摘要
目的探讨肝脏生物钟相关基因的昼夜节律变化在二氢杨梅素改善高脂喂养小鼠肝脏脂代谢中的作用。方法按随机数表法将150只6周龄C57BL/6J雄性小鼠分为普通饲料组(CON)、高脂饲料组(HFD)和高脂饲料+二氢杨梅素100 mg/(kg·d)干预组(HFD+DHM)。干预12周结束后,取小鼠肝脏组织进行油红O染色,检测小鼠肝脏甘油三酯含量;根据给予光照时刻(zeitgeber time,ZT; 8:00给予光照记为ZT0)分别在ZT18、ZT6两个时间点按照两步灌注法提取小鼠肝细胞,立即检测耗氧率(oxygen consumption rate,OCR);每隔4 h分别提取ZT0、ZT4、ZT8、ZT12、ZT16、ZT20共6个时间点的小鼠肝脏组织,采用荧光定量PCR和Western blot分别检测肝脏生物钟相关基因[钟基因(circadian locomotor output cycles kaput,CLOCK),脑和肌肉组织芳香烃受体核转运蛋白的类似蛋白1 (brain and muscle-arnt-like1,BMAL1),周期蛋白2(period2,PER2)]的mRNA及蛋白表达。结果与CON组比较,HFD组肝脏油红O染色见组织间隙有大量脂肪滴蓄积,肝组织甘油三酯含量显著增高(P <0. 05),ZT18和ZT6肝细胞的基础OCR和最大OCR均显著降低(P <0. 05),肝脏组织生物钟基因CLOCK、BMAL1 mRNA和蛋白表达在ZT0、ZT4、ZT8、ZT20显著降低,PER2在ZT4、ZT8、ZT16、ZT20显著降低(P <0. 05)。二氢杨梅素干预显著抑制了高脂喂养诱导的肝脏脂肪蓄积、甘油三酯的升高以及肝细胞ZT18和ZT6的基础OCR和最大OCR的降低(ZT18显著高于ZT6),并且明显拮抗高脂诱导的肝脏生物钟基因mRNA和蛋白的表达。结论二氢杨梅素干预可部分恢复非酒精性脂肪肝小鼠肝脏生物钟相关基因表达节律紊乱,进而改善肝脏脂代谢。
Objective To investigate the possible role of circadian rhythm variation of hepatic clockrelated genes in the improvement of hepatic lipid metabolism in high-fat diet（ HFD）-induced mice after dihydromyricetin（ DHM） treatment. Methods A total of 150 C57 BL/6 J male mice（ 6 weeks old） were randomly divided into normal diet group（ CON）,HFD group and HFD ＋ DHM treatment [100 mg/（ kg·d） ]group. After 12 weeks of intervention,the liver tissues of mice were stained with oil O red,and the liver triglyceride levels were detected. The hepatocytes of each group were extracted at ZT18 and ZT6 by a two-step perfusion method according to the Zeitgeber time（ ZT0 represents the light time point at 8： 00 am）. The oxygen consumption rate（ OCR） was detected immediately. The liver tissues were also harvested at ZT0,ZT4,ZT8,ZT12,ZT16 and ZT20,respectively. The mRNA and protein expression levels of hepatic clockrelated genes,circadian locomotor output cycles kaput（ CLOCK）,brain and muscle-arnt-like 1（ BMAL1）,and Period2（ PER2） were detected by real-time PCR and Western blot analysis. Results The HFD group presented dominant accumulation of fat droplets in the interstitial space after oil O red staining and increased triglyceride content in the liver tissues（ P〈0. 05）,and significantly reduced basic OCR and maximum respiratory OCR level at the ZT18 and ZT6（ P〈0. 05） when compared with the CON group. HFD feeding led to significantly decreased expression of CLOKC and BMAL1 at mRNA and protein levels at ZT0,ZT4,ZT8 and ZT20,and to that of PER2 at ZT4,ZT8,ZT16 and ZT20 in liver tissues（ P〈0. 05）. Furthermore,DHM treatment significantly inhibited HFD-induced fat accumulation and hepatic triglyceride elevation as well as the decreased levels of the basis OCR and maximal respiratory OCR at ZT18 and ZT6 in the hepatocytes（ with that of ZT18 significantly higher than that of ZT6）. Besides,it also significantly antagonized HFDinduced changes of mRNA and protein expression of CLOC
作者
马鑫
时皎皎
陈卡
易龙
朱俊东
糜漫天
MA Xin;SHI Jiaojiao;CHEN Ka;YI Long;ZHU Jundong;MI Mantian(Research Center for Nutrition and Food Safety,Chongqing Key Laboratory of Nutrition and Food Safety,Chongqing Medical Center of Nutrition,Faculty of Military Preventive Medicine,Army Medical University（Third Military Medical University）,Chongqing,400038;Department of Rehabilitation Medicine,3Department of Nutrition,General Hospital of Chengdu Military Command,Chengdu,Sichuan Province,610083,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2018年第21期1956-1961,共6页
Journal of Third Military Medical University
基金
全军后勤科研"十三五"计划重大项目(AWS17J014)~~
关键词
二氢杨梅素
非酒精性脂肪肝
生物钟基因
昼夜节律
dihydromyricetin
non-alcoholic fatty liver disease
clock genes
circadian rhythm
