HDLBP通过调控氧化应激水平和炎性因子表达参与鹅肥肝的形成  

HDLBP Is Involved in Goose Fatty Liver Formation by Regulating the Level of Oxidative Stress and the Expression of Inflammatory Factors

作　　者：袁紫金 王婉昕 邢娅 李家惠 薛颖 葛晶 赵敏孟 刘龙[1] 龚道清[1] 耿拓宇[1] YUAN Zijin;WANG Wanxin;XING Ya;LI Jiahui;XUE Ying;GE Jing;ZHAO Minmeng;LIU Long;GONG Daoqing;GENG Tuoyu(College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《畜牧兽医学报》2024年第9期3897-3913,共17页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(32172717);国家自然科学基金青年基金(32302754);中国博士后面上项目(2023M742960)。

摘　　要：旨在利用活体与细胞模型探究高密度脂蛋白结合蛋白(HDLBP)的亚细胞分布、基因功能及其与鹅肥肝形成的关系。本研究选取70日龄健康朗德鹅公鹅14只,单笼饲养,随机均分为对照组(平均体重为3.71 kg,自由采食)和试验组(平均体重为3.72 kg,填饲20 d)进行活体模型试验。从23日龄朗德鹅胚胎中分离肝细胞并过表达HDLBP基因进行细胞模型试验。首先采用免疫印迹法、免疫荧光技术对鹅原代肝细胞中HDLBP蛋白质进行亚细胞定位分析,其次采用免疫印迹法检测填饲鹅和对照鹅肝脏全细胞HDLBP(wHDLBP)及线粒体中HDLBP(mHDLBP)的蛋白质丰度,然后在鹅原代肝细胞中过表达HDLBP,检测其对细胞中mHDLBP蛋白质丰度、丙二醛(MDA)含量、总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性、活性氧类物质(ROS)和线粒体膜电位水平的影响,最后通过转录组测序分析筛选HDLBP过表达影响的差异表达基因与相关信号通路,并在活体模型中对部分差异表达基因进行定量PCR验证。结果表明:HDLBP可结合到线粒体中;填饲组wHDLBP和mHDLBP蛋白质丰度均显著低于对照组(P<0.01);在鹅原代肝细胞中过表达HDLBP显著增加mHDLBP的蛋白质丰度(P<0.05),增加MDA(P<0.01)和ROS(P<0.05)含量,降低线粒体膜电位(P<0.05)及T-SOD(P<0.05)和GSH-PX(P<0.05)活性;HDLBP过表达所影响的上调差异表达基因主要富集于免疫/炎症相关通路。此外,相对于对照组,填饲组中炎症相关基因IL 1R1、TNFSF10、LTC 4S、NCF1、SFTPA 1及KDR的表达可能受到HDLBP的调控而显著减少(P<0.05,0.01或0.001)。HDLBP能够与线粒体结合,填饲显著降低鹅肝全细胞和线粒体样中HDLBP的蛋白水平,过表达HDLBP导致线粒体功能损伤、氧化应激和炎性因子的表达增强,因此HDLBP可能通过影响线粒体功能、调控氧化应激和炎症反应为鹅肥肝提供保护。The aim of this study was to investigate the subcellular localization and function of high density lipoprotein binding protein(HDLBP),and its relationship with the formation of goose fatty liver using in vivo and cellular models.Fourteen 70-day-old healthy Landes male geese were selected and raised in single cages.They were randomly divided into the control group(average weight 3.72 kg,ad libitum feeding)and the treating group(average weight 3.71 kg,overfed for 20 d)for in vivo experiment.Hepatocytes were isolated from 23-day-old Landes goose embryos and overexpressed with HDLBP gene for cellular experiment.Firstly,the subcellular localization of HDLBP protein in goose primary hepatocytes was determined by immunoblotting and immunofluorescence analyses.Secondly,the protein abundance of total HDLBP(wHDLBP)and mitochondrial HDLBP(mHDLBP)in the liver of the overfed geese and the control geese was determined by immunoblotting analysis.Then,HDLBP was overexpressed in goose primary hepatocytes,which was followed by determining the effects of HDLBP overexpression on the protein abundance of mHDLBP,the content of malondialdehyde(MDA),the activities of total superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-PX),the level of reactive oxygen species(ROS),and mitochondrial membrane potential.Finally,the differentially expressed genes(DEGs)and related signaling pathways affected by HDLBP overexpression were identified by transcriptome sequencing analysis,and some DEGs were verified in the in vivo model by quantitative polymerase chain reaction(PCR).The results showed that HDLBP could bind to mitochondria;the protein abundance of wHDLBP and mHDLBP in the overfeeding group was significantly lower than that in the control group(P<0.01);overexpression of HDLBP in goose primary hepatocytes significantly increased the protein abundance of mHDLBP(P<0.05),increased the levels of MDA(P<0.01)and ROS(P<0.05),and decreased the mitochondrial membrane potential(P<0.05)and the activities of T-SOD(P<0.05)and GSH-PX(P<0.05).The up-reg

关 键 词：鹅 肥肝 HDLBP 线粒体 氧化应激 炎症 

分 类 号：S835.2[农业科学—畜牧学]