淫羊藿苷对小鼠慢性低氧诱导学习记忆功能降低的防护作用及机制

Protective effect and mechanism of icarrin on the decrease of learning and memory function induced by chronic hypoxia in mice

目的探究淫羊藿苷(ICA)对小鼠慢性低氧环境暴露诱导记忆障碍的主要保护作用机制。方法选择8周龄雄性C57BL/6小鼠40只,建立模拟海拔6000 m低氧环境暴露动物模型。40只小鼠随机分至常氧组、常氧+ICA组、低氧组、低氧+ICA组(每组10只)。暴露28 d后进行Y迷宫、新物体识别实验评测小鼠空间记忆功能。利用免疫荧光染色观察小鼠海马组织小胶质细胞活化程度和神经元损伤情况,利用qRT-PCR方法检测海马组织iNOS、IL-1β、TNF-α、Cxcl-1、IL-4等炎性分子表达。使用喹啉酸试剂盒检测海马组织色氨酸犬尿氨酸途径代谢产物喹啉酸含量,使用qRT-PCR方法检测色氨酸犬尿氨酸代谢途径Ido1、Kmo、Haao、Kynu、Kyat3相关分子mRNA表达情况。结果与常氧组相比,Y迷宫及新物体识别实验结果显示低氧组小鼠出现明显空间记忆障碍(P<0.01);免疫荧光染色组织切片可见低氧组较常氧组海马小胶质细胞活化及神经元损伤明显(P<0.01);低氧组海马组织喹啉酸含量较常氧组升高(P<0.01);qRT-PCR检测发现低氧组海马组织炎症相关分子表达较常氧组显著升高(P<0.01),色氨酸犬尿氨酸代谢相关分子Ido1(P<0.01)、Kmo(P<0.01)、Haao(P<0.01)表达较常氧组明显升高,Kynu(P<0.01)、Kyat3(P<0.01)较常氧组表达降低。低氧+ICA组与低氧组小鼠相比,空间记忆能力有所改善(P<0.05),海马区小胶质细胞活化及神经元损伤相对减弱(P<0.05),炎症相关分子(P<0.01)及色氨酸犬尿氨酸代谢相关分子(P<0.01)表达均有相应回调。结论ICA可通过调控小胶质细胞色氨酸犬尿氨酸代谢,抑制小胶质细胞炎性活化和喹啉酸生成,缓解慢性低氧诱导小鼠海马神经元损伤,改善记忆功能减退。

Objective To explore the main protective mechanism of icariin(ICA)on memory impairment induced by chronic hypoxic exposure in mice.Methods Forty 8-week-old male C57BL/6 mice were selected to establish an animal model simulating hypoxic environment exposure at an altitude of 6000 m.Forty mice were randomly divided into normoxia group,normoxia+ICA group,hypoxia group,hypoxia+ICA group(10 mice in each group).After 28 d of exposure,Y-maze and novel object recognition tests were performed to evaluate the spatial memory function of mice.The degree of microglia activation and neuronal damage in the hippocampus of mice were observed by immunofluorescence staining.The expression of iNOS,IL-1β,TNF-α,Cxcl-1 and IL-4 in hippocampus were detected by qRT-PCR.The concentration of quinolinic acid,a metabolite of tryptophane-kynuridine pathway,was detected by quinolinic acid kit in hippocampus,and the mRNA expression of Ido1,Kmo,Haao,Kynu and Kyat3 in tryptophane-kynuridine pathway was detected by qRT-PCR.Results Compared with the normoxia group,Y-maze and novel object recognition tests revealed significant spatial memory impairment in mice of hypoxia group(P<0.01).Immunofluorescence staining demonstrated increased microglia activation and neuronal damage in hypoxia group compared with those in normoxia group(P<0.01).The concentration of quinolinic acid in hippocampus was higher in hypoxia group than that in normoxia group(P<0.01).qRT-PCR analysis indicated significantly elevated expression of inflammation-related molecules in hippocampus of mice in hypoxia group(P<0.01).For those functional molecules related to tryptophan-kynurenine metabolism,Ido1(P<0.01),Kmo(P<0.01)and Haao(P<0.01)were notably increased,while Kynu(P<0.01)and Kyat3(P<0.01)were significantly decreased in hypoxia group compared with those in normoxia group.In comparison with hypoxia group,hypoxia+ICA group had an improvement in spatial memory ability(P<0.05),a reduction in microglial activation and neuronal damage in hippocampus(P<0.05),as well as a decrease in expressions of inflammation-related molecules(P<0.01)and functional molecules involved in tryptophan-kynurenine metabolism(P<0.01).Conclusion ICA can regulate tryptophan-kynurenine metabolism in microglia,inhibit the inflammatory activation of microglia and quinolinic acid production,thereby alleviating hippocampal neuronal damage induced by chronic hypoxia and improving memory function in mice.