CAJ | 학술논문

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,32 P-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。
목적：탐토화악해면유암소( CA)재성신경류세포( N2a)인기tau단백과도린산화적궤제,급퇴흑소대기시부구유보호작용。방법소서성신경류세포( N2a)급여CA 5 nmol·L－1처리,혹동시급여퇴흑소50 mol·L－1,혹동시급여유생소E( Vit E)50 mol·L－1처리12 h,용면역인적기술검측tau단백재Ser422위점적린산화수평,린산화c-jun안기단격매( p-JNK)화린산화사렬원활화단백격매격매( p-MKK4)적함량,면역형광검측p-JNK함량화분포,형광법측정세포내병이철( MDA)함량,32 P-특이저물표기기술검측p38사렬원활화단백격매( p38MAPK)활성。결과 CA재N2a세포인기tau단백Ser422위점린산화수평현저승고(1.70±0.19,1.0, P<0.01),퇴흑소길항CA인기적 tau단백Ser422위점과도린산화(0.98±0.12,1.70±0.19, P<0.01)；CA인기세포내MDA함량승고(μmol·g－1단백,0.241±0.006,0.141±0.006, P<0.01),퇴흑소화Vit E 균가억제 CA 인기적세포내 MDA 함량승고(μmol·g－1단백,0.172±0.004,0.193±0.005,0.241±0.006, P<0.01)；CA 불개변 p38MAPK 활성화단백수평,단인기 p-JNK 함량승고(1.91±0.27,1, P<0.01)화p-MKK4(1.81±0.09, P<0.01)함량승고,퇴흑소억제CA인기적p-JNK함량승고(1.11±0.15,1.91±0.27,P<0.01)화p-MKK4(1.14±0.06,1.81±0.09, P<0.01)함량승고；JNK억제제SP600125가억제CA유도적tau단백과도린산화,MKK4린산화억제제지석파명가소제CA유도적JNK린산화화tau단백과도린산화。결론퇴흑소억제JNK린산화삼여기대CA유도적tau단백Ser422위점과도린산화적예방작용。
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.