2. 中国科学院大学, 北京 100049
2. University of Chinese Academy of Sciences, Beijing 100049, China
新生动物死亡率一直居高不下[1],新生绵羊的平均死亡率高达15%左右[2],新生山羊死亡率在11.5%~37%[3],新生犊牛的死亡率超过30%[4],造成巨大的经济损失。大量研究主要关注病因的探索、治疗等方面,并未从疾病本身出发,幼龄动物死亡率也没得到有效控制[2]。
代谢组位于基因组、转录组和蛋白质组的下游,代谢组的变化可以在功能水平上反映真实的机体状况,已被广泛地用于疾病研究,包括疾病诊断标志物的发现[5-6]。本研究利用高效液相色谱串联质谱仪研究并确定健康羔羊和病危临死羔羊之间的血清代谢物的差异,从代谢的角度理解濒死期新生动物的疾病发展过程,探索新生羔羊疾病诊断标识物。
1 材料与方法 1.1 试验设计动物在新生阶段(0~4周龄)尚未建立主动免疫,死亡的风险相对较高[7]。试验在一羊场规模约为200只基础母羊的小型羊场开展,选取0~4周龄内所有病危临死的山羊,将其与相同日龄的健康山羊进行配对,配对成功的共有9对,采集血清样品。羔羊从发病至濒死期都不给与药物治疗。
本研究采用的健康评分方法改编自威斯康辛大学犊牛健康评分(https://fyi.extension.wisc.edu/heifermgmt/files/2015/02/calf_health_scoring_chart.pdf),采用态度得分、眼分泌物得分、鼻分泌物得分、咳嗽得分和粪便得分指标对新生羔羊进行健康评定。评分标准:0=活跃/无眼分泌物/正常浆液性分泌物/无咳嗽/正常颗粒粪便,1=沉闷/少量眼分泌物/少量单侧浑浊分泌物/诱导单咳嗽/粪便不成形,2=沮丧/中度双侧眼分泌物/双边,浑浊或过多分泌物/诱导反复咳嗽或偶尔自发咳嗽/浆液状粪便,3=无反应/重度双侧眼分泌物/大量双侧黏液性分泌物/反复自发地咳嗽/水样稀便。对于所有健康评分类别,得分为0则代表动物表面上健康,至少3项评分指标得分为3则代表动物病危临死。
1.2 仪器和试剂高效液相色谱串联质谱UPLC-MS/MS、甲醇(Merck)、乙腈(Merck)、乙醇(Merck)、标准品(BioBioPha/Sigma-Aldrich)。
1.3 样品制备在冰水浴中解冻、涡旋后,各血清样本取50 μL置于1.5 mL EP管中,并加入含1 μg ·mL-1 2-氯苯丙氨酸作为内标的150 μL预冷的冰甲醇中。涡旋3 min,并在4 ℃、12 000 r ·min-1条件下离心10 min后,取上清液置于另一新的EP管中。再在相同的条件下离心5 min,取上清液置于进样瓶内衬管中,用于LC-MS/MS分析。
1.4 统计分析采用有监督的偏最小二乘判别分析(PLS-DA)[8],再结合差异倍数值(fold change)筛选差异代谢物,即VIP值>1且fold change≥2或fold change≤0.5的代谢物被认为是差异代谢物。利用KEGG数据库[9]对差异代谢物进行通路注释和富集分析。
2 结果 2.1 OPLS-DA分析OPLS-DA模型得分图(图 1)显示,病态羔羊组和健康羔羊组分别分布在PC1的右侧和左侧, 说明两组样品之间存在一定的差异。
如图 2所示,共鉴定到13个差异代谢物,按性质主要分为核苷酸、氨基酸和脂类代谢产物,主要富集在氨基酸的生物合成和代谢通路上(图 3),包括氨酰基-tRNA的生物合成(aminoacyl-tRNA biosynthesis)、半胱氨酸和蛋氨酸的代谢(cysteine and methionine metabolism)、甘氨酸、丝氨酸和苏氨酸的代谢(glycine, serine and threonine metabolism)等通路上。
疾病发生发展过程中往往伴随着炎症和氧化状态的改变[10-11],造成DNA、蛋白质和脂类损伤[12-13],导致机体核苷酸、氨基酸、脂类等代谢紊乱,本研究结果与之一致,病危临死羔羊体内核苷酸、氨基酸、脂类代谢亦出现异常。氨基酸在新陈代谢过程中起着重要的作用,是健康状况的重要参数指标[14],氨基酸及其代谢产物的变化可用来监测动物机体的健康状况[15-16],本研究主要关注氨基酸的合成和代谢的变化。
据报道磷酸丝氨酸二聚体(2PS)和三聚体在Caco-2细胞中表现出抗氧化应激作用[17-18],2PS还可通过下调NF-κB通路和抑制MAPKs磷酸化发挥抗炎症作用[19]。酪氨酸代谢异常可导致多种氨基酸代谢疾病[15-16],对羟基苯丙酸是酪氨酸的代谢产物[20],可抑制低密度氧化脂蛋白诱导的NF-κB通路的激活,降低促炎细胞因子IL-1β、IL-6的分泌[21]。赖氨酸作为山羊限制性氨基酸之一,具有一定的抗氧化作用[22-23]。在本研究中,病态羔羊血清L-O-磷酸丝氨酸、对羟基苯丙酸分泌显著增多,而L-丙氨酰-赖氨酸分泌显著减少,这一结果也表明氨基酸代谢在维持动物机体健康过程中起重要作用。
谷胱甘肽是动物细胞中主要的水溶性抗氧化剂[24],还可激活T淋巴细胞和多形核白细胞,诱导细胞因子的产生进行免疫应答[25-26]。同型半胱氨酸是半胱氨酸和蛋氨酸代谢通路的中间产物,作为一种主要成分参与机体谷胱甘肽的合成,从而发挥免疫作用[27]。本研究结果显示,差异代谢物主要参与半胱氨酸和蛋氨酸的代谢,这一结果进一步表明氨基酸代谢在动物疾病过程中起重要作用。
甘氨酸和丝氨酸可以提供合成核酸和脂质的前体物质[28],丝氨酸的合成还会直接影响细胞的抗氧化能力[29],在机体炎症反应中也发挥一定的作用[30]。甘氨酸、丝氨酸和苏氨酸代谢通路还涉及到多种氨基酸降解和合成,在维持动物健康方面发挥重要作用。该通路还可以生成乙酰辅酶A,为机体提供能量[31]。氨酰-tRNA主要功能是将氨基酸运转到核糖体合成蛋白质[32],从而完成一系列生命活动。
本研究病态羔羊血清差异代谢物主要富集到氨酰-tRNA生物合成、半胱氨酸和蛋氨酸的代谢以及甘氨酸、丝氨酸和苏氨酸的代谢通路上,进一步说明了氨基酸代谢在动物疾病进程中的重要推动作用,濒死羔羊变化的代谢指标可能作为新生羔羊疾病相关诊断标识物,为构建新生羔羊疾病相关诊断标识物体系提供基础。
4 结论基于UPLC-MS/MS研究方法,研究了新生山羊在濒临死亡的情况下血清代谢物的变化情况,共鉴定出13种差异代谢物,其中,12种上调,1种下调。病危临死山羊与健康山羊相比,其内源性氨基酸合成和代谢出现了改变。
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