四逆散对心理应激肝郁证模型大鼠ERK1/2-CREB信号通路的影响

Effects of Sini powder on the ERK1/2-CREB signal pathway with rat liver stagnation syndrome model

关键词:  四逆散  心理应激  肝郁证模型  ERK1/2-CREB信号通路

Key words:  Sini powder  psychological stress  liver stagnation syndrome  ERK1/2-CREB signal pathway

摘要:

[目的]探讨四逆散对心理应激肝郁证模型大鼠应激相关激素、神经递质及海马和下丘脑部位细胞外信号调节蛋白激酶1/2（ERK）1/2-cAMP反应元件结合蛋白（CREB）信号通路的影响。[方法]采用慢性束缚制动结合孤养的方法制备心理应激肝郁证大鼠模型，根据大鼠一般状态、体质量增长情况、高架十字迷宫实验验证模型成功。将120只大鼠随机分为正常对照组、模型空白组、模型+四逆散组（四逆散组）、模型+艾司唑仑组（艾司唑仑组），各30只。采用酶联免疫吸附（ELISA）法检测大鼠血浆及脑脊液促肾上腺皮质激素释放激素（CRH）、促肾上腺皮质激素（ACTH）、肾上腺素（E）、去甲肾上腺素（NE）、5-羟色胺（5-HT）的含量，半定量PCR法检测大鼠海马和下丘脑部位丝裂原活化蛋白激酶激酶1/2（MEK1/2）、ERK1/2、CREB、c-fos mRNA的表达，蛋白印迹（Western blot）法检测大鼠海马和下丘脑部位磷酸化（p-）MEK1/2、p-ERK1/2、p-CREB、c-fos蛋白的表达。[结果]与正常对照组比较，模型空白组大鼠血浆和脑脊液中CRH、ACTH、E、NE的含量明显增加（P<0.05），5-HT的含量明显减少（P<0.05），海马和下丘脑部位MEK1/2、ERK1/2、CREB mRNA及p-MEK1/2、p-ERK1/2、p-CREB蛋白的表达明显降低（P<0.05）。与模型空白组比较，四逆散和艾司唑仑均可降低血浆和脑脊液中CRH、ACTH、E、NE的水平（P<0.05），升高5-HT的水平（P<0.05），上调海马和下丘脑部位MEK1/2、ERK1/2、CREB mRNA及p-MEK1/2、p-ERK1/2、p-CREB蛋白的表达（P<0.05）。各组之间大鼠海马和下丘脑部位c-fos mRNA和蛋白质的表达差异无统计学意义（P>0.05）。[结论]四逆散能降低心理应激肝郁证模型大鼠血浆和脑脊液CRH、ACTH、E、NE的水平，增加5-HT的含量，提高应激反应能力，其机制可能与上调ERK1/2-CREB信号通路活动有关。

Abstract:

[Objective] To investigate the effects of Sini powder on stress-related hormones, neurotransmitters and ERK1/2-CREB signal pathway in hippocampus and hypothalamus of rats with liver stagnation syndrome.[Methods] One hundred and twenty Rats were randomly divided into normal control group, model group, Sini powder group and estazolam group, 30 rats in each group. The liver stagnation syndrome model was induced by chronic restraint and single cage feeding. The levels of CRH, ACTH, E, NE and 5-HT both in plasma and cerebrospinal fluid were tested by ELISA, and the expression of MEK1/2, ERK1/2, CREB and c-fos mRNA in the hippocampus and hypothalamus of rats were detected by semi-quantitative PCR. The expression of p-MEK1/2, p-ERK1/2, p-CREB and c-fos protein in the hippocampus and hypothalamus of rats were detected by Western blot.[Results] Compared with the normal control group, the levels of CRH, ACTH, E and NE in the plasma and cerebrospinal fluid of the model group were significantly increased (P<0.05), and the level of 5-HT was significantly decreased (P<0.05). The expression of MEK1/2, ERK1/2, CREB, c-fos mRNA and p-MEK1/2, p-ERK1/2, p-CREB, c-fos protein were significantly decreased (P<0.05). However, Sini powder and estazolam could significantly decrease the levels of CRH, ACTH, E and NE, and significantly increase the level of 5-HT both in plasma and cerebrospinal fluid(P<0.05), could significantly increase the expression of MEK1/2,ERK1/2, CREB mRNA and p-MEK1/2, p-ERK1/2, p-CREB protein both in the hippocampus and hypothalamus (P<0.05). There was no significant difference in the expression of c-fos protein both in hippocampus and hypothalamus in each group (P>0.05).[Conclusion] Sini powder can decrease the levels of CRH, ACTH, E and NE both in the plasma andcerebrospinal fluid of rats with liver stagnation syndrome, and increase the level of 5-HT and improve the stress response. The mechanism may be related to the up-regulation of ERK1/2-CREB signal pathway.