枳椇果梗多糖调节谷氨酸代谢和紧密连接蛋白表达改善酒精对小鼠神经行为影响

张玉超; 袁子飞; 刘良禹; 朱思洁; 杨永; 郑代梅; 刘旭东

doi:10.13386/j.issn1002-0306.2024050346

枳椇果梗多糖调节谷氨酸代谢和紧密连接蛋白表达改善酒精对小鼠神经行为影响

Hovenia dulcis Fruit Peduncle Polysaccharides Modulate Glutamate Metabolism and Tight Junction Protein Expressions to Attenuate the Neurotoxic Effects of Alcohol on Mice

摘要

摘要: 目的：本研究旨在明确枳椇果梗多糖（HDPs）对酒精暴露所致的小鼠神经行为异常的改善效果，并探究谷氨酸代谢和紧密连接蛋白表达在其中的作用。方法：雄性C57BL/6小鼠按114 μL/20 g剂量连续酒精灌胃14 d，建立酒精暴露模型，同时设置干预组进行HDPs干预（114 μL/20 g酒精+100 mg/kg HDPs）。应用行为学实验（旷场实验、高架十字迷宫实验）评估神经行为学变化，采用气相色谱法测定小鼠血液中乙醇浓度，γ-H2AX荧光检测小鼠脑海马组织DNA损伤，免疫组化分析检测小鼠脑组织中紧密连接蛋白Claudin-1和ZO-1的表达，并通过超高效液相色谱-四级杆飞行时间质谱法（UPLC-Q-TOF-MS）代谢组学技术对小鼠脑组织代谢物进行分析。结果：HDPs可有效降低酒精暴露小鼠血液乙醇浓度，由4.69±0.29 g/L降至1.64±0.104 g/L；改善酒精暴露所致的小鼠神经行为异常，旷场实验中，与酒精组相比，HDPs干预组总路程显著提升至27340±3304 cm（P<0.05），平均速度显著提升至67.4±13.4 cm/s（P<0.05），不动时间缩短29%（P<0.05）；高架十字迷宫实验中，与酒精组相比，HDPs干预组闭臂停留时间显著减少至195.6±10.3 s（P<0.05），开放臂进入次数显著增加26%（P<0.05））；还可降低酒精诱导的脑组织氧化应激与DNA损伤水平，ROS、MDA分别降低5.4%、29.5%（P<0.05），T-AOC提高10.9%，上调脑海马组织中Claudin-1（2.2倍）和ZO-1（0.1倍）蛋白的表达；并调节脑组织谷氨酸代谢通路，提高甘氨酸（19.7%）、谷光甘肽（25%）、琥珀酸（22.6%）等代谢物水平。结论：HDPs可有效改善酒精对小鼠神经行为的影响，其机制或可能通过抗氧化、保护紧密连接蛋白和调节谷氨酸代谢通路发挥作用，研究结果可为扩展枳椇资源在食品领域中的应用提供理论依据。

Abstract: Objective: This study aimed to investigate the protective effects of Hovenia dulcis fruit peduncle polysaccharides (HDPs) on alcohol-induced neurobehavioral alterations in mice and to elucidate whether HDPs mitigate alcohol-induced neuronal damage by modulating glutamate metabolic pathways and the expression of tight junction proteins. Methods: Male C57BL/6 mice were administered alcohol intragastrically at a dose of 114 μL/20 g for 14 d to establish an alcohol exposure model, and an intervention group was set up for HDPs intervention (114 μL/20 g alcohol+100 mg/kg HDPs). Behavioral experiments (open field test, elevated plus maze test) were used to assess changes in neurobehavior, gas chromatography was used to determine ethanol concentrations in mouse blood. γ-H2AX fluorescence was used to detect DNA damage in mouse hippocampal tissue, and immunohistochemical analysis was used to detect the expression of tight junction proteins Claudin-1 and ZO-1 in mouse brain tissue. Metabolites in mouse brain tissue were analyzed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) metabolomics technology. Results: HDPs effectively reduced blood ethanol concentration in alcohol-exposed mice (from 4.69±0.29 g/L to 1.64±0.104 g/L) and ameliorated alcohol-induced neurobehavioral abnormalities. In the open field test, compared to the alcohol group, HDPs intervention significantly increased total distance traveled (27340±3304 cm, P<0.05) and average velocity (67.4±13.4 cm/s, P<0.05), while reduced immobility time by 29% (P<0.05). The elevated plus maze test revealed that HDPs treatment decreased closed-arm dwelling time (195.6±10.3 s, P<0.05) and increased open-arm entries by 26% (P<0.05) compared to the alcohol group. Additionally, HDPs alleviated alcohol-induced oxidative brain damage by reducing ROS levels (5.4% reduction) and MDA content (29.5% decrease, P<0.05), while enhanced total antioxidant capacity (T-AOC) by 10.9%. It upregulated hippocampal expression of tight junction proteins Claudin-1 (2.2-fold increase) and ZO-1 (10% increase). HDPs also modulated glutamatergic metabolic pathways, elevating brain levels of glycine (19.7% increase), glutathione (25% increase), and succinate (22.6% increase). Conclusion: HDPs can effectively alleviate the neurobehavioral impact of alcohol on mice. The mechanism may involve antioxidant activity, protection of tight junction proteins, and modulation of glutamate metabolic pathways, providing a theoretical basis for the development and application of Hovenia dulcisis resources in the food field.