地锦草提取物通过miR-106-5p/TLR4信号通路改善非酒精性脂肪性肝炎

Euphorbia humifusa extract ameliorates nonalcoholic steatohepatitis through regulating miR-106-5p/TLR4 pathway in mice

研究地锦草(Euphorbia humifusa Willd.)提取物对高脂饲料联合葡聚糖硫酸钠(dextran sodium sulfate,DSS)诱导的非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)的改善作用及其干预的分子机制。将SPF级别C57BL/6小鼠雌雄对半随机分为正常对照组,NASH模型组,地锦草提取物低、高剂量组(n=10)。对照组给予正常饮水和食物。模型组和药物组在实验全程的14 d中均给予高脂饲料喂养,在此期间药物组进行灌胃给药,从第8 d开始给予持续饮用2%DSS水7 d。实验结束后杀小鼠,检测小鼠血清中甘油三酯(TG),谷丙转氨酶(ALT)含量;H&E染色法观察小鼠肝组织病理学变化;根据生物信息学及前期研究预测靶向调节TLR4的miRNA;采用RT-qPCR法检测小鼠肝组织中miR-106-5p、Toll样受体4(TLR4)、IL-6及TNF-α基因的相对表达情况;Western blot法分析小鼠肝脏中TLR4、IL-1β蛋白水平表达变化。与正常组相比,模型组小鼠TG、ALT均有升高。H&E染色结果显示肝脏出现明显脂肪变性和炎症反应。RT-qPCR结果显示TLR4和炎性因子(IL-6、TNF-α)表达明显升高,而靶向调节TLR4的miR-106-5p表达降低(P<0.05)。Western blot结果显示模型组肝脏中TLR4、IL-1β蛋白表达水平升高(P<0.05)。经药物干预后,小鼠TG、ALT均有所下降;肝组织中TLR4和炎性因子mRNA相对表达水平达降低,miR-106-5p表达上升;TLR4、IL-1β蛋白表达水平下降(P<0.05)。地锦草提取物可有效改善高脂饲料联合DSS诱导的NASH的炎症情况及脂肪沉积,其机制可能与调控miR-106-5p/TLR4信号通路有关。

To explore the effect of Euphorbia humifusa Willd.on nonalcoholic steatohepatitis(NASH)induced by high fat diet and dextran sulfate sodium(DSS),and the molecular mechanism of its intervention.Male and female SPF grade C57BL/6 mice were randomly divided into a normal control group,a model group,and low and high dose groups of E.humifusa,10 mice in each group.The normal control group was not given any intervention,while both the model and treatment groups were fed high fat diet throughout the 14 days of the experiment,during which time the E.humifusa were orally administered by gavage daily to the mice of treatment groups.Starting from the 8th day,the model and treatment groups were given 2%DSS water for 5-7 days.Mice were executed at the end of the experiment,the serum triglyceride(TG)and alanine aminotransferase(ALT)contents were detected for all the mice.The pathological changes of mouse liver tissue were observed by H&E staining.The miRNA targeting to TLR4 was predicted based on bioinformatics and pre-experiments.The relative expressions of miR-106-5p,Toll-like receptor 4(TLR4),IL-6 and TNF-αin the liver tissue of mice were detected by RT-qPCR.The expression levels of TLR4 and IL-1βproteins in the liver of mice were analyzed by Western blot.Compared with the normal group,the serum TG and ALT of the model group were increased.H&E staining results showed significant hepatocyte steatosis and inflammatory reaction in the model liver tissues.RT-qPCR results showed that the expressions of TLR4 and inflammatory factors(IL-6,and TNF-α)were significantly increased,while miR-106-5p which targets the TLR4,was reduced in the liver tissues of model mouse(P<0.05).Western blot results further confirmed that the expression levels of TLR4 and IL-1βproteins were increased in the liver of the model group.After the intervention with E.humifusa,the serum TG and ALT were decreased(P<0.05),the relative mRNA expression levels of TLR4 and inflammatory factors(IL-6 and TNF-α)in liver tissues were decreased,while the expression of miR-106-5p was increased compared with model group.Similarly,the expression levels of TLR4 and IL-1βproteins were decreased(P<0.05).The E.humifusa can effectively alleviates the inflammatory condition and fat deposition in NASH induced by high-fat diet combined with DSS,and its mechanism may be related to the regulation of the miR-106-5p/TLR4 signaling pathway.