摘要
旨在研究橙皮苷(Hesperidin, HDN)对高脂饲喂诱导的小鼠肝氧化应激损伤的保护作用及作用机制。将18只雄性C57BL/6(体重20~23 g)小鼠,随机分为对照(control)组、高脂饮食(HFD)组、高脂饮食+橙皮苷(HFD+HDN)组(300 mg·kg^(-1)),每组6只。control组小鼠饲喂常规饲料(脂肪含量10%、碳水化合物含量70%、蛋白质含量20%);HFD组小鼠饲喂高脂饲料(脂肪含量60%、碳水化合物含量20%、蛋白质含量20%);HFD+HDN组在饲喂高脂饲料的同时每天灌胃给药HDN 300 mg·kg^(-1)。16周后腹腔注射3%戊巴比妥钠(60 mg·kg^(-1))麻醉小鼠后进行眼球采血,采血完毕后对小鼠进行脱颈处死并解剖取肝组织;使用试剂盒检测血液中肝功能指标丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)的活性;使用试剂盒检测肝组织中氧化应激指标丙二醛(MDA)、总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-Px)以及总抗氧化能力(T-AOC)的水平;采集肝组织进行转录组学测序,筛选出HFD组和HFD+HDN组的差异表达基因集进行KEGG通路富集分析,以P<0.05作为显著性富集的阈值,据此筛选出HDN干预后影响最显著的一条信号通路;从筛选出的信号通路上挑选显著变化的基因并采取qRT-PCR和蛋白免疫印迹的方法验证转录组学结果;检测mtDNA相对含量和线粒体外膜蛋白(TOMM20)相对表达量;检测肝组织ATP含量。结果显示:与HFD组相比,HDN干预改善了高脂饲喂引起的肝损伤,显著降低了小鼠血液中ALT以及AST活性(P<0.01);显著降低小鼠肝MDA含量(P<0.01);显著升高T-AOC、T-SOD以及GSH-Px水平(P<0.05),KEGG富集分析,筛选出氧化磷酸化(OXPHOS)途径是最显著上调的信号通路(P<0.000 1);与HFD组相比,HDN干预后肝组织Cox8b、Cox6a2、Gm10231、mt-Atp8、mt-Nd4l、Gm11237、Ndufb8、Ndufb10的mRNA相对表达量显著上调(P<0.05),Atp6v0d2、Cox6c2的mRNA的相对表达量显著下调(P<0.05)。Cox6a2、Ndufb8、Ndufb10的蛋白表达量显著升高(P<0.05),Atp6v0d2d蛋白表达量表达量显著降低(P<0.001),与转录组学结果一致;与HFD组相比,HDN干预后TOMM20相对表达量、mtDNA相对含量、ATP含量显著升高(P<0.05)。结果提示,HDN通过调节OXPHOS途径降低高脂饲喂诱导的小鼠肝氧化应激。
The aim of this study was to investigate the protective effect and mechanism of action of hesperidin(HDN)on high-fat feeding-induced hepatic oxidative stress injury in mice.In this study,18 male C57BL/6(body weight 20-23 g)mice were randomly divided into a control group,a high-fat diet(HFD)group,and a high-fat diet+hesperidin(HFD+HDN)group(300 mg·kg^(-1))of six mice each.The mice in the control group were fed with basal diet(10%fat,70%carbohydrate,20%protein);mice in the HFD group were fed with high-fat diet(60%fat,20%carbohydrate,20%protein);and mice in the HFD+HDN group were fed with high-fat diet while HDN was administered by gavage at 300 mg·kg^(-1) per day.After 16 weeks,mice were anaesthetised by intraperitoneal injection of 3%sodium pentobarbital(60 mg·kg^(-1))and then blood was collected from the eyeballs.After blood collection,mice were decapitated and dissected for liver tissues.The activities of alanine aminotransferase(ALT)and aspartate aminotransferase(AST),which are indicators of hepatic function,were detected using kits in the blood;The levels of malondialdehyde(MDA),activities of total superoxide dismutase(T-SOD),glutathione peroxidase(GSH-Px),and the level of total antioxidant capacity(T-AOC),which are indicators of oxidative stress,were detected using the kits in the liver tissues;The liver tissues were also collected for transcriptomic sequencing to screen differentially expressed genes between the HFD and HFD+HDN group for KEGG pathway enrichment analysis,with P<0.05 as the threshold for significant enrichment,and the most significant signalling pathway affected by HDN intervention was screened accordingly.Genes with significant changes from the screened signalling pathway were selected to validate the transcriptomics results by qRT-PCR and protein immunoblotting methods,the relative content of mtDNA and the relative expression of the mitochondrial outer membrane protein(TOMM20),as well as the liver ATP content were detected.The results showed that,compared to the HFD group,the HDN intervention improved high-fat feeding-induced liver damage,significantly reduced ALT and AST activity in the blood of mice(P<0.01);Significantly reduced liver MDA levels in mice(P<0.01);Significantly increased T-AOC,T-SOD and GSH-Px levels(P<0.05);Compared to the HFD group,the HFD+HDN group of which 889 genes were up-regulated(P<0.05)and 405 genes were down-regulated(P<0.05);KEGG enrichment analysis screened the oxidative phosphorylation pathway as the most significantly up-regulated signaling pathway(P<0.0001),Compared with the HFD group,the relative mRNA expression of Cox 8b,Cox 6a2,Gm10231,mt-Atp8,mt-Nd 4l,Gm11237,Ndufb 8,and Ndufb 10 were significantly up-regulated in the liver tissues after the HDN intervention(P<0.05),and the relative mRNA expression of Atp6v 0d2,Cox 6c2 were significantly down-regulated(P<0.05).Protein expression of Cox6a2,Ndufb8,Ndufb10 were significantly increased(P<0.05),and the expression of Atp6v0d2d protein expression were significantly decreased(P<0.01),which was consistent with the transcriptomics results;And the relative expression,relative mtDNA content,and ATP content of TOMM20 were significantly higher after HDN intervention(P<0.05)compared with that of the HFD group.These results indicated that HDN reduces high-fat feeding-induced oxidative stress in mouse liver by modulating the oxidative phosphorylation pathway.
作者
王鑫
聂桐
李阿群
马隽
WANG Xin;NIE Tong;LI Aqun;MA Jun(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;Heilongjiang Key Laboratory of Experimental Animals and Comparative Medicine,Harbin 150030,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2024年第3期1302-1313,共12页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
黑龙江省博士后资助项目(LBH-Z21101)
东北农业大学“学术骨干”项目(20YJXG03)。
关键词
橙皮苷
高脂
转录组测序
KEGG富集分析
氧化磷酸化途径
氧化应激
hesperidin
high-fat
transcriptome sequencing
KEGG enrichment analysis
oxidative phosphorylation pathway
oxidative stress
