铁螯合肽联合索马鲁肽治疗阿尔茨海默病小鼠的研究

Study on Iron Chelating Peptide Combined with Semaglutide Therapy in Alzheimer's Disease Mice

关键词:  阿尔茨海默病  铁离子  铁螯合肽  索马鲁肽  联合治疗

Key words:  Alzheimer's disease  iron ion  iron chelating peptide  semaglutide  combination therapy

摘要:

目的 研究bs-5-YHEDA铁螯合肽联合索马鲁肽对D-半乳糖诱导的阿尔茨海默病(Alzheimer's disease，AD)模型小鼠认知能力和病理特征的影响。方法 将40只8周龄昆明小鼠随机分为5组，分别为健康对照组、PBS组、bs-5-YHEDA铁螯合肽组、联合治疗组和阳性对照组，每组8只，雌雄各半。除健康对照组外，其他各组注射D-半乳糖连续6周诱导构建AD小鼠模型。从第4周开始连续3周，bs-5-YHEDA铁螯合肽组每隔1 d尾静脉注射200 μL bs-5-YHEDA(1 mg·mL^–1)1次；联合治疗组每天交替给予200 μL bs-5-YHEDA铁螯合肽(1 mg·mL^–1)和索马鲁肽(25 nmol·kg^–1·d^–1)1次；阳性对照组，每隔 1 d灌胃给药美金刚(3.3 mg·kg^–1·d^–1)1次；健康对照组和PBS组注射同等剂量PBS。治疗结束后通过Morris水迷宫法检测小鼠学习记忆能力，解剖取全脑和全血，HE染色观察海马区病理改变，免疫组织化学、酶联免疫吸附、免疫印迹检测Aβ表达和Tau蛋白磷酸化水平。结果 在Morris水迷宫空间探索试验结果显示，与健康对照组比较，PBS组小鼠穿越平台次数、目标象限游泳路程比和时间比的差异均具有统计学意义(P<0.05)；与PBS组相比较，联合治疗组目标象限游泳路程比增加，差异具有统计学意义(P<0.05)。HE染色结果显示，与健康对照组小鼠相比，PBS组海马区锥体细胞层次减少，排列紊乱，细胞水肿，细胞核固缩深染，药物治疗后各治疗组海马区细胞排列紊乱、胞核固缩深染及细胞凋亡情况明显改善。免疫组织化学染色和Western blotting结果显示，与健康对照组小鼠相比，PBS组小鼠Aβ表达水平和Tau蛋白磷酸化水平明显升高，药物治疗后各组小鼠Aβ表达水平和Tau蛋白磷酸化水平明显降低，差异具有统计学意义(P<0.01或P<0.001)。结论 bs-5-YHEDA铁螯合肽联合索马鲁肽能够有效改善AD小鼠学习记忆能力和病理特征，但从免疫组织化学和免疫印迹实验结果来看，相较于单一bs-5-YHEDA铁螯合肽组，联合治疗组对AD模型小鼠病理特征的改善并不明显，提示设计的双靶点联合治疗对AD模型鼠认知改善可能存在阈值效应，多靶点组合治疗效果的优化及验证有待进一步的研究。

Abstract:

OBJECTIVE To investigate the effect of bs-5-YHEDA iron chelating peptide combined with semaglutide on the cognitive ability and pathological characteristics of D-Gal-induced Alzheimer's disease(AD) model mice. METHODS Forty mice were randomly divided into 5 groups, namely the healthy control group, PBS group, bs-5-YHEDA iron chelating peptide group, combined treatment group and positive control group, with 8 mice in each group, half of each sex. Except for the healthy control group, D-galactose was injected to induce the AD mice model for 6 weeks. For 3 consecutive weeks starting from the 4th week, the bs-5-YHEDA iron chelating peptide group was injected with bs-5-YHEDA(1 mg·mL^–1) once every other day at 200 μL in the tail vein; the bs-5-YHEDA iron chelating peptide(1 mg·mL^–1) and semaglutide(25 nmol·kg^–1·d^–1) were given alternately once a day in the combination treatment group; the positive control group was given memantine(3.3 mg·kg^–1·d^–1) by gavage every other day. The healthy control group and PBS group were injected with the equal dose of PBS. At the end of treatment, the learning memory ability of mice was detected by the Morris water maze method, whole brain and whole blood were dissected, and pathological changes in hippocampal region were observed by HE staining, and Aβ expression and Tau protein phosphorylation levels were detected by immunohistochemistry, enzyme-linked immunosorbent assay and immunoblotting. RESULTS In the Morris water maze spatial exploration experiment, the differences in the number of times the mice traversed the platform, the ratio of swimming distance to the target quadrant, and the time ratio were statistically significant in each group(P<0.05); compared with the PBS group, the ratio of swimming distance to the target quadrant increased in the combined treatment group, and the differences were statistically significant(P<0.05). The results of HE staining showed that compared with the healthy control mice, the hippocampal area in the PBS group showed reduced levels of pyramidal cells, disorganized arrangement, cell edema, and deep staining of nuclei consolidation. Cellular disorganization, deep staining of nuclei and apoptosis in the hippocampus were significantly improved in each treatment group after drug treatment. Immunohistochemistry and Western blotting results showed that the Aβ expression levels and Tau protein phosphorylation levels were significantly higher in the PBS-administered mice compared with the healthy control mice, and the Aβ expression levels and Tau protein phosphorylation levels were reduced in each group after drug treatment, with statistically significant differences(P<0.01 or P<0.001 ). CONCLUSION The combination of bs-5-YHEDA iron chelating peptide and semaglutide can effectively improve the learning and memory ability and pathological characteristics of AD mice, but from the results of immunohistochemistry and immunoblotting experiments, the improvement of pathological characteristics of AD mice in the combination treatment group is not obvious compared with the single bs-5-YHEDA iron chelating peptide group, suggesting that there may be a threshold effect of our designed dual-target combination treatment on the cognitive improvement of AD mice, and the optimization and validation of the effect of multi-target combination treatment need further study.