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

doi:10.11843/j.issn.0366-6964.2024.09.015

摘要/Abstract

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IL1R1、TNFSF10、LTC4S、NCF1、SFTPA1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。

关键词: 鹅, 肥肝, HDLBP, 线粒体, 氧化应激, 炎症

Abstract:

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-regulated DEGs affected by HDLBP overexpression were mainly enriched in immune/inflammation-related pathways. In addition, compared with the control group, the expression of inflammation-related genes including IL1R1, TNFSF10, LTC4S, NCF1, SFTPA1, and KDR in the overfeeding group might be significantly reduced via HDLBP regulation (P < 0.05, 0.01 or 0.001). HDLBP can bind to mitochondria, and overfeeding significantly reduced the protein levels of wHDLBP and mHDLBP in goose liver. Overexpression of HDLBP leads to mitochondrial dysfunction, oxidative stress and increased expression of inflammatory factors. Therefore, HDLBP may provide protection for goose fatty liver by affecting mitochondrial function, regulating oxidative stress and inflammatory response.

Key words: goose, fatty liver, HDLBP, mitochondria, oxidative stress, inflammation

中图分类号:

S835.2

袁紫金, 王婉昕, 邢娅, 李家惠, 薛颖, 葛晶, 赵敏孟, 刘龙, 龚道清, 耿拓宇. 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[J]. 畜牧兽医学报, 2024, 55(9): 3897-3913.

Zijin YUAN, Wanxin WANG, Ya XING, Jiahui LI, Ying XUE, Jing GE, Minmeng ZHAO, Long LIU, Daoqing GONG, Tuoyu GENG. HDLBP Is Involved in Goose Fatty Liver Formation by Regulating the Level of Oxidative Stress and the Expression of Inflammatory Factors[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 3897-3913.

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基因 Gene	引物序列(5′→3′) Primers sequence	产物大小/bp Product size
HDLBP	F: CCGTGGAGGTGAAGAAGTCC; R: CCTCGCAGTATCACCGTCTC	139
UBC	F: AGGGTGGATTCTTTCTGG; R: ACTGAGTTTGGAGGGAGC	243
GAPDH	F: CTGATGCTCCCATGTTCGT; R: CCACGATGCCAAAGTTGTCA	138
CHP1	F: TCCAGAAGGAGAGGACCAAGT; R: GGAGCTTGTTACTTCGGCTG	134
PCAN1	F: TTGCCTGTGTGGCAGATAGT; R: TGTGCAACTGGACTCTAGCAT	177
LOC106049357	F: TGGCAGATGCAGACACAACC; R: CACCCTGGCCTCTCGTAAAT	140
HRG	F: ACAAGGGATTCTGCAAGGCAC; R: TCTATGTCCATGGTGATGCTGCC	107
ELOVL6	F: GGTGGTCGGCACCTAATGAA； R: TCTGGTCACACACTGACTGC	169
GMPR	F: CTGCCAATCACCCAGAATGC; R: TCAGAGTAGCCGTTTGCCAC	142
ALDH1A1	F: GGCGACAAGGCAGATGTAGA; R: CAAGAGCCTTCCTCGCTCTG	105
MAT1A	F: TGCGCGTTCATACCATTGTG; R: GACCAGTAACACCAGCGTCA	198
PSAT1	F: AGAATGTTGGCTGTGCTGGA; R: AAAGAGCCATTCACCGCTTG	118
LOC106034448	F: ATCTGCTTGCTCCCTGGATT; R: TGTCCCTTTTCCATCTGCCA	187
SLC2A9	F: GCAGGGAAGGGCAATTGGT; R: GACCAGAGGAGAGTCAGGGT	194
IL1R1	F: GAACGCCCAGCTCAGAACAT; R: AAAGGATGGCACGAGTTCCG	140
LTC4S	F: TTCGAGCACAGGTGAACTCC; R: GAGGACGATGCCCTGTATCC	157
TNFSF10	F: TGTCCACAGGATAGCAGCAC; R: GGTATCACCAGCTCGCCATT	180
NCF1	F: GACACCTTCATCCGGCACA; R: TTTCGGTGAGGTCGTTCCAC	106
SFTPA1	F: GGCACACCTCTGAATTACACC; R: TGTGAGGCGATACAGGTTGC	126
KDR	F: ACCTGACGATGAACCCACAC; R: CCACATTCAGCTCCGTCCTT	90
PPAP2B	F: TGCTATCATCACGGGAGAGC; R: TGTGAAGGACTGGCTGATGG	142
SIGLEC1	F: CTTCAGCACCCAAAAGACCG; R: CAGGACATCCGAAACGCTCA	229
CCL20	F: TGAACAGCTCTCCAGTGAAGTC; R: TCCTTAGGGTTTACGCAGGC	87

	基因 Gene	变化倍数的对数 log₂(fold change)	P_adj
上调 Up-regulated	CP	2.97	2.38×10^-168
	TNIP3	4.39	1.00×10^-140
	LOC106046725	2.52	7.01×10^-134
	LOC106046905	2.30	2.85×10^-121
	NOXO1	5.09	3.23×10^-109
	SLCO4C1	4.76	8.57×10^-6
	STEAP4	5.20	3.21×10^-98
	CHN2	2.24	1.44×10^-96
	SLC13A5	9.04	3.98×10^-90
下调 Down-regulated	DPYS	-2.84	3.24×10^-153
	MAP3K7CL	-2.62	6.42×10^-132
	LOC106030908	-3.52	6.02×10^-116
	PCK1	-1.65	4.90×10^-69
	HRG	-1.51	1.52×10^-65
	TPH2	-2.96	2.26×10^-46
	LOC106034448	-1.52	6.91×10^-43
	GPR1	-1.39	1.94×10^-39
	LOC106048076	-2.67	4.63×10^-35
	CRACR2A	-1.43	4.83×10^-34

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