抑制JNK磷酸化参与褪黑素对花萼海绵诱癌素引起的tau蛋白过度磷酸化的保护机制

Inhibiton of JNK phosphorylation involved in melatonin's protection of calyculin A-induced tau hyperphosphorylation

目的探讨花萼海绵诱癌素(CA)在成神经瘤细胞(N2a)引起tau蛋白过度磷酸化的机制,及褪黑素对其是否具有保护作用。方法小鼠成神经瘤细胞(N2a)给予CA 5 nmol·L-1处理,或同时给予褪黑素50 mol·L-1,或同时给予维生素E(Vit E)50 mol·L-1处理12 h,用免疫印迹技术检测tau蛋白在Ser422位点的磷酸化水平,磷酸化c-jun氨基端激酶(p-JNK)和磷酸化丝裂原活化蛋白激酶激酶(p-MKK4)的含量,免疫荧光检测p-JNK含量和分布,荧光法测定细胞内丙二醛(MDA)含量,32P-特异底物标记技术检测p38丝裂原活化蛋白激酶(p38MAPK)活性。结果 CA在N2a细胞引起tau蛋白Ser422位点磷酸化水平显著升高(1.70±0.19,1.0,P<0.01),褪黑素拮抗CA引起的tau蛋白Ser422位点过度磷酸化(0.98±0.12,1.70±0.19,P<0.01);CA引起细胞内MDA含量升高(μmol·g-1蛋白,0.241±0.006,0.141±0.006,P<0.01),褪黑素和Vit E均可抑制CA引起的细胞内MDA含量升高(μmol·g-1蛋白,0.172±0.004,0.193±0.005,0.241±0.006,P<0.01);CA不改变p38MAPK活性和蛋白水平,但引起p-JNK含量升高(1.91±0.27,1,P<0.01)和p-MKK4(1.81±0.09,P<0.01)含量升高,褪黑素抑制CA引起的p-JNK含量升高(1.11±0.15,1.91±0.27,P<0.01)和p-MKK4(1.14±0.06,1.81±0.09,P<0.01)含量升高;JNK抑制剂SP600125可抑制CA诱导的tau蛋白过度磷酸化,MKK4磷酸化抑制剂地昔帕明可消除CA诱导的JNK磷酸化和tau蛋白过度磷酸化。结论褪黑素抑制JNK磷酸化参与其对CA诱导的tau蛋白Ser422位点过度磷酸化的预防作用。

OBJECTlVE To explore the mechanisms of tau hyperphosphorylation induced by calyculin A （ CA） in neuroblastoma cells and the effect of melatonin. METHODS N2a cells were treated with CA 5 nmol·L^-1 , or CA with melatonin 50 μmol·L^-1 , or CA with vitamin E （ Vit E ） 50 μmol·L^-1 for 12 h. The level of tau phosphorylation at Ser422 （ recognized by R145d antibody） site and the level of phosphorylated c-Jun N-terminal kinases （ p-JNK ） and phosphorylated mitogen-activated protein kinase kinase 4 （ p-MKK4 ） were detected with immunoblotting, the level of malondialdehyde （ MDA ） was assayed with fluorimetry, and the activity of p38-mitogen activated protein kinase （ p38MAPK ） was assayed by radioimmunobloting. RESULTS CA treatment increased the level of phosphorylated tau at Ser422 site （1.70±0.19, 1.0, P〈0.01）, and melatonin attenuated the effect of CA （0.98±0.12, 1.70± 0.19, P〈0.01）. ln addition, CA treatment increased the level of MDA （μmol·g-1 protein：0.241±0.006, 0.141±0.006, P〈0.01） and melatonin antagonized the increase of MDA induced by CA （μmol·g^-1 protein：0.172±0.004, 0.193±0.005, 0.241±0.006, P〈0.01） . CA treatment increased the level of p-JNK （1.91±0.27, 1, P〈0.01） and p-MKK4 （1.81±0.09, 1, P〈0.01） and melatonin antagonized the effect of CA induced increase of p-JNK （1.11±0.15, 1.91±0.27, P〈0.01） and p-MKK4 （1.14±0.06, 1.81±0.09, P〈0.01） without changing the level or activity of p38MAPK. Both JNK inhibitor （ SP600125 ） and MKK4/JNK transduction pathway inhibitor antagonized CA induced tau phosphorylation at Ser422 site and JNK phosphorylation. CONCLUSlON lnhibiton of JNK phosphorylation is possibly involved in the protection of melatonin on CA-induced tau hyperphosphorylation at Ser422 site.