泌乳初期奶牛相关脂肪因子及生理生化指标与脂肪肝的相关性
朱颍琨, 肖劲邦, 钱柏霖, 姜思汛, 尤留超, 张钺, 刘红, 马莉, 曹随忠, 余树民, 沈留红*
四川农业大学 动物医学院,动物疫病与人类健康四川省重点实验室/奶牛疾病研究中心,四川 成都 611130
*通信作者,沈留红,E-mail: shenlh@sicau.edu.cn

作者简介:朱颍琨(1979—),男,安徽阜阳人,硕士研究生,从事反刍动物疾病及繁殖新技术研究。E-mail: CrhistianZHu@foxmail.com

摘要

为探究泌乳初期奶牛血清脂肪因子及生理生化指标与脂肪肝的相关性,选取四川某规模化奶牛场1~4胎围产期奶牛64头,分别于产后第7、14天早饲前尾静脉采血,检测产后第14天奶牛血清谷草转氨酶、血糖、游离脂肪酸水平,根据脂肪肝判定公式判定是否发生脂肪肝,检测并分析血清脂肪因子及生理生化指标与肝功能指标和能量代谢指标之间的相关性。结果显示,脂肪肝奶牛血清瘦素、脂联素水平显著低于健康奶牛( P<0.05),TNF-α水平显著高于健康奶牛( P<0.05),IL-6水平差异不显著( P>0.05)。瘦素水平与血糖、白蛋白呈显著正相关( P<0.05),与游离脂肪酸、β-羟基丁酸、谷草转氨酶、总胆红素、直接胆红素、总蛋白呈显著负相关( P<0.05),提示泌乳初期奶牛能量负平衡是脂肪肝发生的重要原因。脂联素水平与游离脂肪酸、β-羟基丁酸、谷草转氨酶呈显著负相关( P<0.05),与总蛋白、白蛋白呈显著正相关( P<0.05),提示泌乳初期脂肪肝奶牛存在着明显的肝功能损伤与脂质代谢紊乱。TNF-α水平与总胆红素、直接胆红素、谷丙转氨酶呈显著正相关( P<0.05),与血糖、白蛋白呈显著负相关( P<0.05);IL-6水平与谷丙转氨酶呈显著正相关( P<0.05),与总蛋白、白蛋白呈显著负相关( P<0.05),提示泌乳初期脂肪肝奶牛存在着明显的炎性反应。血清瘦素与脂联素水平呈显著正相关( P<0.05),与TNF-α呈显著负相关( P<0.05);TNF-α与脂联素呈显著负相关( P<0.05),与IL-6呈显著正相关( P<0.05),提示泌乳初期脂肪肝奶牛存在着糖脂代谢调控紊乱。综上所述,泌乳初期奶牛能量负平衡导致体脂动员,使得奶牛血清脂联素和瘦素水平下降,造成大量脂质合成于肝脏,肝脏功能受损,诱发炎性反应,TNF-α与IL-6水平随之上升,进而导致奶牛脂肪肝的发生,脂联素、瘦素和TNF-α可作为预测产后奶牛脂肪肝发生的指标。

关键词: 奶牛; 脂肪肝; 脂肪因子
中图分类号:S858.23 文献标志码:A 文章编号:1004-1524(2019)05-0722-08
Correlations between adipokine, biochemical indicators in early lactation cows with fatty liver
ZHU Yingkun, XIAO Jinbang, QIAN Bolin, JIANG Sixun, YOU Liuchao, ZHANG Yue, LIU Hong, MA Li, CAO Suizhong, YU Shumin, SHEN Liuhong*
College of Veterinary Medicine, Sichuan Agricultural University, Key Laboratory of Animal Disease and Human Health of Sichuan Province, The Medical Research Center for Cow Disease, Chengdu 611130, China
Abstract

To explore correlations between adipokine, biochemical indicators in early lactation cows with fatty liver, 64 perinatal Chinese Holstein cows from a farm in Sichuan province were used in the present study. Tail vein blood was collected before morning feeding on 7 and 14 days after delivery, serum AST, Glu and NEFA levels of 14 days after delivery were tested. According to fatty liver determination formula, cows were grouped into fatty liver group and control group. Some adipokine and biochemical indicators were tested and their correlations with liver function indexes and energy metabolism indexes were analyzed. The results showed that leptin, adiponectin level of fatty liver early lactation cows were significantly lower than the normal group( P<0.05). TNF-α level of fatty liver early lactation cows was significantly higher than the normal group( P<0.05), IL-6 level appeared no significant difference between fatty liver cows and normal cows( P>0.05). Leptin had significant positive correlations with Glu and ALB levels( P<0.05), significant negative correlations with AST, TBIL, DBIL, TP, NEFA and BHBA levels( P<0.05), indicated that negative energy balance was an important factor of fatty liver. Adiponectin level had significant negative correlations with NEFA, BHBA and AST( P<0.05), significant positive correlations between TP and ALB( P<0.05), speculating liver function and energy metabolism disorders were occurred in fatty liver early lactation cows. TNF-α level had significant positive correlations with TBIL,DBIL and ALT levels( P<0.05), significant negative correlations with Glu and ALB( P<0.05). IL-6 level had a significant positive correlation with ALT level( P<0.05), significant negative correlations with TP and ALB levels( P<0.05), showed that obvious inflammatory stress was appeared in fatty liver cows. Leptin levels showed a significant positive correlation with adiponectin level( P<0.05), significant negative correlation with IL-6 levels, speculating metabolism regulation disorder in fatty liver cows. In summary, the negative energy balance of early lactation cows induced body fat mobilization and downregulated adiponectin and leptin levels, causing lipids accumulate in liver and damaged liver function and triggered inflammatory stress, rising IL-6 and TNF-α levels and induced fatty liver, adiponectin. Thus, leptin and TNF-α levels could be able to be a predicative index of fatty liver in early lactation cows.

Keyword: cows; fatty liver; adipokines

脂肪肝是泌乳初期奶牛常见的一种营养代谢病, 又称肥胖奶牛综合症, 是指泌乳初期母牛(尤其是产后7~14 d)因干物质采食量(dry matter intake, DMI)下降、泌乳的启动及DMI上升速度低于泌乳量上升速度, 机体能量供应不足而引起能量负平衡(negative energy balance, NEB), 导致脂肪分解生成大量游离脂肪酸(non-esterified fatty acid, NEFA)。NEFA被肝脏吸收后, 合成大量甘油三酯(triglyceride, TG), 蓄积于肝脏, 最终导致能量代谢障碍性疾病[1, 2, 3]。目前, 泌乳初期奶牛脂肪肝多发生于高产奶牛, 发病率高达50%, 且呈不断上升趋势[4], 由于其病程长且难以治疗, 严重影响奶牛生产性能、繁殖性能及免疫功能, 给奶牛养殖行业带来了严重的经济损失[5]。脂肪因子是由脂肪等组织分泌的活性物质, 包括脂联素(adiponectin, ADPN)、瘦素(leptin, LP)、抵抗素(resistin)、内脂素(visfatin)、网膜素(omentin)和肿瘤坏死因子α (tumor necrosis factor α , TNF-α )等100余种, 在糖、脂代谢调控中有重要作用, 与能量代谢性疾病有着密切联系[6]。研究表明, ADPN、LP、TNF-α 和白介素6(interleukin 6, IL-6)等在人、小鼠和大鼠的脂肪肝发生和发展中均发挥不同程度的作用[7, 8, 9], 但在奶牛上还未见相关研究报道。因此, 本研究旨在探究泌乳初期脂肪肝奶牛ADPN、LP、TNF-α 和IL-6与肝功能、能量代谢指标的相关性, 为揭示奶牛血清脂肪因子对脂肪肝的作用机理奠定基础。

1 材料与方法
1.1 试验动物

试验选用四川某规模化奶牛场半封闭统一饲舍, 体质量(510± 47)kg、1~4胎次泌乳初期的荷斯坦奶牛64头, 自由采食、饮水, 统一全混合日粮饲喂。

1.2 试验试剂

NEFA、血糖(Glu)、β -羟基丁酸(BHBA)、天门冬氨酸氨基转移酶(AST)、谷丙转氨酶(ALT)、总蛋白(TP)、白蛋白(ALB)、球蛋白(GLB)、总胆红素(TBIL)、直接胆红素(DBIL)、间接胆红素(IBIL)检测试剂盒, 南京建成生物工程研究所; ADPN、LP、TNF-α 、IL-6双抗体夹心酶联免疫吸附检测(ELISA)试剂盒, 北京雅安达生物技术有限公司。

1.3 试验方法

1.3.1 血清收集

于奶牛产后第7、14天, 早饲前尾静脉采血10 mL, 所采血样置于无抗凝剂的离心管中, 室温静置1 h, 3 000 r· min-1离心10 min, 取上层血清, -70 ℃保存, 待检。

1.3.2 脂肪肝奶牛判定

根据Reid等[10]提出的脂肪肝判定公式, 即Y=-0.51-0.003NEFA+2.84Glu-0.0528AST。当Y< 0时为重度脂肪肝; 当0< Y< 1时为中度脂肪肝; 当Y> 1时为正常。产后第14天Y< 1的被纳入脂肪肝组, Y> 1的被纳入对照组, 其中脂肪肝组28头, 对照组36头。

1.3.3 脂肪因子和生化指标检测

按照试剂盒说明书检测相关脂肪因子和血清生化指标。

1.4 数据统计分析

试验数据用Excel2007进行初步处理, 用SPSS19.0软件统计, 检验数据是否符合正态分布, 组间采用独立样本t检验, 相关性使用Pearson相关分析, 以P< 0.05作为差异显著性判断标准。试验数据以平均值± 标准差表示。

2 结果与分析
2.1 血清部分脂肪因子检测结果

产后第7、14天, 脂肪肝组奶牛血清瘦素水平显著低于对照组(P< 0.05), TNF-α 水平显著高于对照组(P< 0.05)。产后第14天, 脂肪肝组奶牛脂联素水平显著低于对照组(P< 0.05)(表1)。

表1 血清中LP、ADPN、TNF-α 与IL-6检测结果 Table 1 Results of the concentration of leptin, adiponectin, TNF-α and IL-6 in serum
2.2 血清生化指标检测结果

产后第7、14天, 脂肪肝组奶牛血清血糖、总蛋白水平显著低于对照组(P< 0.05), 天门冬氨酸氨基转移酶、游离脂肪酸和β -羟基丁酸水平显著高于对照组(P< 0.05)。产后第7天, 脂肪肝组奶牛球蛋白水平显著低于对照组(P< 0.05), 而白球比显著高于对照组(P< 0.05)。产后第14天, 脂肪肝组奶牛白蛋白水平显著低于对照组(P< 0.05), 而总胆红素与直接胆红素水平均显著高于对照组(P< 0.05)(表2)。

表2 血清中部分生化指标检测结果 Table 2 Results of biochemical indexes in serum
2.3 血清瘦素、脂联素、TNF-α 、IL-6间的相关性

奶牛血清中瘦素与脂联素呈显著正相关(P< 0.05), 与TNF-α 呈显著负相关(P< 0.05); 脂联素与TNF-α 呈极显著负相关(P< 0.05); TNF-α 与IL-6呈显著正相关(P< 0.05)(表3)。

表3 血清中瘦素、脂联素、TNF-α 、IL-6间的相关性 Table 3 Correlation between leptin, adiponectin, TNF-α and IL-6 in serum
2.4 血清瘦素、脂联素、TNF-α 、IL-6与部分能量代谢指标的相关性

奶牛血清中瘦素与血糖、TNF-α 与β -羟基丁酸之间呈显著正相关(P< 0.05); TNF-α 与游离脂肪酸、IL-6与游离脂肪酸之间呈显著正相关(P< 0.05); 瘦素与β -羟基丁酸、脂联素与游离脂肪酸、脂联素与β -羟基丁酸、TNF-α 与血糖之间呈显著负相关(P< 0.05); 瘦素与游离脂肪酸呈显著负相关(P< 0.05)(表4)。

表4 血清中瘦素、脂联素、TNF-α 、IL-6与部分能量代谢指标之间的相关性 Table 4 Correlation between leptin, adiponectin, TNF-α , IL-6 and energy metabolism index in serum
2.5 血清瘦素、脂联素、TNF-α 、IL-6与肝功指标的相关性

奶牛血清中瘦素与天门冬氨酸氨基转移酶之间呈显著负相关(P< 0.05); 瘦素与总蛋白、脂联素与总蛋白之间呈显著正相关(P< 0.05); 瘦素与白蛋白、瘦素与白球比、脂联素与白蛋白、TNF-α 与天门冬氨酸氨基转移酶、TNF-α 与直接胆红素、IL-6与谷丙转氨酶之间呈显著正相关(P< 0.05); 瘦素与总胆红素、瘦素与直接胆红素、脂联素与天门冬氨酸氨基转移酶、TNF-α 与总蛋白、TNF-α 与白蛋白、IL-6与总蛋白、IL-6与白蛋白之间呈显著负相关(P< 0.05)(表5)。

表5 血清中瘦素、脂联素、TNF-α 、IL-6与部分肝功指标间的相关性 Table 5 Correlation between leptin, adiponectin, TNF-α , IL-6 and liver function index in serum
3 讨论
3.1 泌乳初期脂肪肝奶牛血清瘦素水平与脂肪肝的相关性

瘦素主要由机体白色脂肪组织产生, 可反映机体能量储备与摄入的急性变化[11], 可通过受体介导的细胞内吞作用穿越血脑屏障到达中枢神经系统。研究表明, 瘦素的其他受体亚型如LepRa等可参与瘦素血脑屏障转运[12]。瘦素作为信号分子, 其水平与机体外周能源供给情况呈正相关, 具有将外周能量状态传递给中枢神经系统, 进而调控食欲和新陈代谢的作用。

奶牛脂肪肝的发病机理尚不明确, 但目前较为认同的假说是能量负平衡可引起体脂动员, 导致大量的甘油三酯合成并蓄积在肝脏而发病。泌乳初期奶牛经历妊娠、分娩和启动泌乳等生理应激, 尤其妊娠后期和泌乳初期奶牛因干物质摄入减少、能量需求增加易导致NEB发生[13]。研究表明, 干物质摄入量越低, 泌乳期能量负平衡越严重, 血液循环瘦素水平越低[14, 15]。本研究中, 产后第7、14天, 脂肪肝组奶牛血清中瘦素水平均显著低于对照组, 暗示奶牛泌乳初期NEB可能是奶牛产生脂肪肝的原因之一。

本研究结果显示, 奶牛血清中瘦素与血糖呈极显著正相关, 与游离脂肪酸、β -羟基丁酸呈显著负相关, 与郑家三等[16]、何宝祥等[17]的研究结果一致。Malloy等[18]的研究结果也表明, 瘦素缺乏小鼠体内游离脂肪酸显著增加。但在研究中使用高脂日粮可诱导小鼠发生肥胖与脂肪肝, 并表现出高血糖、高瘦素抵抗、高血脂、高胰岛素抵抗、高游离脂肪酸, 与奶牛脂肪肝发病时低血糖、低瘦素的状况有较大差别, 提示奶牛泌乳初期脂肪肝与高脂饮食导致的脂肪肝发病原因不同。

瘦素能够调控机体代谢, 限制甘油三酯在非脂肪细胞(包括肝细胞)中的储存, 从而影响肝脏功能[19]。瘦素水平过高, 易引起瘦素抵抗, 使瘦素敏感性降低, 导致机体糖脂代谢紊乱[20]。Okamatsu等[21]的研究表明, 超重儿童瘦素水平与肝脏损伤程度显著高于正常儿童。本研究结果显示, 瘦素与AST、TBIL、DBIL、TP呈显著负相关, 与ALB呈显著正相关, 与之前的研究不完全相同。这进一步提示了泌乳初期奶牛脂肪肝的发病机制与传统意义上的摄入脂质过剩导致的脂肪肝不同, 印证了NEB导致脂肪动员, 脂肪沉积于肝脏而发病的假说, 与此同时, NEB所表现出的血糖降低也会引起瘦素水平降低, 进一步加剧肝脏的TG合成, 使得TG沉积, 引发脂肪肝, 影响肝功能。

3.2 泌乳初期脂肪肝奶牛血清脂联素水平与脂肪肝的相关性

脂联素是由脂肪组织分泌的一种分子量为30 kD的蛋白多聚体[22], 能够促进游离脂肪酸的氧化, 改善胰岛素敏感性, 降低血管内皮细胞炎症反应[23]。Jarrar等[24]和Hui等[25]的研究表明, 非酒精性脂肪肝患者循环脂联素水平显著低于健康人, 认为低血浆脂联素可导致血浆甘油三脂和游离脂肪酸的升高与脂质在肝脏中的蓄积。另有研究发现, 脂联素缺失小鼠体内2种脂肪酸合成的限速酶(乙酰辅酶A羧化酶和脂肪酸合酶)表达量增加, 并且肝脂肪变性相比野生型小鼠更严重[26]。本研究结果显示, 产后第7天, 脂肪肝组奶牛血清中脂联素水平低于对照组, 但差异不显著; 产后第14天, 脂肪肝组血清中脂联素水平显著低于对照组, 与上述学者在人鼠研究报道中趋势一致。

Zou等[27]在人上研究表明, 儿童发育时期脂联素水平下降, 会导致血清游离脂肪酸上升, 进而引发脂肪代谢紊乱, 易导致儿童肥胖症。Lavoie等[28]在小鼠上的研究也有相似的结果。张辉等[29]研究结果显示, 高浓度游离脂肪酸和β -羟基丁酸对牛脂肪细胞中脂联素mRNA表达具有抑制作用。本研究结果也显示, 奶牛血清中脂联素与游离脂肪酸和β -羟基丁酸呈显著负相关, 提示泌乳初期奶牛体内由于脂肪动员导致的循环游离脂肪酸水平上升导致脂联素合成被抑制, 进一步加剧脂肪动员, 提高脂肪肝风险。

脂联素能减轻肝细胞脂质堆积, 正常水平的脂联素意味着较低的肝脏疾病风险[22]。Yokoyama等[30]对791名日本工人的研究发现, AST活性增高者的脂联素水平显著低于AST活性正常的人, 提示脂联素水平与AST活性成负相关。Kusumawocti等[31]对37名肥胖儿童的研究也有相同的结果。本研究结果显示, 泌乳初期奶牛血清中脂联素与AST呈显著负相关, 与TP和ALB呈显著正相关, 推测是由于脂联素水平下降, 导致肝细胞脂质堆积, 进而损伤肝脏功能。

3.3 泌乳初期脂肪肝奶牛血清TNF-α 水平与脂肪肝的相关性

TNF-α 是具有多种生物学功能的细胞因子, 由巨噬细胞、脂肪细胞、T淋巴细胞和成纤维细胞等分泌, 调节机体免疫应答及炎症反应[32], 直到1993年人们才发现其对脂肪代谢也有重要调控作用。Hui等[25]对非酒精性、单纯性脂肪肝及正常肝脏进行检测, 发现非酒精性和单纯性脂肪肝中血清TNF-α 水平较高。Ajmal等[33]对72名脂肪肝患者和32名健康者血清TNF-α 进行检测, 也发现相同的结果。本研究发现, 产后第7、14天, 脂肪肝组奶牛血清TNF-α 水平均显著高于对照组, 提示TNF-α 水平的上升与奶牛脂肪肝发病有较强相关性。TNF-α 可促进脂肪细胞分解和游离脂肪酸的释放, Castro等[34]发现, TNF-α 基因敲除小鼠由于体内缺乏TNF-α , 导致促脂分解作用消失, 游离脂肪酸水平也降低。本研究中, TNF-α 与血糖呈显著负相关, 与游离脂肪酸和β -羟基丁酸显著正相关, 与上述学者在人鼠上报道的趋势一致, 提示奶牛TNF-α 水平上升会加剧脂肪动员, 导致糖脂代谢紊乱, 进而引发脂肪肝。

TNF-α 不仅影响脂质代谢, 还可通过上调肝细胞解偶联蛋白2(uncoupling protein-2, UCP2)的表达影响线粒体能量代谢, 产生大量活性氧簇, 引起脂质过氧化, 损伤肝细胞, 进而引起肝功能异常[35, 36]。韩旭等[37]研究显示, TNF-α 较高时, 肝功能损伤更为严重。Hughes[38]对肝功能异常者使用TNF-α 拮抗剂后, 发现患者肝功能得到明显改善。本研究显示, TNF-α 与AST和DBIL呈显著正相关, 与TP和ALB呈显著负相关, 与上述学者研究结果相一致, 推测TNF-α 水平上升可通过一系列途径影响肝细胞生理活动, 引起肝功能指标的改变。

3.4 泌乳初期奶牛血清IL-6水平与脂肪肝的相关性

IL-6是由脂肪细胞和巨噬细胞等多细胞分泌的一种促炎因子[39, 40]。内脏脂肪是IL-6分泌的重要器官, 其产生的IL-6至少是皮下脂肪的3倍。IL-6在脂肪肝发生及发展过程中的作用尚存争论。金世禄等[41]研究发现, 脂肪肝患者血清IL-6水平显著升高, 且IL-6与胰岛素抵抗呈显著正相关。而Hong等[42]研究结果则显示, 注射IL-6可改善小鼠肥胖和脂肪肝。本研究中, 产后第7、14天, 脂肪肝组奶牛血清中IL-6水平高于对照组, 但差异不显著, 推测主要是检测时间处于疾病初期, 尚未造成严重的肝脏的功能损伤, 因而并未产生显著的IL-6水平差异。此外, 奶牛血清中IL-6与游离脂肪酸呈显著正相关, 与王军英等[43]、陈华等[44]在人和大鼠上研究结果一致, 推测泌乳初期奶牛IL-6水平上升, 可直接或间接的导致胰岛素抵抗, 从而引起机体糖脂代谢紊乱, 血液中游离脂肪酸上升, 过量的游离脂肪酸进入肝脏, 引发脂肪肝。

IL-6不仅参与胰岛素抵抗的发生, 同时也可介导肝脏的巨噬细胞浸润, 损害肝脏功能[45]。韩旭等[37]报道, 血清IL-6水平随肝功能受损程度增加而上升, 且与TBIL、ALT呈显著正相关, 与ALB呈显著负相关。本研究结果显示, IL-6与ALT呈显著正相关, 与TP、ALB呈显著负相关, 与上述学者在人上的报道基本一致, 表明IL-6参与了奶牛脂肪肝肝脏损伤的过程, 但其具体机制还有待进一步研究。

The authors have declared that no competing interests exist.

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