目的:探讨磷酸二酯酶4(PDE4)特异性抑制剂咯利普兰(rolipram)对抗慢性应激诱导的抑郁和焦虑样行为的分子机制.方法:(1)将40只SD大鼠分为4组(每组10只):空白对照组、模型组、阳性对照药氯化锂(LiCl)组及rolipram组,并进行应激前旷场实验.给予慢性束缚应激21 d 后,分别进行强迫游泳、高架十字迷宫及应激后旷场实验.最后一次行为学实验结束后立即处死动物并取双侧海马组织,免疫印迹法检测海马内磷酸化cAMP反应元件结合蛋白(p-CREB)、脑源性神经营养因子(BDNF)、磷酸化丝氨酸残基21糖原合成酶激酶3α(p-Ser21-GSK3α)、磷酸化丝氨酸残基9糖原合成酶激酶3β(p-Ser9-GSK3β)、磷酸化酪氨酸残基279糖原合成酶激酶3α(p-Tyr279-GSK3α)、磷酸化酪氨酸残基216糖原合成酶激酶3β(p-Tyr216-GSK3β)、总糖原合成酶激酶3α(total GSK3α)及总糖原合成酶激酶3β(total GSK3β)的表达.(2)30只SD大鼠平均分为6组,双侧海马CA1区手术埋管并恢复7 d 后进行慢性应激处理21 d,并在每次应激前微量注射蛋白激酶A(PKA)的拮抗剂H89,并腹腔注射LiCl和rolipram.双侧海马用于检测PDE4D、p-CREB和p-Ser9-GSK3β的表达.结果:(1)应激前各组动物自主活动能力无显著差异,应激14 d及21 d后应激组与空白对照组相比体重显著下降,LiCl及rolipram均显著逆转了应激对体重的下调作用.应激显著增加了强迫游泳实验中动物的不动时间,减少了攀爬时间和高架十字迷宫实验中进入开臂的次数及时间,表现出抑郁和焦虑样行为,而LiCl及rolipram均显著逆转了慢性束缚应激诱导的上述行为障碍.免疫印迹结果显示rolipram可显著逆转应激对p-CREB、BDNF、p-Ser21-GSK3α和p-Ser9-GSK3β表达的下调作用,而LiCl仅显著上调p-Ser21-GSK3α及p-Ser9-GSK3β的表达.各组p-Tyr279-GSK3α、p-Tyr216-GSK3β、total GSK3α及total GSK3β的表达无显著差异.(2)慢性应激诱导了大鼠海马内PDE4D表达的上调,PKA、p-CREB及p-Ser9-GSK3β的下调.Rolipram显著逆转了上述效应,且H89显著阻断了Rolipram的药物效应.结论:Rolipram抗抑郁及抗焦虑作用不仅通过CREB/BDNF信号通路,而且还有GSK3 抑制性丝氨酸残基磷酸化信号通路的参与,且CREB介导的信号转导通路对GSK3 抑制性丝氨酸残基磷酸化信号通路发挥重要调节作用.%AIM: To discuss the impact of phosphodiesterase 4 (PDE4) inhibitor rolipram on chronic restraint stress - induced depression - and anxiety - like behaviors in rats. METHODS: (1) Forty SD rats were randomly divided into 4 weight -matched groups: unstressed animals injected with vehicle of lithium chloride (LiCl) and rolipram, restraint - stressed animals injected daily with vehicle prior to stress, restraint stress plus 100 mg/kg LiCl group and restraint stress plus 1 mg/kg rolipram group. The open field test was conducted 24 h before the first stress and drug administration, andrnthen the rats received drugs daily 1 h prior to restraint stress (6 h/d) for 25 d. Daily body weight recording, forced swim-ming test, elevated plus - maze and open field test were conducted to determine the changes of depression - and anxiety -like behaviors. The expression of phosphorylated cAMP response element - binding protein (p - CREB) , brain - derived Reurotrophic factor ( BDNF) , p - Ser21 - glycogen synthase kinase ( GSK) 3α, p - Ser9 - GSK3β, p - Tyr279 - GSK3α, p -Tyr216 - GSK3β, total GSK3α and total GSK3β was measured by Western blotting. (2) Thirty SD rats were randomly divided into 6 groups and the cannula was surgically placed above the CA1 region in the hippocampus. Seven days after the surgery, the restraint stress was conducted for 21 d after microinjection of protein kinase A (PKA) antagonist H89 and in-traperitoneal injection of LiCl and rolipram everyday. The expression of PDE4D, PKA, p - CREB and p - Ser9 - GSK3β was measured by Western blotting. RESULTS: ( 1) No difference of the locomotor activity among all groups before stress was observed. After repeated stress, the body weight,and the crossing, rearing and grooming in open field test were lower than those in control group, and LiCl and rolipram reversed these effects significantly. In addition, in comparison with con-trol group, the immobility in forced swimming test was increased, the climbing in forced swimmming test and the open -arm exploration in elevated plus - maze were decreased and the expression of p - CREB, BDNF, p - Ser21 - GSK3α and p - Ser9 - GSK3β was down - regulated. Stress induced depression - and anxiety - like behaviors, and rolipram reversed these changes. The LiCl showed similar effects as rolipram except for the expression of p - CREB and BDNF. No significant difference of the expression of p - Tyr279 - GSK3α, p -Tyr216 - GSK3β, total GSK3α and total GSK3β among all groups was found. (2)The expression of PDE4D was increased, the expression of PKA, p - CREB and p - Ser9 - GSK3β was de-creased in the hippocampus induce by restraint stress. However, the effect of rolipram on the expression of PKA, p - CREB and p - Ser9 - GSK3β was blocked by PKA inhibitor H89. CONCLUSION: Rolipram significantly reduces the depression -and anxiety - like behaviors, possibly through CREB/BDNF signaling and inhibitory serine - phosphorylation of GSK3 -mediated signaling. Importantly, the CREB/BDNF signaling also plays a key role in the down - regulation of serine - phos-phorylation of GSK3.
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