柴胡疏肝散对围绝经期综合征肝郁证大鼠海马cAMP/Ca^(2+)信号通路上相关基因影响的实验研究

Effects of herbal formula Chaihu Shugan San on the expression of genes in cAMP/Ca^(2+) signal pathway in hippocampus of perimenopausal rats with liver-qi stagnation syndrome

目的:通过比较柴胡疏肝散(CSS)干预前后的围绝经期综合征(PPS)肝郁证大鼠海马区cAMP/Ca2+信号通路上相关基因表达的关系,探求围绝经期综合征肝郁证的生物学基础。方法:以3月龄成年大鼠为正常对照组(A组),13月龄自然衰老大鼠为围绝经期模型,分为围绝经期对照组(B组)、围绝经期对照加中药组(C组),13月龄自然衰老和慢性束缚法建立PPS肝郁证大鼠模型,分为PPS肝郁证加中药组(D组)和PPS肝郁证组(E组)等5组,每组12只,中药干预3周后,予行为学观察,用RT2 Profiler PCR Arrays芯片法检测海马区cAMP/Ca2+信号通路上相关基因表达,ELISA法测CaMKⅡ含量,Fura-2/AM双波长荧光法检测钙离子浓度。结果:E组海马Ca2+浓度显著升高,中药干预后Ca2+浓度下降(P<0.01);E组海马CaMKⅡ含量显著升高,中药干预后CaMKⅡ浓度下降(P<0.01)。PCR芯片显示,E组与B组比较,海马细胞cAMP通路上有6个功能基因(IL-2,NOS2,Th,Calm1,S100g和SSt)有差异性表达(P<0.05,P<0.01),中药干预后,E组与D组比较,SSt有差异性表达(P<0.05)。结论:PPS大鼠肝郁证与海马区Ca2+/CaMKⅡ通路有密切关系,与海马cAMP/Ca2+通路调节神经传导的基因表达变化有关系,cAMP/Ca2+信号通路部分功能基因表达改变可能是PPS肝郁证的分子学机制。

Abstract： Objective： To observe the effects of Chaihu Shugan San （CSS） on perimenopausal syndrome （PPS） and liver- qi stagnation syndrome rat models, and explore the molecular mechanisms through cAMP/Ca2＋ signal pathway in hippocampal cells. Methods： In this experiments, twelve-week-old female rats are used as normal controls （Group A）, 52-week-old nature aging rat as PPS rat. A total of 48 nature aging rats are randomly divided into four groups： PPS controls （Group B, n=12）, PPS controls treated with CSS （Group C, n=12）, PPS＋ liver-qi stagnation model treated with CSS （Group D, n=12）, and PPS＋liver-qi stagnation model （Group E, n=12）. CSS was administered simultaneously when the liver-qi stagnation model being established. The sucrose consumption test and the open field test （OFT） were selected for the liver-qi stagnation behavior test. The CaMK 1I levels in serum and hippocampus were measured by ELISA, Ca2＋ was measured by FUm-2/AM in hippocampus and cAMP/Ca2＋ signal pathway by RTz Profiler PCR Arrays. Results： The E group hippocampal Ca2~ expression increased sharply than D group （P〈0.01）. The change of CaMKⅡ expression was as same as it. PCR Arrays indicated that there was 6 functional genes namely S100g, Th, NOS2, IL-2, Calml and SSt expression change between E group and B group （P〈0.05, P〈0.01）. After CSS administration, only the SSt functional gene changed （P〈0.05）. Conclusion： There is a close relation between Liver-qi stagnation syndrome and Ca2＋/CaMK Ⅱsignal pathway in PPS. There is a close relation between genes related to in cAMP/Ca2＋ signal pathway and liver-qi stagnation syndrome, maybe it is the molecular mechanism of liver-qi stagnation syndrome of PPS