋白激酶(MAPK)的蛋白表达。结果①体内实验:作用12 h,LPS组血浆和腹腔灌洗液中12-HETE以及腹腔灌洗液中炎性因子、血清中IFN-γ、IL-1β含量均较对照组显著增加。与LPS组比较,15 mg/kg和30 mg/kg ML355预处理后血浆和腹腔灌洗液中12-HETE及血清中IFN-γ含量均明显减少〔12-HETE:血浆(mg/L)为11.59±1.80、11.58±1.75比18.59±1.68,腹腔灌洗液(μg/L)为355.80±59.47、196.30±88.98比712.10±44.55;血清中IFN-γ(ng/L):147.60±2.94、114.20±2.94比326.40±2.22,均P<0.05〕,用30 mg/kg ML355预处理后小鼠腹腔灌洗液中IFN-γ水平明显降低(ng/L:228.70±6.94比309.80±16.37,P<0.05);qRT-PCR结果显示,给予LPS后,PMs中IFN-γ、IL-1β的mRNA表达水平明显升高,用15 mg/kg和30 mg/kg ML355作用后均可明显下调IFN-γ及Alox15的mRNA表达水平〔IFN-γmRNA(2^(-ΔΔCt)):1.25±0.25、0.33±0.07比2.32±0.13,Alox15 mRNA(2^(-ΔΔCt)):0.10±0.01、0.18±0.02比0.91±0.18,均P<0.05〕。②体外实验:与对照组比较,LPS刺激PMs 12 h后,小鼠PMs上清液中12-HETE含量呈升高趋势,但差异无统计学意义,给予25μmol/L ML355预处理12 h后小鼠PMs上清液中12-HETE含量较LPS组明显降低(μg/L:39.92±6.22比79.01±9.82,P<0.05);与对照组比较,LPS组小鼠PMs上清液中IFN-γ、IL-1β、TNF-α、IL-6水平均明显升高,给予50μmol/L的ML355预处理后,PMs上清液中IFN-γ明显降低(ng/L:255.30±8.82比303.10±6.76,P<0.05);qRT-PCR和Western blotting结果显示,LPS刺激12 h后,PMs中炎性因子IFN-γ、IL-1β的mRNA表达较对照组明显上调,Alox15 mRNA表达无显著变化,细胞外信号调节微酶(ERK)、p38MAPK、c-Jun氨基末端激酶(JNK)的磷酸化水平及12/15-LOX蛋白表达均较对照组明显增加;与LPS组比较,用50μmol/L ML355预处理后IFN-γ、IL-1β的mRNA表达明显下调〔IFN-γmRNA(2^(-ΔΔCt)):0.16±0.05比1.25±0.03,IL-1βmRNA(2^(-ΔΔCt)):4.57±0.19比7.83±0.56,均P<0.05〕,ERK、JNK的磷酸化水平及12/15-LOX蛋白表达水平均受到抑制,JNK磷酸化水平的降低以50μmol/L ML355作用12 h更显著(p-JNK/JNK:0.96±0.04比1.20±0.04,P<0.05),而25μmol/L和50μmol/L ML355作用12 h后ERK的磷酸化水平均明显降低(p-ERK/ERK:1.49±0.18、1.40±0.07比2.04±0.07,均P<0.05)。结论ML355对小鼠经LPS诱导的炎症反应具有拮抗效应,其机制可能是通过抑制12/15-LOX、p-ERK、p-JNK的蛋白表达水平来实现的。

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.Methods①In vivo experiment:24 adult male C57BL/6 mice were randomly divided into control group[intraperitoneal injection of 100μL 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-1βwere measured by enzyme linked immunosorbent assay(ELISA).The mRNA expressions of IFN-γ,IL-1βand arachidonic 15-lipoxygenase(Alox15)in mice PMs were detected by quantitative real time polymerase chain reaction(qRT-PCR).②In 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 25μmol/L,50μmol/L of ML3551 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-1βand 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.Results①In 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-γand IL-1βin 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-γin 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-γin 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-γand IL-1βin PMs were significantly increased after LPS administration,and the mRNA expressions of IFN-γand Alox15 were significantly down-regulated after 15 mg/kg and 30 mg/kg ML355 pretreatment[IFN-γmRNA(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].②In 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 25μmol/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-αand IL-6 in PMs supernatant of LPS group were significantly increased.After 50μmol/L ML355 pretreatment,the level of IFN-γin 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-γand IL-1βin 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 50μmol/L ML355 pretreatment the mRNA expressions of IFN-γand IL-1βwere significantly down-regulated[IFN-γmRNA(2^(-ΔΔCt)):0.16±0.05 vs.1.25±0.03,IL-1βmRNA(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 50μmol/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 25μmol/L,50μmol/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).Conclusion 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.