下瘀血汤对胆碱蛋氨酸缺乏诱导的小鼠非酒精性脂肪性肝炎的抑制作用

Xiayuxue decoction inhibits methionine-choline-deficient-induced nonalcoholic steatohepatitis in mice

目的探讨下瘀血汤对胆碱蛋氨酸缺乏(methionine-choline-deficient,MCD)诱导的小鼠非酒精性脂肪性肝炎(non-alcoholic steatohepatitis,NASH)的抑制作用。方法将24只雄性C57/BL6小鼠按照1∶2的比例随机采用蛋氨酸胆碱充足(methionine-choline-sufficient,MCS,8只)或MCD饲料喂养(16只)。造模第4周第1天,将采用MCD喂养的16只小鼠随机分为MCD对照组(简称MCD组,8只)及MCD-下瘀血汤(Xiayuxue decoction,XYXD)组(简称XYXD组,8只)。XYXD组小鼠按70 kg成人体重临床用量等体重的10倍量经口灌胃XYXD 3周,MCD对照组小鼠以等量无菌水灌胃,6周末取血清和肝组织,检测各组小鼠的肝功能、甘油三酯(triglyceride,TG)和总胆固醇(total cholesterol,TC)水平。采用免疫印迹和RT-PCR检测核苷酸结合寡聚化结构域蛋白样受体6(nucleotide-binding oligomerization-domain protein-like receptors protein 6,NLRP6)、簇分化抗原68(cluster of differentiation-68,CD68)、单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)及转化生长因子β(transforming growth factor-β,TGF-β)等的表达。采用激光共聚焦检测CD68与NLRP6的共定位。结果与MCS组相比,MCD组小鼠血清ALT、肝内TG和TC水平显著升高(P <0.05),HE及油红染色结果表明:肝组织脂肪变明显,NAS评分显著升高;RT-PCR显示:MCD组小鼠巨噬细胞标志物CD68、MCP-1等炎症相关因子和TGF-β1等致纤维化因子m RNA的相对表达量显著升高(P <0.05);免疫印迹结果显示:MCD组小鼠NLRP6蛋白表达显著下调,而p-NF-κB p65水平显著上调;激光共聚焦结果显示MCD组小鼠NLRP6与CD68共定位显著降低。与MCD组相比,XYXD组小鼠的肝功能显著改善,肝组织炎症及纤维化反应显著降低,NLRP6下调被抑制。结论下瘀血汤对MCD诱导的小鼠NASH有显著改善作用;调控巨噬细胞NLRP6的表达是下瘀血汤改善NASH的作用机制之一。

Objective To explore the inhibitory effects of the Xiayuxue decoction （XYXD） on methionine-choline- deficient （MCD）-induced non-alcoholic steatohepatitis （NASH） in mice. Methods Total of 24 male C57/BL6 mice were randomly fed with methionine-choline-sufficient （MCS） diet （8 mice） and MCD diet （16 mice） according to 1∶2 ratio. At the first day of the fourth week, the mice fed with MCD diet were randomly divided into MCD-water group （MCD group in short, 8 mice） and MCD-XYXD group （XYXD group in short, 8 mice）. Mice in XYXD group were given XYXD intragastrically at a dose of 10 times the body weight of 70 kg of adult once a day for 3 weeks, and mice in MCD group were given the same amount of water intragastrically. At the end of the sixth week, liver function and levels of triglyceride （TG） and total cholesterol （TC） were detected. The expression of nucleotide-binding oligomerization-domain protein-like receptors protein 6 （NLRP6）, cluster of differentiation-68 （CD68）, monocyte chemoattractant protein-1 （MCP-1）and transforming growth factor-β （TGF-β） were detected by RT-PCR and Western blot. Confocal laser scanning was used to detect the co-location of CD68 and NLRP6. Results Compared with mice in MCS group, the levels of serum ALT, liver TG and TC of mice in MCD group increased significantly （P 〈 0.05）; HE and oil red staining showed that the fatty degeneration of liver tissue and NAS score of mice in MCD group increased significantly; RT-PCR showed that the relative expression of mRNA of macrophage markers CD68, MCP-1 and fibrosis factors like TGF-β1 of mice in MCD group increased significantly （P 〈 0.05）; Western blot showed that the expression of NLRP6 decreased significantly, whereas the expression of p-NF-κB p65 up- regulated significantly of mice in MCD group. Laser confocal microscopy showed that the co-localization of NLRP6 and CD68 decreased significantly of mice in MCD group. Compared with mice in MCD group, inflammation and fibrosis in liver tissue reduced significantly and the downregulation of NLRP6 was inhibited in XYXD group. Conclusions Xiayuxue decoction can significantly improve NASH induced by MCD in mice. Regulating the expression of NLRP6 in macrophages is one of the mechanisms of Xiayuxue decoction in improving NASH.