苦荞醋饮及沙棘醋饮在代谢相关脂肪性肝病斑马鱼幼鱼中的应用

Application of tartary buckwheat vinegar and sea⁃buckthorn vinegar in zebrafish larvae with metabolic associated fatty liver disease

目的:探讨苦荞醋饮及沙棘醋饮在代谢相关脂肪性肝病斑马鱼幼鱼中的应用效果。方法:选取受精后5 d斑马鱼幼鱼,适应性喂养3 d后随机分为对照组、高胆固醇饮食组、苦荞醋饮组、沙棘醋饮组、苦荞醋饮联合沙棘醋饮组,每组100条幼鱼。所有实验组和对照组均设置2个平行组。对照组给予标准普通饲料20 mg,高胆固醇组给予高胆固醇饲料20 mg,苦荞醋饮组给予高胆固醇饲料20 mg+0.1%苦荞醋饮,沙棘醋饮组给予高胆固醇饲料20 mg+0.1%沙棘醋饮,苦荞醋饮联合沙棘醋饮组给予高胆固醇饲料20 mg+0.05%苦荞醋饮+0.05%沙棘醋饮。干预1周后,观察斑马鱼幼鱼存活率、体重、体长、三酰甘油、总胆固醇、丙二醛,采用冰冻油红O染色观察肝内脂质积累情况,采用苏木素-伊红(HE)染色观察肝脏组织病理形态,通过荧光摄影判断肝脏内活性氧水平,采用实时荧光定量聚合酶链式反应(qPCR)法检测脂质代谢、氧化应激和炎症相关基因信使核糖核酸(mRNA)表达。结果:与高胆固醇饮食组相比,苦荞醋饮组、沙棘醋饮组、苦荞醋饮联合沙棘醋饮组斑马鱼幼鱼体重较轻(均P<0.05),沙棘醋饮组斑马鱼幼鱼总胆固醇较低(P<0.05),苦荞醋饮联合沙棘醋饮组斑马鱼幼鱼丙二醛较低(P<0.05);苦荞醋饮组、沙棘醋饮组、苦荞醋饮联合沙棘醋饮组肝脏处绿色荧光面积较小,颜色较暗,荧光定量分析显示,苦荞醋饮组和苦荞醋饮联合沙棘醋饮组较低(均P<0.05);qPCR结果显示,沙棘醋饮组斑马鱼幼鱼肿瘤坏死因子α的mRNA表达水平较低(P<0.05);苦荞醋饮组、沙棘醋饮组、苦荞醋饮联合沙棘醋饮组斑马鱼幼鱼白细胞介素1β的mRNA表达水平较低(均P<0.05)。结论:喂食醋饮主要通过减轻氧化应激、抑制炎症反应发挥对代谢相关脂肪性肝病斑马鱼幼鱼的保护作用。

Objective:To explore the application effect of tartary buckwheat vinegar and sea-buckthorn vinegar in zebrafish larvae with metabolic associated fatty liver disease.Methods:Zebrafish larvae with 5 days post fertilization were selected for adaptive feeding for 3 days,then they were randomly divided into control group,high cholesterol diet group,tartary buckwheat vinegar group,sea-buckthorn vinegar group,and tartary buckwheat vinegar combined with sea-buckthorn vinegar group,100 pieces in each group.All experimental groups and control group were set up with 2 parallel groups.The control group was given 20 mg of standard common diet,the high cholesterol group was given 20 mg of high cholesterol diet,the tartary buckwheat vinegar drink group was given 20 mg of high cholesterol diet and 0.1%tartary buckwheat vinegar,the sea-buckthorn vinegar group was given 20 mg of high cholesterol diet and 0.1%sea-buckthorn vinegar,and tartary buckwheat vinegar combined with sea-buckthorn vinegar group was given 20 mg of high cholesterol diet,0.05%tartary buckwheat vinegar and 0.05%sea-buckthorn vinegar.After one week of continuous intervention,survival rate,body weight,body length,triacylglycerol,total cholesterol,malondialdehyde of zebrafish larvae were observed.Lipid accumulation in liver was observed by freeze-oil red O staining and pathological morphology of liver tissue was observed by Hematoxylin-Eosin staining.Reactive oxygen species in liver was judged by fluorescence photography,and the mRNA expression of lipid metabolism,oxidative stress and inflammation were tested by qPCR method.Results:Compared with high cholesterol diet group,the body weight of zebrafish larvae in tartary buckwheat vinegar group,sea-buckthorn vinegar group,and tartary buckwheat vinegar combined with sea-buckthorn vinegar group were lower(all P<0.05),the total cholesterol of zebrafish larvae in sea-buckthorn vinegar group was lower(P<0.05),and malondialdehyde of zebrafish larvae in tartary buckwheat vinegar combined with sea-buckthorn vinegar group was lower(P<0.05).The green fluorescence area of liver in tartary buckwheat vinegar group,sea-buckthorn vinegar group,and tartary buckwheat vinegar combined with sea-buckthorn vinegar group were smaller and the color was darker.Fluorescence quantitative analysis showed that tartary buckwheat vinegar group and tartary buckwheat vinegar combined with sea-buckthorn vinegar group were lower(both P<0.05).qPCR method results showed that mRNA expression level of TNF-αof zebrafish larvae in sea-buckthorn vinegar group was lower(P<0.05),and mRNA expression level of IL-1βof zebrafish larvae in tartary buckwheat vinegar group,sea-buckthorn vinegar group,and tartary buckwheat vinegar combined with sea-buckthorn vinegar group were lower(both P<0.05).Conclusions:Vinegar diet has protective effects on zebrafish larvae with metabolic associated fatty liver disease by reducing oxidative stress and inhibiting inflammatory reaction.