2-氨基甾体化合物对阿尔茨海默病模型大鼠的治疗作用及机制研究

Effects of 2-amino steroid on Alzheimer's disease in rats and its underlying mechanisms

目的淀粉样β蛋白(β-Amyloid,Aβ)致神经毒性和细胞毒性的机制与细胞内钙离子失衡有关。文中旨在观察2-As对海马区注射Aβ的阿尔茨海默病模型大鼠的作用,并探讨其可能的机制。方法体外实验:实验分为Aβ_(1-40)组、Aβ_(1-40)+Cu^(2+)组、Aβ_(1-40)+2-As组、Aβ_(1-40)+2-As+Cu^(2+)组,其中Aβ_(1-40)、Cu^(2+)、2-As的终浓度分别为25、25、50μmol/L,实验反应体系终体积为200μL。各样品置于37℃恒温箱孵育48 h,取5μL待测样本在透射电镜下观察。体内实验:将雄性SD大鼠按随机数字表法分为AβO组、AβO+2-As组、对照组,每组3只。实验组用以15μm/s的速度将10 mmol/L的Aβ_(1-42)溶液5μL缓慢注入,对照组注入等体积无菌等渗盐水,注射完毕留针2 min,缓慢撤针,用牙科泥封堵颅骨,缝合切口皮肤。造模后每天AβO+2-As组大鼠腹腔注射10%(2.5%无水乙醇做溶媒)的2-As(1 m L/100 g),AβO组和对照组大鼠每天予2.5%无水乙醇腹腔注射(1m L/100 g),连续给药7 d;造模后第8天取样。全波长扫描式多功能读数仪检测大鼠血清内丙二醛的含量变化,ELISA法检测脑组织IL-6、TNF-α的表达,Fura-2荧光探针检测脑组织Ca^(2+)含量的表达,RT-PCR检测脑组织L型钙通道mRNA的表达。结果体外实验:电镜下Aβ_(1-40)组发生凝集形成结晶;与Aβ_(1-40)组相比,Aβ_(1-40)+2-As组形成的结晶明显减少,Aβ_(1-40)+Cu^(2+)组形成的结晶增多;单体Aβ_(1-40)+2-As+Cu^(2+)组结晶无明显变化。体内实验:与AβO组大鼠血清丙二醛[(2.923±0.125)μmol/L]、脑组织IL-6[(2.210±0.157)ng/L]、脑组织TNF-α[(1.323±0.106)ng/L]、海马Ca^(2+)[(1.610±0.121)nmol/L]、海马L-Ca通道mRNA[(3.407±0.100)ng/L]比较,对照组、AβO+2-As组丙二醛[(1.077±0.040),(1.067±0.169)μmol/L],IL-6[(1.353±0.049),(1.343±0.264)ng/L],TNF-α[(0.607±0.031),(0.640±0.118)ng/L],Ca^(2+)[(0.303±0.031),(0.703±0.142)nmol/L],L-Ca通道mRNA[(1.713±0.116),(1.943±0.076)ng/L]均显著降低(P<0.05)。结论 2-As对Aβ诱导的AD模型大鼠血清内丙二醛增高及脑组织细胞内IL-6、TNF-α、Ca^(2+)、L-Ca通道mRNA升高均有抑制作用;2-As对AD的作用机制可能与抗Aβ蛋白聚集作用、减轻炎症反应、提高抗氧化能力以及降低海马细胞质内钙离子流等有关。

Objective The neurotoxicity and cytotoxicity induced by p-amyloid （ Ap） are related to the disequilibrium of calcium ion （ Ca2＋ ） . This study was to observe the effect of 2-amino steroid （ 2-As） on Ap-induced Alzheimer^s disease （ AD） in model rats and investigate its possible mechanisms. Methods Nine adult male SD rats were randomly divided into an ApO, an ApO ＋ 2-As, and a control group of equal number. The animals in the experimental group were injected with 10 mmol/L Ap2 40 solution into the den-tate gyrus at the rate of jjini/s, while the controls with the same volume of isotonic saline. Then the rats in the ApO ＋ 2-As group re-ceived daily intraperitoneal injection of 10% 2-As while those in the ApO and control groups received that of 2.5% absolute alcohol at lmL per 100 g of the body weight, all for 7 days. At 8 days after modeling, the concentration of serum malondialdehyde （ MDA） in the rat hippocampus was detected by full-wavelength scanning multifunctional reading, the expressions of interleukin- 6 （IL-6） and tumor necrosis factor a （TNF-a） determined in the brain tissue by ELISA, the concentration of Ca2 ＋ in the hippocampus measured with the Fura-2 fluorescent probe, and the mRNA expression of the L-type calcium channel obtained by RT-PCR. Results Transmission e-lectron microscopy showed aggregation and formation of crystals in the Ap2 40 group, significantly fewer in the Ap2 40＋2-As group, more in the Ap140＋ Ca2＋ group, and with no significant changes in the Ap140＋ 2-As ＋ Ca2＋ group. Compared with the ApO group, the ani-mals in the control and ApO ＋ 2-As groups showed remarkable decreases in the concentration of serum MDA （[ 2.923 ±0.125 ] vs [1.077±0.040] and [ 1.067±0.169] （jimol/L, P〈0.05） , the expressions of IL-6 （[2.210±0.157] [ 1.353± 0.049] and [ 1.343± 0.264] ng/L, P〈0.05） and TNF-a （ [ 1.323±0.106] vs [ 0.607±0.031 ] and [ 0.640±0.118] ng/L, P〈0.05） in the brain tissue, thelevel of Ca2＋ in the hippocampus （ [ 1.610±0.121] vs [ 0.303±0.031 ] and [0.703±0.142] nmol/L, P〈0.05） , and the mRNA expres-sion of the L-calcium channel （[3.407±0.100] vs [ 1.713±0.116] and [ 1.943±0.076] ng/L, P 〈 0 .0 5 ） . Conclusion 2-As can significantly inhibit the increases of the serum MDA concentration, IL-6 and TNF-a expressions in the brain tissue, Ca2＋ level in the hippocampus, and mRNA expression of the L-calcium channel in Ap-induced AD rats, which may be associated with its effects of anti- Ap protein aggregation, relieving the inflammation response, increasing antioxidant capacity, and decreasing calcium influx in the cyto-plasm of the hippocampus.