脂多糖影响奶山羊肝脏代谢的1H—NMR代谢组学和形态学研究

王林枫 贾少丹 杨改青 朱河水 杨国宇

摘 要：为研究脂多糖（lipopolysaccharide，LPS）对奶山羊肝脏营养代谢的影响，找出肝脏在LPS攻击条件下的生物标志物，探索LPS损伤肝脏的机理。该研究选择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）、低剂量组（LPS-L）和高剂量组（LPS-H）。LPS-L和LPS-H分别腹腔注射20、40 μg/kgBW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。0 h，24 h，48 h，分别从各组试验羊静脉采血，制取血清，测定代谢相关生化检测；48 h后活体采集肝脏组织，液氮保存。采用氢核磁共振（1H-nuclear magnetic resonance，1H-NMR）技术对肝脏组织各种代谢物进行检测，采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），结合常规统计学中的显著性检验（t-test）对肝脏代谢物进行差异性分析。血清生化指标表明，LPS-L和LPS-H谷丙转氨酶（ALT）、总胆红素（TBIL）水平较CTL显著升高（P<0.05）；甘油三酯、总胆固醇、高（低）密度脂蛋白、白蛋白和总蛋白含量较CTL显著降低（P<0.05）。利用1H-NMR代谢组学检测方法共检测到69个代谢物（变量）。PLS-DA分析发现，代谢组数据可将CTL、LPS-L组与LPS-H之间分别聚类区分，并找到9种组间差异显著的代谢物。与CTL相比，LPS-L和LPS-H各有6个差异代谢物，LPS-L和LPS-H之间的差异代谢物有3种，这些代谢物主要与蛋白质、脂肪和碳水化合物等代谢途径相关，可作为LPS肝损伤状态下的标志物。1H-NMR可以全面地检测到肝脏在LPS攻击下代谢状态的变化，准确地筛选到LPS致奶山羊肝损伤的标志代谢产物，为判断LPS影响肝脏相关代谢的途径提供信息，为科学全面地阐明LPS影响肝脏的机理提供理论依据。

关键词：代谢组学 脂多糖 肝脏 代谢物 奶山羊

Study of the Effect of Lipopolysaccharide on Hepatic Metabolism in Dairy Goat Liver Based on 1H-NMR and Morphology

Wang Linfeng Jia Shaodan Yang Gaiqing Zhu Heshui Yang Guoyu

（He'nan Agricultural University）

Abstract：In order to research the changes of dairy goat liver metabolites by using 1H-NMR test via molecular markers and explore the influence of lipopolysaccharide （LPS） on dairy goat liver.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， LPS-L group and LPS-H group. Animals could access to total mixed ration feeds and water ad libitum. At beginning of the experiment （0 h）， the goats in LPS-L and LPS-H groups were given 20 and 40 μg/kg 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 treated groups were added LPS solution once again.At the time point of 0 h， 24 h and 48 h， blood sample were drawn from neck venous and were used to extract serum after centrifuging for biochemical determination. 48 h later， the goats liver tissue samples were collected by biopsy and stored in liquid nitrogen. Metabolites in liver tissue were detected by 1H-NMR and analyzed by partial least squares discriminant analysis （PLS-DA） combined with t-test to identify metabolic differences under each treatment.Serum biochemical results showed the concentration of ALT and TBIL were increased significantly （P<0.05）， while TG， TC， TP， ALB， HDL， LDL were markedly decreased （P<0.05） in each LPS treated groups compared with CTL， indicating the goats liver were injured in some degree. Liver metabolism also changed markedly. Totally， there are 69 metabolites were detected via 1H-NMR based on its identification database. When the metabolite variables were analyzed using PLS-DA， it showed that CTL， LPS-L and LPS-H groups could be clustered and distinguished based on the metabolomic data analysis. 9 out of 69 metabolites differ significantly among the three groups. 6 metabolites in LPS-L and LPS-H groups were notable compared with CTL， and 3 metabolites were different significantly between LPS-L and LPS-H. Further analysis indicates that these metabolites were related to amino acid metabolism， fat metabolism and carbohydrate metabolism in hepatic cells.In conclusion， metabolomics profile can effectively distinguish LPS-L and LPS-H from CTL based on 1H-NMR technique. Metabolomic technology could precisely detect characteristic metabolites in liver under LPS as well as provide enough information to diagnose the injured pathway relevant to clarify the mechanism of liver suffering from LPS.

Key Words：Metabolomics； Lipopolysaccharide； Liver； Metabolite； Dairy goat

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