慢性吗啡处理对C6细胞EAAT3蛋白表达的影响及其机制研究

目的探讨慢性吗啡暴露、戒断、再次暴露对C6细胞兴奋性氨基酸转运蛋白3(excitatory amino-acid transporter 3,EAAT3)蛋白表达的影响及可能机制。方法 0.1～10μmol·L-1吗啡作用于C6细胞不同时间,Western blot检测C6细胞EAAT3蛋白表达水平变化,然后用不含吗啡的培养液培养细胞模拟吗啡自然戒断过程,待EAAT3蛋白表达回升后,再次吗啡暴露(吗啡浓度为第1次给药的1/2)模拟吗啡复吸过程,观察吗啡多次暴露对C6细胞EAAT3蛋白表达的影响。最后在吗啡再次暴露前15 min使用纳洛酮,观察纳洛酮对多次吗啡暴露引起的C6细胞EAAT3表达变化的影响。结果 10μmol·L-1吗啡作用于C6细胞至少48 h可下调C6细胞EAAT3蛋白表达(P<0.05)。停用吗啡至少12 h后,EAAT3蛋白表达回升,5μmol·L-1吗啡再次处理C6细胞4 h即可下调C6细胞EAAT3蛋白表达水平(P<0.05)。1μmol·L-1纳洛酮可明显抑制慢性吗啡处理引起的EAAT3蛋白表达下降(P<0.05)。结论慢性吗啡处理可下调C6细胞EAAT3表达水平,停用吗啡EAAT3表达回升,吗啡再次暴露引起EAAT3表达水平下降所需吗啡浓度降低,暴露时间缩短。吗啡通过作用于阿片受体诱导C6细胞EAAT3表达下降。

Effects of chronic morphine treatment on EAAT3 expression in C6 cells and its mechanisms

AimTo explore the effects of chronic morphine exposure,abstinence and reexposure on excitatory amino-acid transporter 3(EAAT3) expression in C6 cells and the mechanisms.Methods Well-grown C6 cells were exposed to chronic morphine treatment.The time-and concentration-dependent manner of EAAT3 protein expression which was induced by morphine in C6 cells was determined by Western blot.After chronic treatment of morphine at the optimal exposure concentration and duration,C6 cells were cultured in medium without morphine,a simulation of the abstinence process,and EAAT3 expressions were recovered.Then C6 cells were retreated with morphine(at half of the primary dosage) for 2,4 or 8 h,and EAAT3 expression level was detected by Western blot.Naloxone was used 15 min before morphine reexposure,and protein within cells was extracted and EAAT3 expression of each group was assayed by Western blot later.Results 10 μmol·L-1 morphine for no less than 48h reduced EAAT3 expression of C6 cells.After at least 12hs of abstinence,the expression level recovered.Reexposure to 5 μmol·L-1 morphine for only 4h lowered the expression again.Naloxone,a non-selective opioid receptor blocker,weakened the declined expression of EAAT3 triggered by morphine reexposure.Conclusions Chronic morphine treatment reduces EAAT3 expression in C6 cells.The expression may resume after morphine treatment stops.Reexposure to morphine for much shorter duration and lower concentration may cause another decline of EAAT3 level.Opioid receptors may play a role in this decrease.