脂多糖对奶山羊肝脏代谢组学的影响

【目的】探寻脂多糖(lipopolysaccharide,LPS)影响奶山羊肝脏营养代谢的特征代谢物(生物标记物),阐明LPS引起肝脏代谢障碍的机制。【方法】选择12—18月龄,体重24—28 kg的关中奶山羊15只,平均分为3组(每组5只),分别为对照组(CTL)、低剂量LPS处理组(LPS-L)和高剂量LPS处理组(LPS-H)。LPS-L和LPS-H分别腹腔注射20、40μg·kg-1 BW的LPS溶液,CTL注射等容量生理盐水。24 h后,LPS-L和LPS-H追加LPS溶液。48 h后,静脉采血,分离血浆和血清,检测血液相关生化指标;然后活体采集肝脏组织,液氮保存。肝脏组织冻干后,用氢核磁共振(1H-nuclear magnetic resonance,1H-NMR)技术检测肝脏组织代谢物,运用数据库对检测到的代谢物变量进行化合物种类鉴别,再用模式识别方法中的偏最小判别二乘分析法(partia1 1east squares discriminant analysis,PLS-DA)筛选生物标记物。【结果】血清生化指标表明,LPS-L和LPS-H处理的谷丙转氨酶(ALT)、总胆红素(TBIL)水平较CTL显著升高(P0.05);甘油三酯(TG)、非酯化脂肪酸(NEFA)、总胆固醇(TC)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、白蛋白(ALB)和总蛋白(TP)的含量较CTL显著降低(P0.05)。用1H-NMR技术共检测到69种代谢物变量,包括氨基酸,醇类,糖类及其它代谢产物。PLS-DA分析发现,代谢物变量可将CTL、LPS-L与LPS-H组之间聚类区分,并找到9种组间差异显著的代谢物,这些代谢物主要与氨基酸、脂肪和碳水化合物等代谢途径相关,可作为肝脏在LPS诱导损伤状态下的标志物。【结论】LPS可显著影响奶山羊肝脏营养代谢,1H-NMR代谢组学可以较为全面地检测到肝脏组织的代谢物,准确地筛选出差异代谢物,通过分析差异代谢物的代谢途径,阐明LPS引起肝脏代谢障碍的机理。

Study on the Effect of Lipopolysaccharide on Hepatic Metabolism in Dairy Goat Liver

【Objective】 The objective of this experiment is to study the effect of lipopolysaccharide (LPS) on metabolism of dairy goat liver, explore the characteristic metabolites in the liver and clarify the mechanism of hepatic dysbolism caused by LPS.【Method】Total 15 Guanzhong dairy goats of 12-month-old with 24-28 kg live body weight (BW) were selected and divided into three groups randomly, i.e. control group (CTL, goat No. 1-5), LPS-L group (LPS-L, goat No. 6-10) and LPS-H group (LPS-H, goat No.11-15). Animals were fed total mixed ration (TMR) made according to NRC (2007). The experimental goats were fed with feeds and water ad libitum. After 14d adaption, the experiment was conducted. At the beginning of the experiment (0 h), the goats in LPS-L and LPS-H were given 20 and 40μg·kg^-1 BW of LPS solution via intraperitoneal injections, respectively. The goats in CTL were received same volume of saline water. 24 h later, goats in each LPS treatment groups were added LPS solution once again. 48 h later from the beginning, blood samples were drawn from jugular vein and were centrifuged to extract plasma for biochemical determination. Subsequently, the goats’ liver tissue samples were collected by biopsy and marked and stored in liquid nitrogen. ¹H-NMR metabonomics was used to research the changes of metabolites in goat liver, the obtained metabolites. The data variables (concentration value of metabolites) were analyzed by the software of partial least squares discriminant analysis (PLS-DA) installed in the equipment to identify metabolic differences between the groups of each treatment. 【Result】Serum biochemical results showed that the concentration of ALT, AST and TBIL increased significantly, while TG, TC, VLDL, NEFA, HDL, LDL, ALB, TP decreased to different degrees in LPS treated groups compared with that of CTL, indicating the goats liver were injured to some degrees and the dysbolism was occurred in goat liver. With the ¹H-NMR metabonomics determination, totally 69 metabolites were detectedbased on its identification database of Chenomx. After the metabolite variables analyzing using PLS-DA, it showed that the CTL, LPS-L and LPS-H groups could be clustered and distinguished based on the metabolomic data analysis. Nine out of 69 metabolites differed significantly among the three groups. Based on the statistics and analysis between any two of the groups, 6 metabolites located in LPS-L and LPS-H groups were notable compared with the CTL, and 3 metabolites were different significantly between LPS-L and LPS-H. Further analysis indicated that these metabolites were related to amino acid metabolism, fat metabolism and carbohydrate metabolism in the liver.【Conclusion】In conclusion, LPS induced significant changes in liver reflected in blood biochemical indices. Metabolomics profile could precisely detect characteristic metabolites and effectively distinguish LPS-L and LPS-H from CTL effectively. Metabolomic technology could clarify the mechanism for liver dysbolism suffering from LPS and provide enough information to diagnose liver injury via analyzing the relevant pathway.