CAJ | 학술논문

目的探讨舒马普坦对大鼠三叉神经节(TG)离体培养后降钙素基因相关肽(CGRP)表达水平的影响.方法采用TG离体培养模型,按数字随机表法将54个TG随机分为新鲜组(6个)、对照组(6个)和实验组(7个亚组,每亚组6个,共42个).实验组TG培养液中分别加入4种不同浓度舒马普坦,细胞外信号调节激酶1/2 (ERK1/2)信号通路阻滞剂U0126和PD98059,c-Jun氨基末端激酶(JNK)信号通路阻滞剂SP600125,孵育24h后免疫组织化学染色检测CGRP免疫反应(CGRP-ir)阳性细胞表达,实时定量PCR检测CGRP-mRNA表达量,Western blot定量磷酸化ERK1/2( pERK1/2)和JNK (pJNK)蛋白水平.结果离体培养24h后,TG内CGRP-ir(+)细胞表达明显增高,0.1和0.5 mg/ml舒马普坦组CGRP-ir(+)细胞百分比、阳性面积、累积吸光度、平均吸光度、CGRP-mRNA水平较对照组明显下降(tPCP=8.652、26.382,tares=6.220、13.917,tIA=5.606、15.904,tM14=2.661、21.748,tmRNA=8.032、15.675,均P＜0.05)；而0.02和2.50 mg/ml舒马普坦与对照组CGRP表达差异无统计学意义.Western blot结果显示:0.50 mg/ml浓度舒马普坦显著降低TG内pERKl/2、pJNK水平,降低程度分别接近于10μmol/L的U0126、PD98059和SP600125.结论一定浓度舒马普坦通过细胞内ERKl/2、JNK信号通路下调大鼠TG离体培养后CGRP的过度表达.
목적 탐토서마보탄대대서삼차신경절(TG)리체배양후강개소기인상관태(CGRP)표체수평적영향.방법 채용TG리체배양모형,안수자수궤표법장54개TG수궤분위신선조(6개)、대조조(6개)화실험조(7개아조,매아조6개,공42개).실험조TG배양액중분별가입4충불동농도서마보탄,세포외신호조절격매1/2 (ERK1/2)신호통로조체제U0126화PD98059,c-Jun안기말단격매(JNK)신호통로조체제SP600125,부육24h후면역조직화학염색검측CGRP면역반응(CGRP-ir)양성세포표체,실시정량PCR검측CGRP-mRNA표체량,Western blot정량린산화ERK1/2( pERK1/2)화JNK (pJNK)단백수평.결과 리체배양24h후,TG내CGRP-ir(+)세포표체명현증고,0.1화0.5 mg/ml서마보탄조CGRP-ir(+)세포백분비、양성면적、루적흡광도、평균흡광도、CGRP-mRNA수평교대조조명현하강(tPCP=8.652、26.382,tares=6.220、13.917,tIA=5.606、15.904,tM14=2.661、21.748,tmRNA=8.032、15.675,균P＜0.05)；이0.02화2.50 mg/ml서마보탄여대조조CGRP표체차이무통계학의의.Western blot결과현시:0.50 mg/ml농도서마보탄현저강저TG내pERKl/2、pJNK수평,강저정도분별접근우10μmol/L적U0126、PD98059화SP600125.결론 일정농도서마보탄통과세포내ERKl/2、JNK신호통로하조대서TG리체배양후CGRP적과도표체.
Objective To explore the effects of sumatriptan on the modulation of calcitonin generelated peptide(CGRP) expression and its involving intracellular signaling transduction mechanisms in rat trigeminal ganglion(TG) after organ culture.Methods Using organ culture in vitro model,54 isolated TGs of SD rats were randomly divided into fresh group ( n =6 ),control group ( n =6 ) and experimental group (n =42,6 TGs for each subgroup).Experimental group included seven subgroups,which were respectively pretreated with four different concentrations of sumatriptan,specific inhibitors of extracellular signalregulated kinases 1/2 (ERK1/2) pathway (U0126 and PD98059 ),and the inhibitor of c-Jun N-terminal kinase (JNK) (SP600125).After co-cultured with above intervention agents for 24 h,CGRP-immunoreactivity (CGRP-ir) positive neurons and CGRP-mRNA expression levels were quantified by immunohistoehemistry stain and real-time polymerase chain reaction,respectively.Phosphorylated ERK1/2 (pERK1/2) and JNK (pJNK) proteins levels were determined by Western-blotting method.Results The CGRP-ir ( + ) neurons expression levels were significantly increased after 24 h organ culture.However,0.10 and 0.50 mg/ml concentrations of sumatriptan remarkably decreased the CGRP-ir ( + ) neurons expression levels.The positive cell percentage,positive optic area,integrated optical density,mean optical density and CGRP-mRNA expression level in TG were significantly reduced than control groups (tPCP =8.652,26.382; tarea =6.220,13.917; tIA =5.606,15.904; tM14 =2.661,21.748; tmRNA =8.032,15.675.P ＜ 0.05 ).The CGRP-mRNA expressions were significantly down-regulated after co-incubation with concentration of 0.50 mg/ml sumatriptan for 24 h in TG of SD rat ( P ＜0.05 ).The levels of pERK1/2 and pJNK protein kinase detected by Western-blotting were significantly reduced by 0.50 mg/ml concentration of sumatriptan,the degrees of which were closed to the ERK1/2 and JNK pathway specific blockers.Conclusion It suggests that the optimal concentration of sumatriptan significantly down-regulates CGRP over-expression via intracellular ERK1/2 and JNK signaling transduction pathways in TG after organ culture.