山茱萸水提物对Aβ_(25-35)诱导阿尔茨海默病小鼠脑损伤和神经炎症的作用

Effect of aqueous extract of Corni Fructus on Aβ_(25-35)-induced brain injury and neuroinflammation in mice with Alzheimer′s disease

探究山茱萸水提物对β-淀粉样蛋白片段_(25-35)(β-amyloid protein_(25-35),Aβ_(25-35))诱导阿尔茨海默病(Alzheimer′s disease,AD)小鼠脑损伤和神经炎症的作用,为山茱萸治疗AD提供实验依据。将60只C57BL/6J雄性小鼠随机分为假手术组、模型组、阳性药石杉碱甲组(0.2 mg·kg^(-1))以及山茱萸水提物低(1.3 g·kg^(-1))、中(2.6 g·kg^(-1))、高(5.2 g·kg^(-1))剂量组,除假手术组外,其他组采用Aβ_(25-35)侧脑室注射的方法构建AD动物模型,之后灌胃给药24 d。在动物解剖前1周进行行为学检测;苏木精-伊红(hematoxylin-eosin,HE)染色观察海马区神经元形态;流式细胞术检测小鼠原代海马细胞凋亡水平;ELISA试剂盒检测小鼠脑组织中β-淀粉样蛋白1-42(β-amyloid protein 1-42,Aβ1-42)及磷酸化微管相关蛋白Tau(phosphorylated microtubule-associated protein Tau,p-Tau)的水平;免疫荧光及Western blot检测小鼠脑组织中相关蛋白的表达;MTT法检测山茱萸中化合物对Aβ_(25-35)诱导N9细胞损伤的作用;分子对接分析咖啡酸、反式对羟基桂皮酸、异落叶松树脂醇-9′-O-β-D-吡喃葡萄糖苷、马栗树皮素和(+)-南烛木树脂酚与Aβ前体蛋白(β-amyloid precursor protein,APP)、白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)之间的相互作用。山茱萸水提物通过增加Aβ_(25-35)诱导小鼠自主活动时间、自主交替率、偏好系数和辨别系数改善了其学习记忆能力,并通过升高脑组织中白细胞介素-10(interleukin-10,IL-10)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)蛋白表达水平,降低Aβ1-42、p-Tau、IL-6、TNF-α、胱天蛋白酶3(cysteine aspartate-specific protease 3,caspase-3)、胱天蛋白酶9(cysteine aspartate-specific protease 9,caspase-9)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)的表达水平,减少胶质细胞活化数量,显著减轻了Aβ_(25-35)诱导小鼠脑损伤和神经炎症。细胞实验结果显示马栗树皮素和(+)-南烛木树脂酚可改善Aβ_(25-35)诱导N9细胞损伤,分子对接结果表明咖啡酸、反式对羟基桂皮酸、异落叶松树脂醇-9′-O-β-D-吡喃葡萄糖苷、马栗树皮素和(+)-南烛木树脂酚与APP有较好的结合活性,与IL-6、TNF-α结合较弱。山茱萸水提物通过减少脑内细胞凋亡和胶质细胞活化数量,降低炎症因子表达水平来改善Aβ_(25-35)诱导小鼠的认知功能障碍和脑损伤。其中咖啡酸、反式对羟基桂皮酸、异落叶松树脂醇-9′-O-β-D-吡喃葡萄糖苷、马栗树皮素和(+)-南烛木树脂酚可能是山茱萸发挥抗AD作用的物质基础。

The purpose of this study was to investigate the effect of aqueous extract of Corni Fructus onβ-amyloid protein 25-35(Aβ_(25-35))-induced brain injury and neuroinflammation in Alzheimer′s disease(AD)mice to provide an experimental basis for the treatment of AD by aqueous extract of Corni Fructus.Sixty C57BL/6J male mice were randomly divided into a sham group,a model group,a positive control group(huperizine A,0.2 mg·kg^(-1)),a low-dose aqueous extract of Corni Fructus group(1.3 g·kg^(-1)),a medium-dose aqueous extract of Corni Fructus group(2.6 g·kg^(-1)),and a high-dose aqueous extract of Corni Fructus group(5.2 g·kg^(-1)).The AD model was induced by lateral ventricular injection of Aβ_(25-35)in mice except for those in the sham group,and AD model mice were treated with corresponding drugs by gavage for 24 days.The behavioral test was performed one week before animal dissection.Hematoxylin-eosin(HE)staining was performed to observe the morphology of neurons in the hippocampal region.Flow cytometry was used to detect the apoptosis level of primary hippocampal cells in mice.ELISA kits were used to detect the levels ofβ-amyloid protein 1-42(Aβ_(1-42))and phosphorylated microtubule-associated protein Tau(p-Tau)in mouse brain tissues.Immunofluorescence and Western blot were used to detect the expression of related proteins in mouse brain tissues.MTT assay was used to detect the effect of compounds in aqueous extract of Corni Fructus on Aβ_(25-35)-induced N9 cell injury.Molecular docking was employed to analyze the interactions of caffeic acid,trans-p-hydroxy cinnamic acid,isolariciresinol-9′-O-β-D-glucopyranoside,esculetin,and(+)-lyoniresinol withβ-amyloid precursor protein(APP),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α).Aqueous extract of Corni Fructus could improve the learning and memory abilities of Aβ_(25-35)-induced mice by increasing the duration of the autonomous activity,the rate of autonomous alternation,the preference coefficient,and the discrimination coefficient,and reduce Aβ_(25-35)-induced brain injury and neuroinflammation in mice by increasing the expression levels of interleukin-10(IL-10)and B-cell lymphoma-2(Bcl-2)in brain tissues,decreasing the expression levels of Aβ_(1-42),p-Tau,IL-6,TNF-α,cysteine aspartate-specific protease 3(caspase-3),cysteine aspartate-specific protease 9(caspase-9),and Bcl-2-associated X protein(Bax),and decreasing the number of activated glial cells in brain tissues.The results of cell experiments showed that esculetin and(+)-lyoniresinol could improve Aβ_(25-35)-induced N9 cell injury.Molecular docking results showed that caffeic acid,trans-p-hydroxy cinnamic acid,isolariciresinol-9′-O-β-D-glucopyranoside,esculetin,and(+)-lyoniresinol had good binding affinity with APP and weak binding affinity with IL-6 and TNF-α.Aqueous extract of Corni Fructus could ameliorate cognitive dysfunction and brain damage in Aβ_(25-35)-induced mice by reducing the number of apoptotic cells and activated glial cells in the brain and decreasing the expression level of inflammatory factors.Caffeic acid,trans-p-hydroxy cinnamic acid,isolariciresinol-9′-O-β-D-glucopyranoside,esculetin,and(+)-lyoniresinol may be the material basis for the anti-AD effect of aqueous extract of Corni Fructus.