阿魏酸钠对淀粉样β蛋白片段1～40引起的大鼠海马MAP激酶信号传导通路及凋亡蛋白表达的影响

Effects of sodium ferulate on amyloid-β protein fragment 1-40-induced changes of MAP kinase signal transduction and proapoptotic protein expression in rat hippocampus

目的观察阿魏酸钠(SF)是否对淀粉样β蛋白(Aβ)所致的海马损伤具有保护作用及其信号转导机制。方法大鼠灌胃给予SF(50,100和250mg.?-1),连续应用3周,左侧脑室内单次注射Aβ1-40(10μL,1.0mmol.L-1)制备大鼠痴呆动物模型,注射后6h,取海马CA1区。Western印迹观察有丝分裂原活化蛋白p38(p38MAP)激酶、丝裂原激活的蛋白激酶的激酶4(MKK4)、c-Jun氨基末端激酶(JNK1/2)、P53蛋白、FasL和半胱氨酸天冬氨酸蛋白酶-3(caspase3)蛋白表达。应用caspase3活性测定试剂盒分析caspase3活性变化。结果脑室内注射Aβ1-40可引起大鼠海马CA1区磷酸化p38MAP激酶表达明显增加,从正常0.127±0.018增加到0.95±0.12(n=4,P<0.01)。也可使磷酸化MKK4和JNK1/2表达明显增加。这些MAPK信号传导通路改变伴随有促凋亡蛋白P53和FasL表达明显增加,caspase3蛋白表达和活性均明显增加,P53从正常0.053±0.012增加到0.30±0.07(n=4,P<0.01),FasL从正常0.25±0.04增加到0.51±0.05(n=4,P<0.01),caspase3从正常0.036±0.007增加到0.63±0.041(n=4,P<0.01),caspase3活性从正常0.38±0.04增加到0.86±0.09(n=4,P<0.01)。SF(50、100和250mg.kg-1)连续应用3周,能明显抑制Aβ1-40引起的p38MAP激酶、磷酸化MKK4和JNK1/2表达明显增加,同时促凋亡蛋白p53和FasL表达明显增加,降低caspase3的活性。结论SF通过抑制Aβ1-40引起的p38MAP激酶和JNK1/2信号传导通路,产生抗凋亡作用,保护海马免除Aβ1-40引起的神经毒性。

AIM To investigate whether sodium ferulate（SF） has protective effects on amyloid-β（Aβ） 1-40-induced neurotoxicity and the mechanisms of MAP kinase signal transduction. METHODS The rats were randomly divided into five groups ： control group, Aβ1-40 group, Aβ1-40 ＋ SF groups （50, 100 and 250 mg·kg^-1 ）. Rats were given SF （ 50, 100 and 250 mg·kg^-1 daily, ig） for 3 weeks prior to icy single dose of Aβ1-40 （ 10μL, 1.0 mmol ·L^-1 ）. Six hours after injection, Western blotting was used to determine the expressions of phosphop38 MAP kinase （p38 MAPK）, phospho-MAP kinase kinase （MKK） 4, c-Jun N-terminal kinase 1/2（ JNK1/2）, P53 protein, Fas ligand （Fas L）, and caspase-3 in hippocampal CA1 regions. The caspaseo3 activation was measured using a caspase-3 colorimetric activity assay kit. RESULTS Intracerebroventricular injection of Aβ1-40 induced an increase in phosphorylated p38 MAP kinase expressions in hippocampal CAl. The density ratio of phosphorylated p38 MAP kinase to β-actin increased from 0.127 ±0.018 to 0.95±0.12 （n=4, P〈 0.01）. In addition, Aβ1-40 induced increase in phosphorylated MKK4 and JNK1/2 expression. These changes were accompanied by increased expressions of the proapoptotic protein P53, Fas L, and caspase-3 in rat hippocampus. The density ratio of P53 to β-actin increased from 0. 053 ± 0. 012 to 0.30 ± 0.07 （n =4, P 〈 0.01 ）, The density ratio of Fas L to β-actin increased from 0. 25 ± 0. 04 to 0.51 ±0.05 （n =4, P 〈0.01）,The density ratio of caspase 3 to β-actin increased from 0.036±0.007 to 0.63 ±0.041 （n=4, P〈 0.01）, The activity of caspase 3 increased from 0. 38 ±0. 04 to 0. 86 ±0. 09 （n=4, P〈 0.01）. SF （50, 100 and 250 mg·kg^-1 daily, 3 weeks ） significantly prevented Aβ1-40-indueed inereases in phosphorylated p38 MAP kinase, MKK4, and JNK1/2 expression. In parallel with these findings, Aβ1-40-induced increase of the proapoptotie protein P53, Fas L, and easpase-3 were inhibited by SF. CONCLUSION SF prevents Aβ1-40 induced neurotoxicity through suppression of p38 MAP kinase and MKK4/JNK1/2 signal transduction pathway.