慢性醋酸铅染毒对小鼠焦虑样行为和血清代谢组学的影响及相关关系研究
Effects of chronic lead acetate exposure on neurobehavior and serum metabolomics in mice
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摘要:
背景 长期低浓度接触铅可损害中枢神经系统，引起焦虑样行为，这一过程与血清代谢物改变的关系尚不清楚。
目的 探究不同剂量慢性醋酸铅（PbAc）暴露对小鼠焦虑样行为及血清代谢物的影响，从代谢组学角度探讨铅暴露对小鼠神经行为损伤的作用机制。
方法 4周龄SPF级C57BL/6J小鼠共64只，雌雄各半。小鼠根据性别随机分为4组，每组16只：对照组（正常饮水）、20 mg·L−1 PbAc组、100 mg·L−1 PbAc组、500 mg·L−1 PbAc组。小鼠自由饮用含不同浓度PbAc水10周后，采用旷场实验检测小鼠焦虑样行为改变。麻醉小鼠后摘眼球取血，分离血清，采用液相色谱-质谱联用技术结合主成分分析和偏最小二乘判别分析比较不同剂量铅暴露对小鼠血清中代谢物的影响。对差异代谢物进行京都基因与基因组百科全书（KEGG）富集分析，并且对差异代谢物与行为学指标进行相关性分析。
结果 旷场实验结果显示，与对照组比较，100 mg·L−1和500 mg·L−1 PbAc组在中心区域运动时间减少，500 mg·L−1 PbAc组中心区域穿越次数减少，100 mg·L−1和500 mg·L−1 PbAc组排便次数增加（P＜0.05）。在正离子和负离子模式下，与对照组比较，20 mg·L−1 PbAc组筛选出157种差异代谢物，其中上调的差异代谢物80种，下调的差异代谢物77种；100 mg·L−1 PbAc组筛选出172种差异代谢物，其中上调的差异代谢物57种，下调的差异代谢物115种；500 mg·L−1 PbAc组筛选出119种差异代谢物，其中上调的差异代谢物42种，下调的差异代谢物77种。对差异代谢物进行KEGG富集分析，发现代谢通路的改变主要涉及初级胆汁酸的生物合成、胆汁分泌、胆固醇代谢。差异代谢物中，炔诺酮与中心区域穿越次数呈正相关（r=0.406，P<0.05），二氢胸腺嘧啶与中心区域穿越次数呈负相关（r=−0.346，P<0.05），溶血磷脂酰胆碱22∶1和溶血磷脂14∶0与中心区域停留时间呈负相关（r=−0.429，P<0.05；r=−0.374，P<0.05）。
结论 慢性铅暴露可引起小鼠焦虑样行为，这种行为改变与血清中代谢产物改变相关，差异代谢物主要富集到初级胆汁酸生物合成、胆汁酸分泌和胆固醇代谢通路。
Abstract:Background Chronic low-level exposure to lead can damage the central nervous system and cause anxiety-like behavior. However, whether changes of blood metabolites occur in this process and its relationship with lead-induced neurobehavioral disorder remain unclear.
Objective To explore the effects of chronic lead acetate (PbAc) exposure at different concentrations on anxiety-like behavior and serum metabolites and their relationships in mice, as well as the mechanism of lead exposure on neurobehavioral injury in mice from the perspective of metabolomics.
Methods A total of 64 healthy 4-week-old C57BL/6J mice, half male and half female, were randomly divided into four groups: control group (normal drinking water), 20 mg·L−1 PbAc group, 100 mg·L−1 PbAc group, and 500 mg·L−1 PbAc group. After 10 weeks of free drinking of water containing designed concentrations of PbAc, the mice were tested for anxiety-like behavioral changes by open field experiment. After the mice were anesthetized, blood was collected from the eyes, the serum was separated, and the effects of designed doses of lead exposure on metabolites in the serum of mice were compared by liquid chromatography with tandem mass spectrometry combined with principal component analysis and partial least squares discrimination analysis.
Results The results of the open field experiment showed that the reductions in movement time spent in central area in the 100 mg·L−1 and 500 mg·L−1 PbAc groups compared with the control group were of statistical significance (P<0.05); the reduction in crossing times of central region in the 500 mg·L−1 PbAc group was statistically significant compared with the control group (P<0.05); the increases in defecation frequency in the 100 mg·L−1 and 500 mg·L−1 PbAc groups were statistically significant compared to the control group (P<0.05). In both positive and negative ion modes, compared with the control group, 157 differential metabolites were screened out in the 20 mg·L−1 PbAc group, of which 80 were up-regulated and 77 were down-regulated; 172 differential metabolites were screened out in the 100 mg·L−1 PbAc group, of which 57 were up-regulated and 115 were down-regulated; 119 differential metabolites were screened out in the 500 mg·L−1 PbAc group, of which 42 were up-regulated and 77 were down-regulated. The results of the KEGG enrichment analysis on the differential metabolites revealed alterations in metabolic pathways mainly involving primary bile acid biosynthesis, bile secretion, and cholesterol metabolism. Among the differential metabolites, norethisterone was positively correlated with the number of central region crossings (r =0.406, P<0.05); dihydrothymine was negatively correlated with the number of central region crossings (r=−0.346, P<0.05); lysophosphatidylcholine 22∶1 and lysophospholipid 14∶0 were negatively correlated with time spent in central region (r=−0.429, P<0.05;r=−0.374, P<0.05).
Conclusion Chronic lead exposure induces anxiety-like behavior in mice, and this altered behavior is associated with altered metabolites in serum, with differential metabolites enriched primarily in the metabolic pathways of primary bile acid biosynthesis, bile acid secretion, and cholesterol metabolism.
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