基于代谢组学分析海分枝杆菌对巨噬细胞的影响

Evaluation of effects of Mycobacterium marinum on macrophages through a metabolomics analysis

目的通过靶向代谢组学分析海分枝杆菌刺激巨噬细胞引起的能量代谢和氧化脂质代谢变化,为了解巨噬细胞抵御海分枝杆菌感染的免疫机制提供线索。方法从小鼠双侧股骨中获取小鼠骨髓来源巨噬细胞,培养后将其分为活菌组和灭活菌组;采用临床分离培养的海分枝杆菌制备菌悬液,活菌组加活海分枝杆菌刺激12 h,灭活菌组加灭活的海分枝杆菌刺激12 h。显微镜下观察两组细胞的形态并测量巨噬细胞长度。收集两组细胞的裂解液,利用液相色谱串联质谱法检测能量代谢物和氧化脂质代谢物。采用t检验比较组间巨噬细胞长度,采用主成分分析、正交偏最小二乘法-判别分析等筛选差异代谢物。结果镜下可以见活菌组巨噬细胞形成更多的肉芽肿样细胞聚集,灭活菌组巨噬细胞[(439.52±91.67)μm]较活菌组[(289.96±70.11)μm]更细长,差异有统计学意义(P<0.001)。巨噬细胞能量代谢和氧化脂质代谢的主成分分析、正交偏最小二乘法-判别分析均显示两组分离度好。能量代谢方面共发现12种差异代谢物,以氨基酸代谢变化最为显著,其中瓜氨酸的含量明显升高,亮氨酸和丝氨酸的含量下降。氧化脂质代谢方面共发现20种差异代谢物,以花生四烯酸代谢变化最为显著。结论相较于灭活菌,海分枝杆菌活菌刺激的巨噬细胞氨基酸代谢和花生四烯酸代谢发生改变,瓜氨酸代谢水平升高、亮氨酸和丝氨酸代谢水平降低、花生四烯酸类代谢水平改变。

Objective To analyze changes in energy metabolism and oxylipin metabolism in macrophages after stimulation by Mycobacterium marinum(M.marinum)using targeted metabolomics,and to provide insights into the mechanisms underlying the immune defense by macrophages against M.marinum infections.Methods Mouse bone marrow-derived macrophages were obtained from the bilateral femurs of mice,and cultured cells were divided into two groups:the active M.marinum group and the inactivated M.marinum group.Bacterial suspensions were prepared using M.marinum clinical isolates;the active M.marinum group was treated with live M.marinum suspensions for 12 hours,while the inactivated M.marinum group with inactivated M.marinum suspensions for 12 hours.Cell morphology was observed through microscopy,and cell length was measured.Cell lysates collected from both groups were subjected to liquid chromatography-tandem mass spectrometry analysis to detect energy and oxylipin metabolites.A t-test was utilized to compare the lengths of macrophages between the two groups,while principal component analysis and orthogonal partial least squares-discriminant analysis were conducted to identify differential metabolites.Results Under the microscope,macrophages in the active M.marinum group formed more granuloma-like cell aggregates compared with those in the inactivated M.marinum group;the macrophages were significantly thinner and longer in the inactivated M.marinum group(439.52±91.67μm)than in the active M.marinum group(289.96±70.11μm,P<0.001).Principal component analysis and orthogonal partial least squares-discriminant analysis of energy metabolism and oxylipin metabolism in macrophages demonstrated good separation between the two groups.As for the energy metabolism,a total of 12 differential metabolites were identified,with the amino acid metabolism showing the most significant changes.Specifically,there was a significant increase in the content of L-citrulline,while the content of L-leucine and serine decreased.As for the oxylipin metabolism,20 differential metabolites were identified,with the arachidonic acid metabolism showing the most significant changes.Conclusions Macrophages stimulated by live M.marinum exhibited altered amino acid metabolism and arachidonic acid metabolism compared with those stimulated by inactivated M.marinum,characterized by an increase in L-citrulline content,a decrease in L-leucine and serine levels,and alterations in arachidonic acid content.