5-脂氧合酶激活蛋白选择性抑制剂MK886对酒精性肝病小鼠模型的影响

目的 旨在探究5-脂氧合酶激活蛋白(FLAP)的选择性抑制剂MK886对小鼠酒精性肝病(ALD)的影响。方法 48只雄性昆明种小鼠随机分为4组,ALD组、ALD/MK886组给予Tipoe-Nanji酒精液体饲料,6周后酒精灌胃1次形成慢加急性ALD模型,对照组给予不含酒精的对照饲料,正常组给予普通饲料。小鼠开始摄入酒精2 d后,ALD/MK886组小鼠腹腔开始注射MK886(0.01 mg/10 g,1次/d)。ALD组及ALD/MK886组酒精灌胃后9 h处死小鼠。检测血清中AST、ALT、乳酸脱氢酶(LDH)、TG及肝组织中TG和丙二醛(MDA)水平,肝组织行HE染色并进行病理评分,Western Blot法测定肝组织/Thp-1细胞中FLAP和5-脂氧合酶(5-LO)的表达水平,流式细胞法检测Thp-1细胞的凋亡水平。多组间比较采用单因素方差分析,方差齐时用LSD- t 检验进行两两比较,方差不齐时采用Tamhane’T2进行检验。结果 ALD组、ALD/MK886组在造模第1周体质量减轻,随后逐渐增加,至造模结束时ALD组小鼠体质量和肝指数明显低于正常组和对照组( P 值均<0.05),ALD/MK886组小鼠体质量和肝指数明显高于ALD组(P 值均<0.05)。ALD组小鼠血清AST、ALT、LDH、TG及肝组织TG、MDA较正常组和对照组显著升高( P 值均<0.05);与ALD组比较,ALD/MK886组小鼠相关血清及肝组织生化指标明显降低(P 值均<0.05)。ALD组小鼠肝组织脂肪变评分明显高于正常组和对照组(P 值均<0.05),与ALD组比较,ALD/MK886组小鼠肝组织脂肪变评分明显降低(P <0.05)。ALD组肝脏5-LO、FLAP表达较正常组和对照组显著增加(P 值均<0.05),ALD/MK886组较ALD组明显降低(P <0.05)。脂多糖(LPS)上调Thp-1细胞FLAP和5-LO的表达,MK886减轻了LPS的作用(P 值均<0.05)。MK886促进Thp-1细胞凋亡,LPS减轻MK886促进细胞凋亡的作用( P 值均<0.01)。结论 MK886可通过抑制5-LO的表达从而诱发Kupffer细胞凋亡,发挥对小鼠ALD的治疗作用。

Role of the specific inhibitor of 5-lipoxygenase-activating protein MK886 in inhibiting alcoholic liver disease in mice

Objective To investigate the effect of MK886, a specific inhibitor of 5-lipoxygenase-activating protein (FLAP), on alcoholic liver disease (ALD) in mice. Methods A total of 48 male Kunming mice were randomly divided into ALD group, ALD/MK886 group, control group, and normal group. The mice in the ALD group and the ALD/MK886 group were fed with Tipoe-Nanji alcohol liquid diet and were given alcohol by gavage 6 weeks later to establish a model of acute ALD. The mice in the control group were given control diet without alcohol, and those in the normal group were given normal diet. After 2 days of alcohol intake, the mice in the ALD/MK886 group were given intraperitoneally injected MK886 (0.01 mg/10 g) once a day. The mice were sacrificed at 9 hours after the administration of alcohol by gavage. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and triglyceride (TG) and the levels of TG and malondialdehyde (MDA) in liver tissue were measured. HE staining was performed for liver tissue and pathological score was determined. Western blot was used to measure the expression of FLAP and 5-lipoxygenase (5-LO) in liver tissue and Thp-1 cells, and flow cytometry was used to measure the apoptosis of Thp-1 cells. One-way ANOVA was used for comparison between groups;the least significant difference t -test was used for comparison of data with homogeneity of variance, and the Tamhane’s T2 test was used for comparison of data with heterogeneity of variance. Results The ALD group and the ALD/MK886 group had a reduction in body weight within the first week of modeling, followed by a gradual increase;at the end of modeling, the ALD group had significantly lower body weight and liver index than the normal group and the control group (all P <0.05), and the ALD/MK886 group had significantly higher body weight and liver index than the ALD group (both P <0.05). Compared with the normal group and the control group, the ALD group had significant increases in the levels of AST, ALT, LDH, and TG in serum and the levels of TG and MDA in liver tissue (all P <0.05), and compared with the ALD group, the ALD/MK886 group had significant reductions in the above indices (all P <0.05). The ALD group had a significantly higher score of hepatic steatosis than the normal group and the control group (both P <0.05), and compared with the ALD group, the ALD/MK886 group had a significant reduction in this score ( P <0.05). Compared with the normal group and the control group, the ALD group had significant increases in the expression of 5-LO and FLAP in the liver (all P <0.05), and the ALD/MK886 group had significant reductions compared with the ALD group ( P <0.05). Lipopolysaccharide (LPS) upregulated the expression of FLAP and 5-LO in Thp-1 cells, and MK886 alleviated such effect of LPS (all P <0.05);MK886 promoted the apoptosis of Thp-1 cells, and LPS alleviated the effect of MK886 in promoting cell apoptosis (all P <0.01). Conclusion MK886 can inhibit the expression of 5-LO, induce the apoptosis of Kupffer cells, and thus exert a therapeutic effect on ALD in mice.