脂多糖影響奶山羊肝臟代謝的1H—NMR代謝組學(xué)和形態(tài)學(xué)研究

王林楓 賈少丹 楊改青 朱河水 楊國宇

摘 要：為研究脂多糖（lipopolysaccharide，LPS）對奶山羊肝臟營養(yǎng)代謝的影響，找出肝臟在LPS攻擊條件下的生物標(biāo)志物，探索LPS損傷肝臟的機(jī)理。該研究選擇12～18月齡，體重24～28 kg的關(guān)中奶山羊15只，平均分為3組（每組5只），分別為對照組（CTL）、低劑量組（LPS-L）和高劑量組（LPS-H）。LPS-L和LPS-H分別腹腔注射20、40 μg/kgBW的LPS溶液，CTL注射等容量生理鹽水。24 h后，LPS-L和LPS-H追加LPS溶液。0 h，24 h，48 h，分別從各組試驗(yàn)羊靜脈采血，制取血清，測定代謝相關(guān)生化檢測；48 h后活體采集肝臟組織，液氮保存。采用氫核磁共振（1H-nuclear magnetic resonance，1H-NMR）技術(shù)對肝臟組織各種代謝物進(jìn)行檢測，采用模式識別方法中的偏最小判別二乘分析法（partia1 1east squares discriminant analysis，PLS-DA），結(jié)合常規(guī)統(tǒng)計(jì)學(xué)中的顯著性檢驗(yàn)（t-test）對肝臟代謝物進(jìn)行差異性分析。血清生化指標(biāo)表明，LPS-L和LPS-H谷丙轉(zhuǎn)氨酶（ALT）、總膽紅素（TBIL）水平較CTL顯著升高（P<0.05）；甘油三酯、總膽固醇、高（低）密度脂蛋白、白蛋白和總蛋白含量較CTL顯著降低（P<0.05）。利用1H-NMR代謝組學(xué)檢測方法共檢測到69個(gè)代謝物（變量）。PLS-DA分析發(fā)現(xiàn)，代謝組數(shù)據(jù)可將CTL、LPS-L組與LPS-H之間分別聚類區(qū)分，并找到9種組間差異顯著的代謝物。與CTL相比，LPS-L和LPS-H各有6個(gè)差異代謝物，LPS-L和LPS-H之間的差異代謝物有3種，這些代謝物主要與蛋白質(zhì)、脂肪和碳水化合物等代謝途徑相關(guān)，可作為LPS肝損傷狀態(tài)下的標(biāo)志物。1H-NMR可以全面地檢測到肝臟在LPS攻擊下代謝狀態(tài)的變化，準(zhǔn)確地篩選到LPS致奶山羊肝損傷的標(biāo)志代謝產(chǎn)物，為判斷LPS影響肝臟相關(guān)代謝的途徑提供信息，為科學(xué)全面地闡明LPS影響肝臟的機(jī)理提供理論依據(jù)。

關(guān)鍵詞：代謝組學(xué) 脂多糖 肝臟 代謝物 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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