脓毒症小鼠髓源性抑制细胞氨基酸代谢特点

doi:10.19723/j.issn.1671-167X.2022.03.020

摘要/Abstract

摘要：

目的: 探讨脓毒症小鼠髓源性抑制细胞(myeloid-derived suppressor cells, MDSCs)氨基酸代谢组学特点。方法: 采用盲肠结扎穿孔术(cecal ligation and puncture, CLP)制备脓毒症小鼠模型，将小鼠随机分为假手术组(sham组, n = 10)和CLP组(n = 10)。术后第7天在各组存活小鼠中随机选取5只，分离小鼠骨髓MDSCs，采用安捷伦Seahorse XF技术测量MDSCs细胞的氧气消耗速率(oxygen consumption rate, OCR)，采用超高效液相色谱-串联质谱联用技术靶向检测细胞内氨基酸及寡肽含量。通过单维和多维检验分析差异代谢物和潜在生物标志物，并对潜在生物标志物进行通路富集分析。结果: CLP组小鼠骨髓中MDSCs比例(75.53% ± 6.02%)显著大于sham组的MDSCs比例(43.15% ± 7.42%, t = 7.582, P < 0.001)，且CLP组小鼠骨髓中MDSCs的基础呼吸速率[(50.03±1.20) pmol/min]、最大呼吸速率[(78.07±2.57) pmol/min]和腺嘌呤核苷三磷酸(adenosine triphosphate, ATP)产生[(25.30±1.21) pmol/min]均显著大于sham组的基础呼吸速率[(34.53±0.96) pmol/min, (t = 17.41, P < 0.001)]、最大呼吸速率[(42.57±1.87) pmol/min, (t = 19.33, P < 0.001)]和ATP产生[(12.63±0.96) pmol/min, (t = 14.18, P < 0.001)]。亮氨酸、苏氨酸、甘氨酸等17种氨基酸含量显著增加(均P < 0.05)，是脓毒症MDSCs的潜在生物标志物。增加的氨基酸主要富集在苹果酸-天冬氨酸穿梭、氨回收、丙氨酸代谢、谷胱甘肽代谢、苯丙氨酸和酪氨酸代谢、尿素循环、甘氨酸和丝氨酸代谢、β-丙氨酸代谢、谷氨酸代谢、精氨酸和脯氨酸代谢等代谢途径。结论: CLP组小鼠MDSCs中线粒体氧化磷酸化增强，苹果酸-天冬氨酸穿梭和丙氨酸代谢增强，可能为线粒体有氧呼吸提供大量原料，从而促进MDSCs发挥免疫抑制功能，阻断上述代谢途径或将有助于调节MDSCs功能，为改善脓毒症预后提供新思路。

关键词: 脓毒症, 髓源性抑制细胞, 有氧呼吸, 氨基酸代谢组学

Abstract:

Objective: To explore the amino acid metabolomics characteristics of myeloid-derived suppressor cells (MDSCs) in mice with sepsis induced by the cecal ligation and puncture (CLP). Methods: The sepsis mouse model was prepared by CLP, and the mice were randomly divided into a sham operation group (sham group, n = 10) and a CLP model group (n = 10). On the 7th day after the operation, 5 mice were randomly selected from the surviving mice in each group, and the bone marrow MDSCs of the mice were isolated. Bone marrow MDSCs were separated to measure the oxygen consumption rate (OCR) by using Agilent Seahorse XF technology and to detect the contents of intracellular amino acids and oligopeptides through ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) technology. Different metabolites and potential biomarkers were analyzed by univariate statistical analysis and multivariate statistical analysis. The major metabolic pathways were enriched using the small molecular pathway database (SMPDB). Results: The proportion of MDSCs in the bone marrow of CLP group mice (75.53% ± 6.02%) was significantly greater than that of the sham group (43.15%± 7.42%, t = 7.582, P < 0.001), and the basal respiratory rate [(50.03±1.20) pmol/min], maximum respiration rate [(78.07±2.57) pmol/min] and adenosine triphosphate (ATP) production [(25.30±1.21) pmol/min] of MDSCs in the bone marrow of CLP group mice were significantly greater than the basal respiration rate [(34.53±0.96) pmol/min, (t = 17.41, P < 0.001)], maximum respiration rate [(42.57±1.87) pmol/min, (t = 19.33, P < 0.001)], and ATP production [(12.63±0.96) pmol/min, (t = 14.18, P < 0.001)] of sham group. Leucine, threonine, glycine, etc. were potential biomarkers of septic MDSCs (all P < 0.05). The increased amino acids were mainly enriched in metabolic pathways, such as malate-aspartate shuttle, ammonia recovery, alanine metabolism, glutathione metabolism, phenylalanine and tyrosine metabolism, urea cycle, glycine and serine metabolism, β-alanine metabolism, glutamate metabolism, arginine and proline metabolism. Conclusion: The enhanced mitochondrial oxidative phosphorylation, malate-aspartate shuttle and alanine metabolism in MDSCs of CLP mice may provide raw materials for mitochondrial aerobic respiration, thereby promoting the immunosuppressive function of MDSCs. Blocking the above metabolic pathways may reduce the risk of secondary infection in sepsis and improve the prognosis.

Key words: Sepsis, Myeloid-derived suppressor cell, Aerobic respiration, Amino acid metabolomics

中图分类号:

R363.1

马媛,张玥,李蕊,邓书伟,秦秋实,朱鏐娈. 脓毒症小鼠髓源性抑制细胞氨基酸代谢特点[J]. 北京大学学报（医学版）, 2022, 54(3): 532-540.

Yuan MA,Yue ZHANG,Rui LI,Shu-wei DENG,Qiu-shi QIN,Liu-luan ZHU. Characteristics of amino acid metabolism in myeloid-derived suppressor cells in septic mice[J]. Journal of Peking University (Health Sciences), 2022, 54(3): 532-540.

图/表 6

图1

图2

表1

单维检验分析sham组与CLP组小鼠MDSCs中氨基酸及寡肽含量"

Amino acids	Sham group (n=5)	CLP group (n=5)	t/U value	P	log₂FC
Glycine	386.660±77.645	995.180±65.567	13.390	< 0.001	1.364
Leucine	219.580±34.095	587.560±79.165	9.546	< 0.001	1.420
Aspartic acid	122.680±44.956	555.440±118.618	7.628	0.001	2.179
Lysine	224.020±47.647	666.820±134.086	6.958	0.001	1.574
Histidine	98.200±16.996	202.380±36.155	5.831	0.001	1.043
Asparagine	64.500±28.406	140.580±23.324	4.629	0.002	1.124
Valine	195.400±45.947	548.020±136.323	5.481	0.003	1.488
Threonine	112.160±5.280	269.220±40.306	0.000	0.008	1.322
Serine	444.380±60.045	1 251.500±187.629	0.000	0.008	1.676
Phenylalanine	116.560±11.250	301.400±51.594	0.000	0.008	1.563
Tyrosine	89.080±14.074	243.940±47.185	0.000	0.008	1.393
Glutamate	522.220±186.936	2 490.800 (1 678.900, 2 631.900)	0.000	0.008	2.475
4-hydroxyproline	217.480±54.856	729.100±189.424	0.000	0.008	1.995
Tryptophan	24.700±9.100	103.200±30.850	0.000	0.008	2.405
Alanine	921.900 (870.000, 948.200)	3 029.320±780.214	0.000	0.008	1.801
Proline	96.800 (95.400, 104.800)	375.700±121.282	0.000	0.008	2.085
Taurine	4 646.620±2 425.105	8 336.720±739.823	0.000	0.008	0.472
Citrulline	49.140±18.249	124.040±39.186	3.874	0.009	1.336
Methionine	62.860±20.195	127.240±6.354	0.000	0.012	1.122
5-hydroxylysine	2.516±2.567	15.620±8.233	0.000	0.012	2.204
Aminoadipic acid	14.500 (13.800, 14.900)	42.380±13.225	1.000	0.016	1.814
Glutamine	297.100 (277.400, 300.600)	1 452.940±559.859	1.000	0.016	2.448
α-aminobutyric acid	27.740±14.200	83.200±34.351	3.336	0.019	1.585
Arginine	137.380±68.239	305.500±105.690	2.988	0.021	1.153
Cystine	0.180±0.000	12.376±12.628	2.500	0.025	6.207
Ornithine	91.960±58.453	312.800±160.012	2.899	0.033	1.766
Kynurenine	8.520±1.359	10.340±1.383	2.099	0.069	0.279
Carnosine	20.138±15.920	40.700±24.508	1.573	0.160	1.015
Glutathione	273.980±94.760	189.800 (187.300, 268.800)	7.000	0.310	-0.591

表1

图3

表2

图4

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