目的 利用基因敲除技术探究Plin1基因在槲皮素干预MAFLD模型小鼠脂代谢和AMPK/SREBP1通路相关mRNA及蛋白表达变化过程中可能发挥的关键作用。方法 实验共分为6组：24只4 w龄雄性C57BL/6J小鼠分为3组：对照组（C）、MAFLD模型组（M）、400 mg/(kg·bw·d)槲皮素干预组（400Q）;24只4 w龄雄性C57BL/6J Plin1基因敲除小鼠分为3组：敲除对照组（KO-C）、敲除模型组（KO-M）、400 mg/(kg·bw·d)槲皮素干预敲除模型组（KO-400Q）,每组8只。C组和KO-C组饲以普通饲料,其他四组饲以60%高脂饲料,400Q组和KO-400Q组用40 mg/ml槲皮素溶液灌胃,其他四组用生理盐水灌胃,各组灌胃溶液体积据小鼠体重10 μl/g算得。喂养12 w后处死,比较各组小鼠体重,称量肝重,计算肝指数;检测小鼠血清和肝脏TC、TG、ALT、AST含量;油红O染色观察肝脏脂滴情况;检测肝组织中PLIN1、AMPK、SREBP1、FASN、SCD1、ACC1蛋白及相关mRNA的表达水平。结果 与M组相比,槲皮素干预后体重下降,血清及肝脏TC、TG、ALT、AST下降,肝脏脂滴减少,Plin1 mRNA及其蛋白表达上升（P<0.05）;Plin1基因敲除后,血清TC、TG、AST及肝脏TC、TG、ALT、AST水平升高（P<0.05）,400Q和KO-C组AMPK mRNA表达上升,SREBP1及其下游脂质合成基因mRNA表达下降,各蛋白与之变化吻合（P<0.05）,KO-400Q组小鼠血清和肝脏TC、TG、ALT、AST水平升高,肝重及肝指数增高,肝脏脂滴增加,AMPK mRNA表达下降,SREBP1及其下游脂质合成基因mRNA表达升高,各蛋白与之变化吻合（P<0.05）。结论 槲皮素可以改善高脂饮食诱导的MAFLD模型小鼠体内的肝脏脂质蓄积,敲除Plin1基因消除了槲皮素带来的保护性效应,反向验证槲皮素可能通过调节Plin1基因表达影响MAFLD模型小鼠AMPK/SREBP1通路相关mRNA和蛋白的表达来发挥它的保护性作用。
Abstract
Objective To explore the possible key role of Plin1 gene in the action of quercetin on lipid metabolism and AMPK/SREBP1 pathway related mRNA and protein expressions in MAFLD mice. Methods Twenty-four 4 w-old male C57BL/6J mice were divided into three groups: control group (C), MAFLD model group (M), and 400 mg/(kg·bw·d )quercetin treatment group (400Q). Twenty-four C57BL/6J male Plin1 knockout mice were divided into three groups: knockout control group (KO-C), knockout model group (KO-M), and 400 mg/(kg·bw·d) quercetin knockout model group (KO-400Q), with 8 mice in each group. Group C and KO-C were fed a normal diet, and the other four groups were fed a 60% high-fat diet. The 400Q and KO-400Q groups were given 40 mg/ml quercetin solution by gavage, and the other four groups were given normal saline by gavage. The volume of quercetin solution was calculated as 10 μl/g according to the body weight of the mice. The mice were sacrificed after feeding for 12 weeks. The body weight and liver weight were recorded. The levels of TC, TG, ALT and AST in serum and liver were detected. Liver lipid droplets were checked after oil red Ο staining. The protein and mRNA expression levels of PLIN1, AMPK, SREBP1, FASN, SCD1 and ACC1 in the liver were measured. Results Compared with the group M, the body weight, serum and liver TC, TG, ALT, AST and lipid droplets in the liver were decreased, and the expressions of Plin1 mRNA and protein were increased after quercetin intervention (P<0.05). After Plin1 gene knockout, serum and liver TC, TG, ALT, AST levels were increased (P<0.05). The AMPK mRNA expression was increased and the mRNA expression of SREBP1 and its downstream lipid synthesis genes decreased in the 400Q and KO-C groups (P<0.05). The serum and liver TC, TG, ALT and AST levels, liver weight and index of the KO-400Q group were increased. The liver lipid droplets were increased, while the expression of AMPK mRNA was decreased, and the mRNA expression of SREBP1 and its downstream lipid synthesis genes were increased (P<0.05). Conclusion Quercetin treatment improves liver lipid accumulation in MAFLD mice. Knockout of Plin1 abolished the protective effect of quercetin. Quercetin exerts its protective effects possibly by regulating Plin1 gene expression and affecting AMPK/SREBP1 pathway related mRNA and protein expression in MAFLD mice.
关键词
槲皮素 /
代谢相关脂肪性肝病 /
Plin1 基因敲除 /
小鼠
Key words
quercetin /
metabolic associated fatty liver disease /
Plin1 gene knockout /
mice
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基金
山西省自然科学基金 (No.201901D111182)
