薄荷醇抑制β淀粉样蛋白聚合保护神经细胞的实验研究

摘要

Objective To determine whether menthol can inhibit amyloid-β protein(Aβ) polymerization to protect neurocytes.Methods The docking construction between menthol and Aβ was structured by computer modeling technique.The combination between menthol and Aβ was observed by transmission electron microscope using 30 μmol/L Aβ co-cultivating with 200 μmol/L menthol for 72 h;30 μmol/L Aβ polymerized alone and cultivated with 10％ dimethyl sulfoxide were set as control.Human neuroblastoma cell line SH-SY5Y was co-cultivated with 30 μmol/L Aβ or 30 μmol/L Aβ ± 200 μmol/L menthol,and cell proliferation was tested after 12,24,36,72 h by Cell Counting Kit-8 (CCK8).Twenty 6 months old Aβ precursor protein(APP)/presenilin-1 (PS1) transgenic mice were randomly divided into experimental group(n =10) treated by 1 mg/g menthol lavage and control group(n =10) treated by 0.9％ sodium chloride injection lavage till the mice were 10 months old;the formation of senile plaques in hippocampus was tested by immunohistochemistry.Results Computer modeling showed 2 stable docking positions between menthol and Aβ.Transmission electron microscopy showed that Aβ polymerization could be significantly depressed by menthol co-cultivation.CCK8 test showed that menthol could inhibit the neurotoxicity induced by Aβ;the activity of SH-SY5Y cells had significant differences between Aβ group and Aβ ± menthol group[12 h:(0.85 ±0.12) vs (1.13 ±0.14),P ＜0.05;24 h:(0.77 ±0.09) vs (1.09 ±0.11),P ＜0.05;36h:(0.67±0.13) vs (1.01 ±0.11),P＜0.05;72 h:(0.49 ±0.07) vs (0.92±0.10),P＜0.01].Immunohistochemistry test showed that menthol could reduce senile plaque formation in hippocampus of mice.Conclusion Menthol can inhibit Aβ monomer translating to oligomer to prevent nerve cell injury.%目的 观察薄荷醇是否能通过抑制β淀粉样蛋白(Aβ)聚合从而保护神经细胞.方法 利用计算机模拟技术,预测薄荷醇单体与Aβ单体的结构关系.将30 μmol/L Aβ单独孵育、或与10％二甲基亚砜孵育、或与200 μmol/L薄荷醇共同孵育72 h,透射电镜下观察Aβ聚合情况.以入神经母细胞瘤细胞SH-SY5Y为靶细胞,与30μmol/L Aβ或30 μmol/L Aβ±200 μmol/L薄荷醇共同培养,分别于12、24、36、72 h后通过细胞计数试剂盒(CCK8)实验检测SH-SY5Y的细胞增殖活性.将20只6月龄Aβ前体蛋白(APP)/早老素蛋白1(PS1)转基因小鼠完全随机分为观察组和对照组,每组10只;观察组给予1 mg/g薄荷醇灌胃,对照组给予等体积0.9％氯化钠注射液灌胃,隔天1次,至10月龄时处死,免疫组织化学方法检测小鼠海马区老年斑形成情况.结果 通过计算机模拟实验,可发现薄荷醇与Aβ存在2个稳定的对接位置,该2处对接可阻碍nβ聚合.透射电镜下观察证实,薄荷醇与Aβ共同孵育可明显抑制Aβ聚合物的形成.CCK8细胞活性实验显示,薄荷醇可以拮抗Aβ对SH-SY5Y细胞的损伤,并且随着时间的延长,Aβ组与Aβ±薄荷醇组测得吸光度的差异越来越明显[12 h:(0.85±0.12)比(1.13±0.14),P＜0.05;24 h:(0.77±0.09)比(1.09±0.11),P＜0.05;36 h:(0.67±0.13)比(1.01±0.11),P＜0.05;72 h:(0.49±0.07)比(0.92±0.10),P＜0.01].免疫组织化学检测结果显示,观察组薄荷醇给药后APP/PS1小鼠海马区老年斑形成较对照组明显减少.结论 薄荷醇可以通过抑制Aβ聚合防止神经细胞损伤.