基于斑马鱼模型的熊果酸治疗非酒精性脂肪肝病的作用及其机制研究

Effect and mechanism of ursolic acid in non-alcoholic fatty liver disease based on zebrafish model

基于斑马鱼模型,探讨熊果酸(ursolic acid,UA)对斑马鱼非酒精性脂肪肝病(non-alcoholic fatty liver disease,NAFLD)的治疗作用,研究其对NAFLD治疗的分子学机制。随机挑选野生型AB品系斑马鱼分为正常组,模型组,熊果酸组(5、10、20μg/mL)。正常组给予3.5%葡萄糖,模型组给予3.5%果糖96 h,诱导斑马鱼非酒精性脂肪肝模型。向3.5%果糖的培养液中分别给予相应低、中、高浓度的熊果酸液作为给药组,每天进行1次换液,连续96 h。收集幼鱼,检测幼鱼体重、体长以及肥满度(condition factor)以反映幼鱼的肥瘦程度,进行整体油红O染色、苏木素-伊红(HE)染色观察肝脏脂质变性情况和病理组织学损伤程度,甘油三酯(TG)、总胆固醇(TC)试剂盒检测斑马鱼体脂含量,实时荧光定量PCR(RT-qPCR)检测肝组织mTOR/SREBP-1c/ACC1/Fasn mRNA的表达。与正常组比较,模型组斑马鱼肝脏脂质沉积程度严重,斑马鱼体内TG、TC及mTOR、SREBP-1c、ACC1、Fasn mRNA的表达量均上升(P<0.05),与模型组相比,熊果酸组斑马鱼肝脏脂质沉积减少,斑马鱼体内TG、TC及的mTOR、SREBP-1c、ACC1和Fasn mRNA的表达量均降低(P<0.01),说明熊果酸对非酒精性脂肪肝具有一定的保护作用,其机制可能与调控脂质代谢有关,进而抑制肝脏脂质的合成。

Based on the zebrafish model,the therapeutic effect of ursolic acid(UA) on non-alcoholic fatty liver disease(NAFLD) in zebrafish was explored,and the molecular mechanism of NAFLD treatment was studied.The wild type AB strain zebrafish were randomly selected and divided into normal group,model group and UA group(5,10,20 μg/mL).The normal group was given 3.5% glucose,and the model group was given 3.5% fructose for 96 h to induce a non-alcoholic fatty liver model in zebrafish.The 3.5% fructose was administered with the corresponding concentration of 5,10 and 20 μg/mL UA as the administration group,and the liquid was changed once a day.Juvenile fish were collected,and the juvenile fish body weight,body length and condition factor were used to reflect the fatness of juvenile fish.Whole oil red O staining and hematoxylin-eosin(HE) staining were used to observe the liver lipid degeneration and the degree of histopathological damage.Triglyceride and total cholesterol test kits were used to detect the body fat content of zebrafish.RT-qPCR was used to detect the expression of mTOR/SREBP-1 c/ACC1/Fasn mRNA in zebrafish.Compared with the normal group,the model group had severe liver lipid deposition,and the expression levels of TG,TC,mTOR,SREBP-1 c,ACC1 and Fasn mRNA in zebrafish were increased(P<0.05).Compared with the model group,the lipid deposition in the liver of zebrafish was decreased after administration,and the expressions of TG,TC and mTOR,SREBP-1 c,ACC1 and Fasn mRNA were decreased(P<0.01),which suggested that UA has a certain protective effect on NAFLD,and the mechanism may be related to regulating lipid metabolism,thereby inhibiting liver lipid synthesis.