围绝经期综合征肝郁病理特征及生物学机制研究
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摘要
本研究从临床和动物研究两个方面,通过研究围绝经期综合征(Perimenopausal Syndrome, PPS)的中医病理特征以及肝郁病理与雌激素(E2)、雌激素受体(Estrogen Receptor, ER)、单胺类神经递质表达关系；肝郁病理与Ca2+/cAMP信号通路功能基因表达关系,探讨PPS肝郁病理的生物学基础和分子学机制。
目的
(1)通过研究PPS症状、证素病理及兼杂特征,柴胡疏肝散治疗前后的PSS肝郁证患者肝郁积分、Kupperman法症状评分变化,肝郁病理分级与雌激素、神经递质关系,探讨PPS肝郁病理的部分生物学基础。
(2)通过柴胡疏肝散干预PPS肝郁证大鼠,从E2、ER、神经递质变化及大鼠海马雌激素受体介导的Ca2+/cAMP信号通路探讨其分子机制。
方法
(1)临床研究方面收集PPS患者161例,以证素辨证的方法,分析PPS的症状分布频次、病位病性证素及其兼杂的特点。选取PPS患者中肝郁积分≥70分的患者100例,根据肝郁病理积分分为3级,予以4周的柴胡疏肝散干预,观察干预前后的肝郁病理积分、Kupperman法症状评分变化,肝郁病理分级与E2、神经递质5-羟色胺(5-HT),去甲肾上腺素(NE),多巴胺(DA)关系,探讨PPS肝郁证的生物学基础。
(2)动物研究方面以3月龄成年大鼠为正常对照组(A组),13月龄自然衰老和慢性束缚法建立PPS肝郁证大鼠模型,分为围绝经期对照组(B组)、围绝经期对照组加中药组(C组)、PPS肝郁证加中药组(D组)和PPS肝郁证组(E组)等五组,药物干预3周后,予行为学观察,以实时定量PCR法检测海马区雌激素受体ERa, ERβ, GPR30(G protein-coupled receptor30) mRNA表达,用Fura-2/AM双波长荧光法测定海马细胞内游离钙浓度,用RT2Profiler PCR Arrays芯片检测海马Ca2+/cAMP通路上相关基因差异性表达,用ELISA法检测海马及血清雌激素及递质5-HT, NE,DA,血清钙调蛋白依赖性蛋白激酶Ⅱ (CaMKⅡ)表达。
结果
(1)临床研究部分
1.PPS的症状分布频次前10位是阵发性烘热,汗出,急躁易怒,情绪抑郁或焦虑,月经紊乱,胸闷、失眠,喜太息,心悸,咽部异物感,各占91.93%,91.93%,85.09%,81.99%,78.26%,69.56%,67.70%,55.28%,52.17%,39.75%。
2.PPS的病位证素涉及肝、肾、脾、心,主要是肝和肾,分别占91.93%和52.17%,与其它病位相比有显著性差异,P<0.01,肝的病位积分最高。
3.PPS的病性证素主要涉及气滞、阴虚、气虚、阳虚、血虚,其中气滞所占的比例最高为90.1%,与其它病性所占比例相比有显著性差异,p<0.01,气滞的病性积分最高。
4.PPS证素病位兼杂中,以肝肾最多见,其次为脾肾、肝脾,病性兼杂以气滞兼阴虚最为多见。
5.临床干预前PPS患者肝郁病理0级0人,1级50人,2级34人,3级16人,干预后0级70人,1级22人,2级3人,3级5人,干预前后肝郁病理分级人数有显著性改变P<0.01。
6.干预前后,Kupperman法症状评分有显著性改变,P<0.01。
7.干预前后,肝郁病理分级与E2变化有显著相关关系,R=-0.497,P==0.001。
8.干预前后,5-HT, NE, DA变化有显著性差异P<0.01。肝郁病理分级与神经递质变化有显著相关关系：与5-HT R=-0.417,P=001;与NER=-0.352P=0.001;与DAR=-0.609P=0.000。
(2)动物研究部分
1.E组与D组比,旷场实验水平得分和垂直得分有显著性差异,P<0.01。糖水嗜好实验,E组与D组比较,糖水饮用显著下降,P<0.01。E组与D组比较体重明显下降,P<0.01。
2.海马区E组ERamRNA较B组减少,ERβ较B组升高但无显著性差异,GPR30mRNA组间没有显著性差异；E组ERamRNA/ERβmRNA比值较B组低,柴胡疏肝散干预后D组明显升高,P<0.01；E组E2较A组低,干预后D组升高,但无显著性差异；E组5-HT, DA较B组明显下降,NE, DA经干预后升高但无显著性差异,5-HT明显增高,P<0.01。血清中,E组5-HT较B组明显高,经干预后明显下降P<0.01；E组E2, DA, NE较B组低,经干预DA后上升明显,P<0.01,E2,NE有上升趋势,但无显著性差异P>0.05。3.E组海马Ca2+浓度显著升高,干预后Ca2+浓度下降P<0.01。E组CaMKII浓度较D组显著升高,干预后下降,P<0.01。
4.PCR芯片显示,E组与B组比,在海马细胞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或P<0.01,柴胡疏肝散干预后,E组与D组比较,SSt有差异性表达P<0.05。
结论
1.PPS的病位证素以肝为主,涉及肾、脾、心,病性证素以气滞为主,涉及阴虚、血虚、气虚,证素分布呈现多脏分布、虚实夹杂特点,肝郁是其主要病理特点,症状以阵发性烘热,汗出,急躁易怒,情绪抑郁或焦虑,月经紊乱,胸闷、失眠,喜太息,心悸,咽部异物感等为主。
2.PPS患者干预前后Kupperman法症状评分及肝郁病理积分显著改善,肝郁病理分级与血清E2有显著性负性相关关系,与5-HT, NE, DA有显著性差异,呈负相关,说明肝郁是PPS主要病理,E2和神经递质改变是肝郁证的生物学基础。
3.大鼠行为学实验和糖水嗜好实验、体重变化及血清雌激素变化等证明自然衰老和慢性束缚PPS肝郁证大鼠造模成功。
4.PPS大鼠肝郁证与海马及周围血的E2神经递质的表达与有密切关系,与海马细胞的雌激素、雌激素受体ERamRNA/ERβmRNA比值表达有密切关系。
5.PPS大鼠肝郁证与海马区Ca2+/CaMKII通路有密切关系,与海马Ca2+/cAMP通路调节神经传导的基因表达变化有关系,Ca2+/cAMP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。
Perimenopausal syndrome (PPS) is a systematic disease occurred in perimenopausal women include imbalance in the spirit of the nemo-psychological, endocrine and metabolic, attributing to the change of the central and peripheral nerve neurotransmitter, vasomotor factor, cytokines free radicals. Liver-Qi stagnation plays an important role in perimenopausal syndrome, the priority of treatment is to soothe the liver and regulate the Qi and dispel the stagnation, such as Chaihu-Shugan-San(CSS), this study from the clinical and animal experiments explore the TCM pathological feature of perimenopausal syndrome and the relationship of Liver-Qi stagnation syndrome and strogen, estrogen receptors and monoamine neurotransmitters expression, to explore the relationship of gene expression in Ca2+/cAMP signaling pathway.
Objective
To explore the Liver-Qi stagnation syndrome's TCM pathology and syndrome element mixed characteristics in PPS. To observe the relationship of Liver-Qi Stagnation score change and estrogen, neurotransmitters after treating with CSS in PSS Liver-Qi stagnation syndrome rats and to explore the biology basis of Liver-Qi stagnation in PPS.
Methods
We collected161patients of PPS to analyse the main locations and characteristic of the disease throught differentiation of syndrome elements method and syndrome differentiation to analyse main location and characteristic of the disease.100cases of PPS whose Liver-Qi Stagnation score exceed70are collected, after4weeks treatment with CSS, the relationship of Liver-Qi Stagnation score change and estrogen, neurotransmitters are observed. To observe the effective of CSS on PPS and Liver-Qi stagnation syndrome rat models, and explore the molecular mechanisms throught Ca2+/cAMP signaling pathway in hippocampal cell. In this experiments, twelve-week-old female rats (250-280g, n=12) are used as normal controls (Group A),52-week-old nature aging rat as PPS rat. A total of48nature aging rats (52-week-old,370-380g) are randomly assigned into four groups; PPS controls (Group B, n=12), PPS controls treate with CSS (Group C, n=12), PPS+Liver-Qi stagnation model treate with CSS (Group D, n=12), and PPS+Liver-Qi stagnation model (Group E, n=12). CSS is administered simultaneously when the Liver-Qi stagnation model being established. We selecte the sucrose consumption test and the open field test (OFT) for the Liver-Qi stagnation behavior test.We measure E2, neurotransmitters, CaMKII levels in serum and hippocampus by ELISA, Ca2+is measured by Fura-2/AM in hippocampus and estrogen receptors of hippocampal mRNA expression are measured by quantitative real-time PCR and Ca2+/cAMP signaling pathway by RT2Profiler PCR Arrays.
Result
The part of clinical Study
1.The clinical symptoms distribution frequency of the top ten are hot flashes, sweating, irritability, depression or anxiety, menstrual disorders, chest distress, deep sigh, palpitations, globus hystericus, that is91.93%,91.93%,85.09%,81.99%,78.26%,69.56%,67.70%,55.28%,52.17%,39.75%.
2.The locations of PPS are liver, kidney, spleen, heart, but mainly in the liver(91.93%) and kidney(52.17%), P<0.01.The score of liver locations is the highest.
3. The nature syndrome elements include Qi stagnation and Yin deficiency, Yang deficiency, blood deficiency. Qi stagnation and Yin deficiency are in a high proportion, the ration is90.06%and71.43%。 The score of Qi stagnation is the highest.
4. The mixed syndrome elements relationship of the locations mixed mainly are liver and kidney, spleen and kidney, liver and spleen, nature of mixed syndrome elements are Qi stagnation mixed Yin deficiency and Qi stagnation mixed blood deficiency.
5.Before treatment, the cases of pathological Liver-Qi stagnation grade1is50, grade2is34, grade3is16, after treatment, the cases of pathological Liver-Qi stagnation grade0is70, grade1is22, grade2is3, grade3is5. The pathological classification changes significantly,P<0.01.
6. Before and after treatment with CSS, the Kupperman score change significantly P <0.01.
7. Before and after treatment with CSS, there is significant correlation relationship between Liver-Qi stagnation score and estrogen expression, r=-0.479P=0.001.
8. Before and after treatment with CSS, there is significant correlation relationship between Liver-Qi stagnation score and neurotransmitters expression,(5-HT, R=-0.417,P=0.001;NE, R=-0.352P=0.001;DA, R=-0.609P=0.000)
Part II The part of Basical Study
1. The open field test and the sucrose consumption test, the dynamic changes of BW. The BW of group E decrease sharply, while that of group D decreased slowly. We found the sucrose intake in group E are significantly lower than that in group B, p<0.01), whereas the CSS treatment ameliorate the sucrose intake (group D vs. group E, p<0.01).
2. CSS administration effects on hippocampal ERa, ERβ, or GPR30mRNA expression in aging rats or Liver-Qi stagnation group rats.No significant difference is observed in ERβ mRNA expression between the four groups, thereby indicating that CSS administration does not affect the ERβ mRNA expression in groups B and C or groups D and E. Similarly, GPR30mRNA expression does not differ significantly between treatment groups. The ERa/ERβ ratio in group E is markedly decrease compared with the other groups, p<0.01. CSS administration increase ERa/ERβ ratio.E2levels in A group rats were significantly higher than those in the group B. No significant difference is observed between the four aging rat groups, indicating that CSS does not affect E2levels in aging rats. Hippocampal5-HT, NE, DA levels in A group are significantly higher than those aging group, after CSS treatment NE, DA levels increase. The change of serum NE and DA are the same as of hippocampus except5-HT.
3. The E group hippocampal Ca2+expression increase sharply than D group, P<0.01. The change of CaMKII expression is the same.
4. PCR Arrays indicate there are6functional gene namely S100g, Th, NOS2, IL-2, Calml and SSt expression change between E group and B group (P<0.05or P<0.01), after CSS administration only the SSt functional gene change P<0.05.
Conclusion
1. The main clinical symptoms of PSS is Liver-Qi stagnation symptoms.The main locations of PPS are liver. The characteristic of syndrome elements in PPS is multiple organ involvement, mixed excessiveness and deficiency. Liver-Qi stagnation is the main pathology. The main clinical symptoms of the PSS are hot flashes, sweating, irritability, depression or anxiety, menstrual disorders, chest distress, deep sigh, palpitations, globus hystericus.
2. There are significant differences in Liver-Qi Stagnation score and Kupperman score before and after treatment. There is significant difference between Liver-Qi Sagnation score and estrogen before and after treatment. There are significant negative correlation between the pathology grade and estrogen. There are significant differences between Liver-Qi stagnation score and neurotransmitters5-HT, NE, DA before and after treatment. There are significant negative correlation between the pathology grade and neurotransmitters5-HT, NE, DA.
3. The open field test and the sucrose consumption test, the dynamic changes of BW and E2content in serum and hippocampus verify the PPS+Liver-Qi Stagnation syndrome rats model is good.
4. It indicates that there is close relation between ERa/ERβ ratio and Liver-Qi Stagnation syndrome in PPS rats. There is close relation between neurotransmitters and Liver-Qi stagnation syndrome in PPS.
5. There is close relation between Liver-Qi stagnation syndrome and Ca2+/CaMKII signal pathway in PPS. There is close relation between genes relating to neurotransmitter in Ca2+/cAMP signaling pathway and Liver-Qi stagnation syndrome, maybe it was the molecular mechanisms of Liver-Qi stagnation syndrome of PPS.
目的
(1)通过研究PPS症状、证素病理及兼杂特征,柴胡疏肝散治疗前后的PSS肝郁证患者肝郁积分、Kupperman法症状评分变化,肝郁病理分级与雌激素、神经递质关系,探讨PPS肝郁病理的部分生物学基础。
(2)通过柴胡疏肝散干预PPS肝郁证大鼠,从E2、ER、神经递质变化及大鼠海马雌激素受体介导的Ca2+/cAMP信号通路探讨其分子机制。
方法
(1)临床研究方面收集PPS患者161例,以证素辨证的方法,分析PPS的症状分布频次、病位病性证素及其兼杂的特点。选取PPS患者中肝郁积分≥70分的患者100例,根据肝郁病理积分分为3级,予以4周的柴胡疏肝散干预,观察干预前后的肝郁病理积分、Kupperman法症状评分变化,肝郁病理分级与E2、神经递质5-羟色胺(5-HT),去甲肾上腺素(NE),多巴胺(DA)关系,探讨PPS肝郁证的生物学基础。
(2)动物研究方面以3月龄成年大鼠为正常对照组(A组),13月龄自然衰老和慢性束缚法建立PPS肝郁证大鼠模型,分为围绝经期对照组(B组)、围绝经期对照组加中药组(C组)、PPS肝郁证加中药组(D组)和PPS肝郁证组(E组)等五组,药物干预3周后,予行为学观察,以实时定量PCR法检测海马区雌激素受体ERa, ERβ, GPR30(G protein-coupled receptor30) mRNA表达,用Fura-2/AM双波长荧光法测定海马细胞内游离钙浓度,用RT2Profiler PCR Arrays芯片检测海马Ca2+/cAMP通路上相关基因差异性表达,用ELISA法检测海马及血清雌激素及递质5-HT, NE,DA,血清钙调蛋白依赖性蛋白激酶Ⅱ (CaMKⅡ)表达。
结果
(1)临床研究部分
1.PPS的症状分布频次前10位是阵发性烘热,汗出,急躁易怒,情绪抑郁或焦虑,月经紊乱,胸闷、失眠,喜太息,心悸,咽部异物感,各占91.93%,91.93%,85.09%,81.99%,78.26%,69.56%,67.70%,55.28%,52.17%,39.75%。
2.PPS的病位证素涉及肝、肾、脾、心,主要是肝和肾,分别占91.93%和52.17%,与其它病位相比有显著性差异,P<0.01,肝的病位积分最高。
3.PPS的病性证素主要涉及气滞、阴虚、气虚、阳虚、血虚,其中气滞所占的比例最高为90.1%,与其它病性所占比例相比有显著性差异,p<0.01,气滞的病性积分最高。
4.PPS证素病位兼杂中,以肝肾最多见,其次为脾肾、肝脾,病性兼杂以气滞兼阴虚最为多见。
5.临床干预前PPS患者肝郁病理0级0人,1级50人,2级34人,3级16人,干预后0级70人,1级22人,2级3人,3级5人,干预前后肝郁病理分级人数有显著性改变P<0.01。
6.干预前后,Kupperman法症状评分有显著性改变,P<0.01。
7.干预前后,肝郁病理分级与E2变化有显著相关关系,R=-0.497,P==0.001。
8.干预前后,5-HT, NE, DA变化有显著性差异P<0.01。肝郁病理分级与神经递质变化有显著相关关系：与5-HT R=-0.417,P=001;与NER=-0.352P=0.001;与DAR=-0.609P=0.000。
(2)动物研究部分
1.E组与D组比,旷场实验水平得分和垂直得分有显著性差异,P<0.01。糖水嗜好实验,E组与D组比较,糖水饮用显著下降,P<0.01。E组与D组比较体重明显下降,P<0.01。
2.海马区E组ERamRNA较B组减少,ERβ较B组升高但无显著性差异,GPR30mRNA组间没有显著性差异；E组ERamRNA/ERβmRNA比值较B组低,柴胡疏肝散干预后D组明显升高,P<0.01；E组E2较A组低,干预后D组升高,但无显著性差异；E组5-HT, DA较B组明显下降,NE, DA经干预后升高但无显著性差异,5-HT明显增高,P<0.01。血清中,E组5-HT较B组明显高,经干预后明显下降P<0.01；E组E2, DA, NE较B组低,经干预DA后上升明显,P<0.01,E2,NE有上升趋势,但无显著性差异P>0.05。3.E组海马Ca2+浓度显著升高,干预后Ca2+浓度下降P<0.01。E组CaMKII浓度较D组显著升高,干预后下降,P<0.01。
4.PCR芯片显示,E组与B组比,在海马细胞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或P<0.01,柴胡疏肝散干预后,E组与D组比较,SSt有差异性表达P<0.05。
结论
1.PPS的病位证素以肝为主,涉及肾、脾、心,病性证素以气滞为主,涉及阴虚、血虚、气虚,证素分布呈现多脏分布、虚实夹杂特点,肝郁是其主要病理特点,症状以阵发性烘热,汗出,急躁易怒,情绪抑郁或焦虑,月经紊乱,胸闷、失眠,喜太息,心悸,咽部异物感等为主。
2.PPS患者干预前后Kupperman法症状评分及肝郁病理积分显著改善,肝郁病理分级与血清E2有显著性负性相关关系,与5-HT, NE, DA有显著性差异,呈负相关,说明肝郁是PPS主要病理,E2和神经递质改变是肝郁证的生物学基础。
3.大鼠行为学实验和糖水嗜好实验、体重变化及血清雌激素变化等证明自然衰老和慢性束缚PPS肝郁证大鼠造模成功。
4.PPS大鼠肝郁证与海马及周围血的E2神经递质的表达与有密切关系,与海马细胞的雌激素、雌激素受体ERamRNA/ERβmRNA比值表达有密切关系。
5.PPS大鼠肝郁证与海马区Ca2+/CaMKII通路有密切关系,与海马Ca2+/cAMP通路调节神经传导的基因表达变化有关系,Ca2+/cAMP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。
Perimenopausal syndrome (PPS) is a systematic disease occurred in perimenopausal women include imbalance in the spirit of the nemo-psychological, endocrine and metabolic, attributing to the change of the central and peripheral nerve neurotransmitter, vasomotor factor, cytokines free radicals. Liver-Qi stagnation plays an important role in perimenopausal syndrome, the priority of treatment is to soothe the liver and regulate the Qi and dispel the stagnation, such as Chaihu-Shugan-San(CSS), this study from the clinical and animal experiments explore the TCM pathological feature of perimenopausal syndrome and the relationship of Liver-Qi stagnation syndrome and strogen, estrogen receptors and monoamine neurotransmitters expression, to explore the relationship of gene expression in Ca2+/cAMP signaling pathway.
Objective
To explore the Liver-Qi stagnation syndrome's TCM pathology and syndrome element mixed characteristics in PPS. To observe the relationship of Liver-Qi Stagnation score change and estrogen, neurotransmitters after treating with CSS in PSS Liver-Qi stagnation syndrome rats and to explore the biology basis of Liver-Qi stagnation in PPS.
Methods
We collected161patients of PPS to analyse the main locations and characteristic of the disease throught differentiation of syndrome elements method and syndrome differentiation to analyse main location and characteristic of the disease.100cases of PPS whose Liver-Qi Stagnation score exceed70are collected, after4weeks treatment with CSS, the relationship of Liver-Qi Stagnation score change and estrogen, neurotransmitters are observed. To observe the effective of CSS on PPS and Liver-Qi stagnation syndrome rat models, and explore the molecular mechanisms throught Ca2+/cAMP signaling pathway in hippocampal cell. In this experiments, twelve-week-old female rats (250-280g, n=12) are used as normal controls (Group A),52-week-old nature aging rat as PPS rat. A total of48nature aging rats (52-week-old,370-380g) are randomly assigned into four groups; PPS controls (Group B, n=12), PPS controls treate with CSS (Group C, n=12), PPS+Liver-Qi stagnation model treate with CSS (Group D, n=12), and PPS+Liver-Qi stagnation model (Group E, n=12). CSS is administered simultaneously when the Liver-Qi stagnation model being established. We selecte the sucrose consumption test and the open field test (OFT) for the Liver-Qi stagnation behavior test.We measure E2, neurotransmitters, CaMKII levels in serum and hippocampus by ELISA, Ca2+is measured by Fura-2/AM in hippocampus and estrogen receptors of hippocampal mRNA expression are measured by quantitative real-time PCR and Ca2+/cAMP signaling pathway by RT2Profiler PCR Arrays.
Result
The part of clinical Study
1.The clinical symptoms distribution frequency of the top ten are hot flashes, sweating, irritability, depression or anxiety, menstrual disorders, chest distress, deep sigh, palpitations, globus hystericus, that is91.93%,91.93%,85.09%,81.99%,78.26%,69.56%,67.70%,55.28%,52.17%,39.75%.
2.The locations of PPS are liver, kidney, spleen, heart, but mainly in the liver(91.93%) and kidney(52.17%), P<0.01.The score of liver locations is the highest.
3. The nature syndrome elements include Qi stagnation and Yin deficiency, Yang deficiency, blood deficiency. Qi stagnation and Yin deficiency are in a high proportion, the ration is90.06%and71.43%。 The score of Qi stagnation is the highest.
4. The mixed syndrome elements relationship of the locations mixed mainly are liver and kidney, spleen and kidney, liver and spleen, nature of mixed syndrome elements are Qi stagnation mixed Yin deficiency and Qi stagnation mixed blood deficiency.
5.Before treatment, the cases of pathological Liver-Qi stagnation grade1is50, grade2is34, grade3is16, after treatment, the cases of pathological Liver-Qi stagnation grade0is70, grade1is22, grade2is3, grade3is5. The pathological classification changes significantly,P<0.01.
6. Before and after treatment with CSS, the Kupperman score change significantly P <0.01.
7. Before and after treatment with CSS, there is significant correlation relationship between Liver-Qi stagnation score and estrogen expression, r=-0.479P=0.001.
8. Before and after treatment with CSS, there is significant correlation relationship between Liver-Qi stagnation score and neurotransmitters expression,(5-HT, R=-0.417,P=0.001;NE, R=-0.352P=0.001;DA, R=-0.609P=0.000)
Part II The part of Basical Study
1. The open field test and the sucrose consumption test, the dynamic changes of BW. The BW of group E decrease sharply, while that of group D decreased slowly. We found the sucrose intake in group E are significantly lower than that in group B, p<0.01), whereas the CSS treatment ameliorate the sucrose intake (group D vs. group E, p<0.01).
2. CSS administration effects on hippocampal ERa, ERβ, or GPR30mRNA expression in aging rats or Liver-Qi stagnation group rats.No significant difference is observed in ERβ mRNA expression between the four groups, thereby indicating that CSS administration does not affect the ERβ mRNA expression in groups B and C or groups D and E. Similarly, GPR30mRNA expression does not differ significantly between treatment groups. The ERa/ERβ ratio in group E is markedly decrease compared with the other groups, p<0.01. CSS administration increase ERa/ERβ ratio.E2levels in A group rats were significantly higher than those in the group B. No significant difference is observed between the four aging rat groups, indicating that CSS does not affect E2levels in aging rats. Hippocampal5-HT, NE, DA levels in A group are significantly higher than those aging group, after CSS treatment NE, DA levels increase. The change of serum NE and DA are the same as of hippocampus except5-HT.
3. The E group hippocampal Ca2+expression increase sharply than D group, P<0.01. The change of CaMKII expression is the same.
4. PCR Arrays indicate there are6functional gene namely S100g, Th, NOS2, IL-2, Calml and SSt expression change between E group and B group (P<0.05or P<0.01), after CSS administration only the SSt functional gene change P<0.05.
Conclusion
1. The main clinical symptoms of PSS is Liver-Qi stagnation symptoms.The main locations of PPS are liver. The characteristic of syndrome elements in PPS is multiple organ involvement, mixed excessiveness and deficiency. Liver-Qi stagnation is the main pathology. The main clinical symptoms of the PSS are hot flashes, sweating, irritability, depression or anxiety, menstrual disorders, chest distress, deep sigh, palpitations, globus hystericus.
2. There are significant differences in Liver-Qi Stagnation score and Kupperman score before and after treatment. There is significant difference between Liver-Qi Sagnation score and estrogen before and after treatment. There are significant negative correlation between the pathology grade and estrogen. There are significant differences between Liver-Qi stagnation score and neurotransmitters5-HT, NE, DA before and after treatment. There are significant negative correlation between the pathology grade and neurotransmitters5-HT, NE, DA.
3. The open field test and the sucrose consumption test, the dynamic changes of BW and E2content in serum and hippocampus verify the PPS+Liver-Qi Stagnation syndrome rats model is good.
4. It indicates that there is close relation between ERa/ERβ ratio and Liver-Qi Stagnation syndrome in PPS rats. There is close relation between neurotransmitters and Liver-Qi stagnation syndrome in PPS.
5. There is close relation between Liver-Qi stagnation syndrome and Ca2+/CaMKII signal pathway in PPS. There is close relation between genes relating to neurotransmitter in Ca2+/cAMP signaling pathway and Liver-Qi stagnation syndrome, maybe it was the molecular mechanisms of Liver-Qi stagnation syndrome of PPS.
引文
[1]郑锦,李佶,张利.上海地区围绝经期综合征妇女流行病学调查[J].中西医结合学报,2009,7(9)：827-830.
[2]Yen JY, Yang MS, Wang MH, et al.The associations between menopausal syndrome and depression during pre-, peri-, and postmenopausal period among Taiwanese female aborigines [J].Psychiatry Clin Neurosci,2009,63(5):678-684.
[3]张绍芬.绝经-内分泌与临床[M].第一版.北京：人民卫生出版社,2005：177-180.
[4]Sturgeon SR, Volpe SL, Puleo E, et al. Effect of flaxseed consumption on urinary levels of estrogen metabolites in postmenopausal women[J]. Nutr Cancer,2010,62(2):175-180.
[5]梁文娜,李灿东,甘慧娟,等.围绝经期综合征肝郁病理与雌性激素的相关性研究.中华中医药杂志,2011,26(9)：3299-3301.
[6]梁文娜,李灿东,李西海.围绝经期综合征证素规律的临床研究.福建中医学院学报,2009,19(6)：3-5.
[7]乐杰.妇产科学[M].第6版.北京：人民卫生出版社,2004：350-351.
[8]贝政平,来佩琍, 张斌.妇产科疾病诊断标准[M].北京：科学出版社,2007：136-148.
[9]吴宏进,周昌乐,许家佗,等.围绝经期综合征中医证候分布特点文献研究.中国中医药信息杂志[J].2012,19(3)：22-25.
[10]张满风,刘静君.中医药治疗围绝经综合征的研究进展[J].现代中医药,2007,27(3)：72-74.
[11]李红,任林,李灿东,等.围绝经期综合征肝气郁结证雌激素受体β基因多态性分析[J].中华中医药杂志,2010,25(6)：830-832.
[12].刘宏奇,杜彩风,宗惠等.279例更年期综合征常见证候、证候要素临床调查研究[J].山东中医杂志,2012,31(2)：92-94.
[13]马哲.王平教授运用补肾疏肝法治疗更年期综合征的经验[J].光明中医,2011,26(10)：1982-1983.
[14]袁立霞,刘刚.从肝论治妇女更年期综合征[J].山东中医杂志,2011,26(7)：439-440.
[15]蔡群慧.补肾兼疏肝治疗更年期综合征38例临床观察[J].中医杂志,2010,51(增2)：115-116.
[16]罗峰,孟肖飞.柴胡的药理分析及应用[J].中医学报,2012,27(107)：863-864.
[17]邵继红,韩珍,杨艳,等.白芍抗抑郁作用的实验研究[J].宁夏医学杂志,2008,30(6)：490-491.
[18]周海虹,陆沉,陈艳玲,等.柴郁温胆汤对大鼠抑郁模型行为学及脑内单胺类神经递质的影响[J].中华中医药杂志,2008,23(10)：931-934.
[19]王景霞,张建军,李伟,等.白芍提取物对慢性应激抑郁模型大鼠行为学及大脑皮质单胺类神经递质的影响[J].中华中医药杂志,2010,25(11)：1895-1897.
[20]李苒,高杉,于春泉.柴胡、白芍药对的药理学研究柴胡和白芍配伍抗抑郁作用的研究进展[J].中国实验方剂学杂志,2012,18(14)：313-316.
[21]刘冈,王欢.枳壳研究现状[J].九江学院学报,2010,90(3)：93-96.
[22]周江.川芎有效成分及其药理作用研究概况[J].浙江中医杂志,2007,42(10)：615-616.
[23]曹铭希.陈皮中橙皮苷的提取及其药理活性的研究进展[J].中国医药指南,2012,10(12)：452-454.
[24]周中流,刘永辉.香附提取物的抗抑郁活性及其作用机制研究[J].中国实验方剂学杂志,2012,18(7)：191-193.
[25]金卫东,邢葆平,王鹤秋,等.柴胡疏肝散治疗抑郁症对照研究临床疗效的Meta分析[J].中华中医药学刊,2009,27(7)：1397-1399.
[26]黄佩珊.柴胡疏肝散联合抗抑郁药物治疗抑郁障碍的研究进展[J].中医学报,2012,27(168)：627-629.
[27]蔡群慧.补肾兼疏肝治疗更年期综合征38例临床观察[J].中医杂志,2010,5(增2)：115-116.
[28]邱珍国.胡疏肝散加减治疗胃脘痛80例疗效观察[J].中国中医基础医学杂志,2010,16(10)：949.
[29]吴慧,徐有水,刘日才.柴胡疏肝散在消化系统疾病中应用举隅[J].浙江中西医结合杂志, 2012,22(4)：273-275
[30]严彦彪,尹吉恒.柴胡疏肝散加味治疗脂肪肝68例[J].河北中医,2005,27(12)：927.
[31]林冰,夏进.加味柴胡疏肝散治疗抑郁症临床研究[J].新中医,2011,43(8)：36-37.
[32]傅俏芳.柴胡疏肝散加减治疗顽固性失眠60例[J].江西中医药,2012,43(352)：39-40.
[33]华刚,孟静.柴胡疏肝散加减治疗乳腺增生120例[J].陕西中医,2005,26(3)：214.
[34]杨慧敏,李跃华,杨京,等.抑郁症中医辨证分型与外周血神经递质的关系[J].中国中医药信息杂志,2011,18(4)：14-16.
[35]马莉,程为平,梅晨健,等.加强扬刺百会穴对抑郁症患者体内单胺类神经递质代谢影响的研究[J].中西医结合心脑血管病杂志,2012,10(5)：562-563.
[36]李素敏,邱如卿.经前期综合征肝气郁证与血清性激素及单胺类神经递质的相关性研究[J].福建中医学院学报,2009,19(4)：20-21.
[37]毛海燕,叶林,叶向荣,等.肝郁证大鼠中枢神经递质变化的观察[J].福建中医药,2002,33(2)：17.
[38]Bloch M, Schmidt P, Danaceau M, et al. Effects of gonadal steroids in women with a history of postpartum depression[J].Am J Psychiatry,2000,157(6):924-930.
[39]Soares C, Almeide OP,Joffe H, et al. Efficacy of estradiol for the treatment of depressive disorders in perimenopausal women:a double-blind, randomized, placebo-controlled trial[J].Arch Gen Psychiatry,2001,58(6):529-534.
[40]Waif AA, Frye CA. Administration of estrogen receptor beta-specific selective estrogen receptor modulators to the hippocampus decrease anxiety and depressive behavior of ovariectomized rats[J]. Pharmacol Biochem Behav,2007,86(2):407-414.
[41]Walf AA, Koonce CJ, Frye CA. Estradiol or diarylpropionitrile decrease anxiety-like behavior of wild type, but not estrogen receptor beta knockout mice[J]. Behavioral Neuroscience,2008, 122(5):974-981.
[1]陈家旭,李伟,赵歆等.慢性束缚应激大鼠海马脑啡肽mRNA和前强啡肽mRNA表达及中药复方的影响[J].中国应用生理学杂志,2005,21(2)：121-125.
[2]Porsolt RD, Le Pichon M, Jalfre M.Depression:a new animal model sensitive to antidepressant treatments [J].Nature.1977,266(5604):730-732.
[3]Russo E, Citraro R, Davoli A,et al.Ameliorating effects of aripiprazole on cognitive functions and depressive-like behavior in a genetic rat model of absence epilepsy and mild-depression comorbidity [J]. Neuropharmacology,2013,64(1):371-379.
[4]Dildy JE, Leslie SW. Ethanol inhibits NMDA-induced increases in free intracellular Ca2+in dissociated brain cells [J]. Brain Res.1989,499(2):383-387.
[5]Gebhart JB, Rickard DJ, Barrett TJ, et al. Expression of estrogen receptor isoforms alpha and beta messenger RNA in vaginal tissue of premenopausal and postmenopausal women[J]. American Journal Obstet Gyneco 1,2001,185(6):1325-1330.
[6]Romano-Torres, Fernandez-Guasti A, Estradiol valerate elicits antidepressant-like effects in middle-aged female rats under chronic mild stress [J]. Behav Pharmacol.2010,21(2):104-111.
[7]Joffe H, Hall JE, Gruber S, et al. Estrogen therapy selectively enhances prefrontal cognitive processes: a randomized, double-blind, placebo-controlled study with functional magnetic resonance imaging in perimenopausal and recently postmenopausal women [J]. Menopause,2006,13(3):411-422.
[8]Greendale GA, Chu J, Ferrell R, et al.The association of bone mineral density with estrogen receptor gene polymorphisms [J].American Journal Of the Medical Sciences,2006,119 (9):79-86.
[9]李贺琦,陈霞.围绝经期综合征与神经递质(5-羟色胺)相关性的临床研究进展[J].中华中医药学刊,2011,29(2)：375-377.
[10]Alicia A. Walf, Chery A. Frye. Estradiol reduces anxiety-and depression-like behavior of aged female mice [J]. Physiology and Behavior,2010,99(2):169-174.
[11]李红,任林,李灿东,等.围绝经期综合征肝气郁结证患者雌激素受体β基因多态性分析[J].中华中医药杂志,2010,25(6)：830-832.
[12]Brzezinski A, Bcnshlrshan A. Neuropeptides, neurotransmitters, body weight and menopause [J]. Menopause,2000,7(3):137-139.
[l3]Nishizawa S, Benkelfat C, Young SN.Differences between males and females in rates of serotonin synthesis in human brain [J]..Proc Natl Acad Sci USA,1997,94 (10):5308-5313.
[14]Moret C, Briley M.The importance of norepinephrine in depression [J]. Neuropsychiatric Disease and Treatment,2011,7(Suppl):9-13.
[15]Isgor C, Watson SJ. Estrogen receptor a and β mRNA expressions by proliferating and differentiating cells in the adult rat dentategyrus and subventricular zone [J].Neuroscience,2005,134(3):847-56.
[16]Kronmuller KT, Pantel J, Kohler S, et al. Hippocampal volume and 2-year outcome in depression[J]. The British Journal of Psychiatry,2008,192(6):472-473.
[17]Videbech P. Ravnkilde B. Hippocampal volume and depression:a meta-analysis of MRI studies [J]. The American Journal of Psychiatry,2004,161(11):1957-1966.
[18]Shors TJ, Leuner B. Estrogen-mediated effects on depression and memory formation in females [J]. Journal of Affective Disorders,2003,74(1):85-96.
[19]. Conrad CD, Jackson JL, Wieczorek L, et al. Acute stress impairs spatial memory in male but not female rats:influence of estrous cycle [J]. Pharmacology Biochemistry and Behavior,2004,78(3): 569-579.
[20]索玉平,王宝迎,王丹青,等.坤宁安对围绝经期综合征大鼠下丘脑β-内啡肽及神经递质的影响[J].中国药物与临床,2006,6(1)：21-24.
[21]魏盛,侯金良,巢玉,等.郁怒诱发经前期综合征肝气郁证猕猴模型血清单胺类神经递质含量分析[J].中西医结合学报,2012,10(8)：925-931.
[22]石秦东,蔡云,邱根全,等.脑脉康颗粒剂对大脑中动脉缺血再灌注大鼠脑的保护作用[J].北京中医药大学学报,2002,25(3)：39-41.
[23]Volman V, Gerkin R C, Lau P M, et al. Calcium and synaptic dynamics underlying reverberatory activity in neuronal networks [J].Phys Biol,2007,4(2):91-103.
[24]Xu J, He L, Wu LG. Role of Ca2+ channels in short term synaptic plasticity [J]. CurrOpin Neurobiol, 2007,17(3):352-359.
[25]钟晓明,毛庆秋,黄真,等.金丝桃提取物对应激动物模型的抗抑郁作用及其机理研究[J].浙江中医药大学学报,2006,30(2)214-216.
[26]钊,晓明,毛庆秋,黄真,等.苏郁胶囊对慢性应激抑郁模型大鼠海马神经细胞凋亡的影响[J].中国现代应用药学杂志,2006,23(8)：733-736.
[27]Orihuela PA, Zuniga LM, Rios M, et al. Mating changes the subcellular distribution and the f unctionality of estrogen receptors in the rat oviduct[J].Reprod Biol Endocrinol,2009,30(7):132-139.
[28]Barnett P. Somatostatin and Somatostatin Receptor Physiology [J].Endocrine,2003,20(3):255-264.
[29]冯劫.四逆散对抑郁模型大鼠血浆NPY、 SP、 SS含量的影响[J].浙江中医杂志,2011,46(8)：566-567.
[30]White BC, Sullivan JM, DeGracia DJ, et al. Brain ischemia and reperfusion:molecular mechanisms of neuronal injury [J]. J Neurol Sci,2000, 1(179S1-2):1-33.
[31]Mutlu O, Ulak G, Laugeray A, et al. Effects of neuronal and inducible NOS inhibitor 1-[2-(trifluoromethyl) phenyl] imidazole (TRIM) in unpredictable chronic mild stress procedure in mice[J]. Pharmacol Biochem Behav,2009,92(1):82-87.
[32]Yun-Li Peng, Yu-Ning Liu, Lei Liu, et al. Inducible nitric oxide synthase is involved in the modulation of depressive behaviors induced by unpredictable chronic mild stress [J]. Journal of Neuroinflammation,2012,9(6):75-87.
[33]Duncko P, Kiss A, Skultetyova I, et al. Corticotropin releasing hormone mRNA levels in response to chronic mild stress rise in male but not in female rats while tyrosine hydroxylase mRNA decrease in both sexes[J].Psychoneur oendocrinology,2001,26(1):77-89.
[34]侯秀娟,唐启盛,李小黎.脑缺血后抑郁模型大鼠下丘脑酪氨酸羟化酶的表达及中药干预作用[J].中华中医药学刊,2008,26(8)：1692-1694.
[35]姚辉,肖红,张晓斌,等.抑郁症患者血浆白细胞介素与单胺代谢产物的研究[J].神经疾病与精神卫生,2003,3(1)：66-67.
[36]Seidel A, Arolt V, Hunstiger M, et al. Cytokine production and serum proteins in depression[J], Scand J Immunol,1995,41(6):534-538.
[37]Yasui T, Maegawa M, Tomita J, et al. Changes in serum cytokine concentrations during the menopausal transition[J]. Maturitas,2007,56(4):396-403.
[1]Kessler RC, McGonagle KA, Nelson CB, et al.Sex and depression in the National Comorbidity Survey. Ⅱ:Cohort effects[J]. J Affect Disord,1994,30(1):15-26.
[2]董胜莲,吴庆文,安子薇.女性更年期抑郁症相关因素分析.中国健康心理学杂志,2012,20(8)：1160-1162.
[3]宋美英,夏薇,黄民,等.雌激素对抑郁大鼠海马单胺类神经递质含量的影响及缰核在其中的作用[J].中国老年学杂志.2011,31(19)：3766-3768.
[4]姚迪,胡欣妍,苏兴国,等.舒肝解郁饮对抑郁大鼠脑组织单胺类神经递质及其代谢产物的影响[J].吉林大学学报(医学版),2007,33(4)：704-707.
[5]Smith LJ, HendersonJ A, AbellCW, et al. Effects of ovarian steroidsand raloxifene on proteins that synthesiz, transport, and degrade serotonin in the rapheregion of macaques[J]. Neuropsycho pharmacology,2004,29(11):2035-2045.
[6]BetheaCL, MirkesSJ, Michelson D. Effects of oral estrogen, raloxifene and arzoxifene on gene expression in serotonin neurons of macaques[J]. Psychoneuroendocrinology,2002,27(4):431-445.
[7]GundlahC, LuNZ.BetheaCL. Ovarian steroid regulation of monoamineoxidase-A and B mRNAs in the macaque dorsalraphe and hypothalamic nucle[J]. Psychopharmacology,2002,160(3):271-282.
[8]Rivera HM, Santollo J, Nikonova LV, et al. Estradiol increases the anorexia associated with increased 5-HT(2C) receptor activation in ovariectomized rats[J].Physiol Behav,2012,105(2):188-94.
[9]Chavez C, Hollaus M, Scarr E, et al. The effect of estrogen on dopamine and serotonin receptor and transporter levels in the brain:an autoradiography study[J].Brain Res,2010,19(1321):51-59.
[10]Hiroi R, Neumaier JF. Estrogen decreases 5-HT1B autoreceptor mRNA in selective subregion of rat dorsal raphe nucleus:inverse association between gene expression and anxiety behavior in the open field[J]. Neuroscience,2009,158(2):456-64.
[11]Mcewen B. Estrogen actions throughout the brain[J]. Recent Prog HormRes,2002,57(1):357-384.
[12]张吉强,姚青,蔡文琴.卵巢切除对小鼠基底前脑内雌激素α受体表达的影响[J].第三军医大学学报,2003,25(3)：261-263.
[13]GundlahC, LuNZ, MirkesSJ, et al. Estrogen receptor beta (ER(3) mRNA and protein in serotonin neurons of macaques[J].Brain ResMol Brain Res,2001,91 (1-2):14-22.
[14]Walf AA, Frye CA. Administration of estrogen receptor beta-specific selective estrogen receptor modulators to the hippocampus decrease anxiety and depressive behavior of ovariectomized rats[J].Pharmacol Biochem Behav,2007,86(2):407-414.
[15]Walf AA, Koonce CJ, Frye CA. Estradiol or diarylpropionitrile decrease anxiety-like behavior of wildtype, but not estrogen receptor beta knockout mice [J].Behavioral Neuroscience,2008,122(5): 974-981.
[16]Serova LI, Harris HA, Maharjan S, et al. Modulation of responses to stress by estradiol benzoate and selective estrogen receptor agonists[J]. J Endocrinol,2010,205(3):253-262.
[17]Le Saux M, Di Paolo T. Influence of oestrogenic compounds on monoamine transporters in rat striatum[J]. J Neuroendocrinol,2006,18(1):25-32.
[18]A1-Sweidi S, Morissette M, Di Paolo T. Effect of oestrogen receptors on brain NMDA receptors of 1-methy1-4-pheny1-1,2,3,6-tetrahydropyridine mice[J].2012,24(11):1375-1385.
[19]Maharjan S, Serova L, Sabban EL.Transcriptional regulation of tyrosine hydroxylase by estrogen: opposite effects with estrogen receptors alpha and beta and interactions with cyclic AMP[J].J Neurochem,2005,93(6):1502-1514.
[20]Soare.Soares for the treatment of depressive disorders in perimenopausal women:a double-blind, randomized, placebo controlled trial[J]. ArchGen Psychiatry,2001,58:529-534.
[21]罗阳,何国平.性激素及神经递质与产后抑郁症关系的Meta分析[J].中国妇幼保健,2007,22(8)：1020-1023.
[22]罗元恺.妇女更年期综合征的中医治疗[J].新中医,1992,24(1)：16-17.
[23]叶燕萍.更年期综合征辨证分型的研究[J].新中医,2000,32(9)：36-37.
[24]李汉明.辨证分型治疗更年期综合征75例[J].湖南中医学院学报,1995,15(2)：311.
[25]孙静.胥受天.从肝论治更年期综合征经验[J].江西中医药,2003,34(1)：9-10.
[26]袁立霞,刘刚.从肝论治妇女更年期综合征[J].山东中医杂志,2011,26(7)：439-440.
[27]张丽丽,姚秀英.绝经前后诸症从肝论治58例[J].新中医,2007,39(3)：65-66.
[28]杜巧琳,王小云.舒肝胶囊治疗更年期妇女抑郁症36例临床观察[J].新中医,2008,40(10)：61-62.
[29]唐凤.丹栀逍遥散临床应用[J].辽宁中医杂志,2006,33(10)：1344.
[30]谈勇,许小风,卢苏.绝经前后诸证心肾病机探析[J].南京中医药大学报,2001,17(6)：340.
[31]池雷.宁心敛阴汤治疗更年期综合征20例[J].河南中医,2009,29(12)：1201.
[32]李力,任婕,杜彩风,等.更年期综合征中医证候及证候要素分布特点的文献分析[J].中华中医药杂志,2008,23(3)：194-197.
[33]陈家旭,万霞,胡立胜.围绝经期综合征辨证分型的文献研究[J].中华中医药杂志,2006,21(11)：649-651.
[34]申春悌,张华强,朱雄华,等.400例更年期综合征临床证候辨证标准现场调查分析[J].中国中西医结合杂志,2004,24(6)：517-520.
[35]辛意,王天芳,杜彩凤,等.基于经验辨证的更年期综合征围绝经期和绝经后期常见证候及证候要素分布[J].中西医结合学报,2009,7(6)：522-526.
[36]汪涛,秦锋.逍遥散对产后抑郁大鼠海马中单胺类神经递质含量的影响[J].中西医结合学报,2010,8(11)：1075-1079.
[37]严亨秀,任昉,顾健.柴胡疏肝散对实验性肝郁证大鼠的影响[J].中药药理与临床,2006,22(6)：5-6.
[38]王桐生,谢鸣,张艳霞,等.肝郁模型大鼠行为学与脑单胺递质的变化及柴胡的干预作用[J].中华中医药杂志,2008,23(10)：934-937.
[2]Yen JY, Yang MS, Wang MH, et al.The associations between menopausal syndrome and depression during pre-, peri-, and postmenopausal period among Taiwanese female aborigines [J].Psychiatry Clin Neurosci,2009,63(5):678-684.
[3]张绍芬.绝经-内分泌与临床[M].第一版.北京：人民卫生出版社,2005：177-180.
[4]Sturgeon SR, Volpe SL, Puleo E, et al. Effect of flaxseed consumption on urinary levels of estrogen metabolites in postmenopausal women[J]. Nutr Cancer,2010,62(2):175-180.
[5]梁文娜,李灿东,甘慧娟,等.围绝经期综合征肝郁病理与雌性激素的相关性研究.中华中医药杂志,2011,26(9)：3299-3301.
[6]梁文娜,李灿东,李西海.围绝经期综合征证素规律的临床研究.福建中医学院学报,2009,19(6)：3-5.
[7]乐杰.妇产科学[M].第6版.北京：人民卫生出版社,2004：350-351.
[8]贝政平,来佩琍, 张斌.妇产科疾病诊断标准[M].北京：科学出版社,2007：136-148.
[9]吴宏进,周昌乐,许家佗,等.围绝经期综合征中医证候分布特点文献研究.中国中医药信息杂志[J].2012,19(3)：22-25.
[10]张满风,刘静君.中医药治疗围绝经综合征的研究进展[J].现代中医药,2007,27(3)：72-74.
[11]李红,任林,李灿东,等.围绝经期综合征肝气郁结证雌激素受体β基因多态性分析[J].中华中医药杂志,2010,25(6)：830-832.
[12].刘宏奇,杜彩风,宗惠等.279例更年期综合征常见证候、证候要素临床调查研究[J].山东中医杂志,2012,31(2)：92-94.
[13]马哲.王平教授运用补肾疏肝法治疗更年期综合征的经验[J].光明中医,2011,26(10)：1982-1983.
[14]袁立霞,刘刚.从肝论治妇女更年期综合征[J].山东中医杂志,2011,26(7)：439-440.
[15]蔡群慧.补肾兼疏肝治疗更年期综合征38例临床观察[J].中医杂志,2010,51(增2)：115-116.
[16]罗峰,孟肖飞.柴胡的药理分析及应用[J].中医学报,2012,27(107)：863-864.
[17]邵继红,韩珍,杨艳,等.白芍抗抑郁作用的实验研究[J].宁夏医学杂志,2008,30(6)：490-491.
[18]周海虹,陆沉,陈艳玲,等.柴郁温胆汤对大鼠抑郁模型行为学及脑内单胺类神经递质的影响[J].中华中医药杂志,2008,23(10)：931-934.
[19]王景霞,张建军,李伟,等.白芍提取物对慢性应激抑郁模型大鼠行为学及大脑皮质单胺类神经递质的影响[J].中华中医药杂志,2010,25(11)：1895-1897.
[20]李苒,高杉,于春泉.柴胡、白芍药对的药理学研究柴胡和白芍配伍抗抑郁作用的研究进展[J].中国实验方剂学杂志,2012,18(14)：313-316.
[21]刘冈,王欢.枳壳研究现状[J].九江学院学报,2010,90(3)：93-96.
[22]周江.川芎有效成分及其药理作用研究概况[J].浙江中医杂志,2007,42(10)：615-616.
[23]曹铭希.陈皮中橙皮苷的提取及其药理活性的研究进展[J].中国医药指南,2012,10(12)：452-454.
[24]周中流,刘永辉.香附提取物的抗抑郁活性及其作用机制研究[J].中国实验方剂学杂志,2012,18(7)：191-193.
[25]金卫东,邢葆平,王鹤秋,等.柴胡疏肝散治疗抑郁症对照研究临床疗效的Meta分析[J].中华中医药学刊,2009,27(7)：1397-1399.
[26]黄佩珊.柴胡疏肝散联合抗抑郁药物治疗抑郁障碍的研究进展[J].中医学报,2012,27(168)：627-629.
[27]蔡群慧.补肾兼疏肝治疗更年期综合征38例临床观察[J].中医杂志,2010,5(增2)：115-116.
[28]邱珍国.胡疏肝散加减治疗胃脘痛80例疗效观察[J].中国中医基础医学杂志,2010,16(10)：949.
[29]吴慧,徐有水,刘日才.柴胡疏肝散在消化系统疾病中应用举隅[J].浙江中西医结合杂志, 2012,22(4)：273-275
[30]严彦彪,尹吉恒.柴胡疏肝散加味治疗脂肪肝68例[J].河北中医,2005,27(12)：927.
[31]林冰,夏进.加味柴胡疏肝散治疗抑郁症临床研究[J].新中医,2011,43(8)：36-37.
[32]傅俏芳.柴胡疏肝散加减治疗顽固性失眠60例[J].江西中医药,2012,43(352)：39-40.
[33]华刚,孟静.柴胡疏肝散加减治疗乳腺增生120例[J].陕西中医,2005,26(3)：214.
[34]杨慧敏,李跃华,杨京,等.抑郁症中医辨证分型与外周血神经递质的关系[J].中国中医药信息杂志,2011,18(4)：14-16.
[35]马莉,程为平,梅晨健,等.加强扬刺百会穴对抑郁症患者体内单胺类神经递质代谢影响的研究[J].中西医结合心脑血管病杂志,2012,10(5)：562-563.
[36]李素敏,邱如卿.经前期综合征肝气郁证与血清性激素及单胺类神经递质的相关性研究[J].福建中医学院学报,2009,19(4)：20-21.
[37]毛海燕,叶林,叶向荣,等.肝郁证大鼠中枢神经递质变化的观察[J].福建中医药,2002,33(2)：17.
[38]Bloch M, Schmidt P, Danaceau M, et al. Effects of gonadal steroids in women with a history of postpartum depression[J].Am J Psychiatry,2000,157(6):924-930.
[39]Soares C, Almeide OP,Joffe H, et al. Efficacy of estradiol for the treatment of depressive disorders in perimenopausal women:a double-blind, randomized, placebo-controlled trial[J].Arch Gen Psychiatry,2001,58(6):529-534.
[40]Waif AA, Frye CA. Administration of estrogen receptor beta-specific selective estrogen receptor modulators to the hippocampus decrease anxiety and depressive behavior of ovariectomized rats[J]. Pharmacol Biochem Behav,2007,86(2):407-414.
[41]Walf AA, Koonce CJ, Frye CA. Estradiol or diarylpropionitrile decrease anxiety-like behavior of wild type, but not estrogen receptor beta knockout mice[J]. Behavioral Neuroscience,2008, 122(5):974-981.
[1]陈家旭,李伟,赵歆等.慢性束缚应激大鼠海马脑啡肽mRNA和前强啡肽mRNA表达及中药复方的影响[J].中国应用生理学杂志,2005,21(2)：121-125.
[2]Porsolt RD, Le Pichon M, Jalfre M.Depression:a new animal model sensitive to antidepressant treatments [J].Nature.1977,266(5604):730-732.
[3]Russo E, Citraro R, Davoli A,et al.Ameliorating effects of aripiprazole on cognitive functions and depressive-like behavior in a genetic rat model of absence epilepsy and mild-depression comorbidity [J]. Neuropharmacology,2013,64(1):371-379.
[4]Dildy JE, Leslie SW. Ethanol inhibits NMDA-induced increases in free intracellular Ca2+in dissociated brain cells [J]. Brain Res.1989,499(2):383-387.
[5]Gebhart JB, Rickard DJ, Barrett TJ, et al. Expression of estrogen receptor isoforms alpha and beta messenger RNA in vaginal tissue of premenopausal and postmenopausal women[J]. American Journal Obstet Gyneco 1,2001,185(6):1325-1330.
[6]Romano-Torres, Fernandez-Guasti A, Estradiol valerate elicits antidepressant-like effects in middle-aged female rats under chronic mild stress [J]. Behav Pharmacol.2010,21(2):104-111.
[7]Joffe H, Hall JE, Gruber S, et al. Estrogen therapy selectively enhances prefrontal cognitive processes: a randomized, double-blind, placebo-controlled study with functional magnetic resonance imaging in perimenopausal and recently postmenopausal women [J]. Menopause,2006,13(3):411-422.
[8]Greendale GA, Chu J, Ferrell R, et al.The association of bone mineral density with estrogen receptor gene polymorphisms [J].American Journal Of the Medical Sciences,2006,119 (9):79-86.
[9]李贺琦,陈霞.围绝经期综合征与神经递质(5-羟色胺)相关性的临床研究进展[J].中华中医药学刊,2011,29(2)：375-377.
[10]Alicia A. Walf, Chery A. Frye. Estradiol reduces anxiety-and depression-like behavior of aged female mice [J]. Physiology and Behavior,2010,99(2):169-174.
[11]李红,任林,李灿东,等.围绝经期综合征肝气郁结证患者雌激素受体β基因多态性分析[J].中华中医药杂志,2010,25(6)：830-832.
[12]Brzezinski A, Bcnshlrshan A. Neuropeptides, neurotransmitters, body weight and menopause [J]. Menopause,2000,7(3):137-139.
[l3]Nishizawa S, Benkelfat C, Young SN.Differences between males and females in rates of serotonin synthesis in human brain [J]..Proc Natl Acad Sci USA,1997,94 (10):5308-5313.
[14]Moret C, Briley M.The importance of norepinephrine in depression [J]. Neuropsychiatric Disease and Treatment,2011,7(Suppl):9-13.
[15]Isgor C, Watson SJ. Estrogen receptor a and β mRNA expressions by proliferating and differentiating cells in the adult rat dentategyrus and subventricular zone [J].Neuroscience,2005,134(3):847-56.
[16]Kronmuller KT, Pantel J, Kohler S, et al. Hippocampal volume and 2-year outcome in depression[J]. The British Journal of Psychiatry,2008,192(6):472-473.
[17]Videbech P. Ravnkilde B. Hippocampal volume and depression:a meta-analysis of MRI studies [J]. The American Journal of Psychiatry,2004,161(11):1957-1966.
[18]Shors TJ, Leuner B. Estrogen-mediated effects on depression and memory formation in females [J]. Journal of Affective Disorders,2003,74(1):85-96.
[19]. Conrad CD, Jackson JL, Wieczorek L, et al. Acute stress impairs spatial memory in male but not female rats:influence of estrous cycle [J]. Pharmacology Biochemistry and Behavior,2004,78(3): 569-579.
[20]索玉平,王宝迎,王丹青,等.坤宁安对围绝经期综合征大鼠下丘脑β-内啡肽及神经递质的影响[J].中国药物与临床,2006,6(1)：21-24.
[21]魏盛,侯金良,巢玉,等.郁怒诱发经前期综合征肝气郁证猕猴模型血清单胺类神经递质含量分析[J].中西医结合学报,2012,10(8)：925-931.
[22]石秦东,蔡云,邱根全,等.脑脉康颗粒剂对大脑中动脉缺血再灌注大鼠脑的保护作用[J].北京中医药大学学报,2002,25(3)：39-41.
[23]Volman V, Gerkin R C, Lau P M, et al. Calcium and synaptic dynamics underlying reverberatory activity in neuronal networks [J].Phys Biol,2007,4(2):91-103.
[24]Xu J, He L, Wu LG. Role of Ca2+ channels in short term synaptic plasticity [J]. CurrOpin Neurobiol, 2007,17(3):352-359.
[25]钟晓明,毛庆秋,黄真,等.金丝桃提取物对应激动物模型的抗抑郁作用及其机理研究[J].浙江中医药大学学报,2006,30(2)214-216.
[26]钊,晓明,毛庆秋,黄真,等.苏郁胶囊对慢性应激抑郁模型大鼠海马神经细胞凋亡的影响[J].中国现代应用药学杂志,2006,23(8)：733-736.
[27]Orihuela PA, Zuniga LM, Rios M, et al. Mating changes the subcellular distribution and the f unctionality of estrogen receptors in the rat oviduct[J].Reprod Biol Endocrinol,2009,30(7):132-139.
[28]Barnett P. Somatostatin and Somatostatin Receptor Physiology [J].Endocrine,2003,20(3):255-264.
[29]冯劫.四逆散对抑郁模型大鼠血浆NPY、 SP、 SS含量的影响[J].浙江中医杂志,2011,46(8)：566-567.
[30]White BC, Sullivan JM, DeGracia DJ, et al. Brain ischemia and reperfusion:molecular mechanisms of neuronal injury [J]. J Neurol Sci,2000, 1(179S1-2):1-33.
[31]Mutlu O, Ulak G, Laugeray A, et al. Effects of neuronal and inducible NOS inhibitor 1-[2-(trifluoromethyl) phenyl] imidazole (TRIM) in unpredictable chronic mild stress procedure in mice[J]. Pharmacol Biochem Behav,2009,92(1):82-87.
[32]Yun-Li Peng, Yu-Ning Liu, Lei Liu, et al. Inducible nitric oxide synthase is involved in the modulation of depressive behaviors induced by unpredictable chronic mild stress [J]. Journal of Neuroinflammation,2012,9(6):75-87.
[33]Duncko P, Kiss A, Skultetyova I, et al. Corticotropin releasing hormone mRNA levels in response to chronic mild stress rise in male but not in female rats while tyrosine hydroxylase mRNA decrease in both sexes[J].Psychoneur oendocrinology,2001,26(1):77-89.
[34]侯秀娟,唐启盛,李小黎.脑缺血后抑郁模型大鼠下丘脑酪氨酸羟化酶的表达及中药干预作用[J].中华中医药学刊,2008,26(8)：1692-1694.
[35]姚辉,肖红,张晓斌,等.抑郁症患者血浆白细胞介素与单胺代谢产物的研究[J].神经疾病与精神卫生,2003,3(1)：66-67.
[36]Seidel A, Arolt V, Hunstiger M, et al. Cytokine production and serum proteins in depression[J], Scand J Immunol,1995,41(6):534-538.
[37]Yasui T, Maegawa M, Tomita J, et al. Changes in serum cytokine concentrations during the menopausal transition[J]. Maturitas,2007,56(4):396-403.
[1]Kessler RC, McGonagle KA, Nelson CB, et al.Sex and depression in the National Comorbidity Survey. Ⅱ:Cohort effects[J]. J Affect Disord,1994,30(1):15-26.
[2]董胜莲,吴庆文,安子薇.女性更年期抑郁症相关因素分析.中国健康心理学杂志,2012,20(8)：1160-1162.
[3]宋美英,夏薇,黄民,等.雌激素对抑郁大鼠海马单胺类神经递质含量的影响及缰核在其中的作用[J].中国老年学杂志.2011,31(19)：3766-3768.
[4]姚迪,胡欣妍,苏兴国,等.舒肝解郁饮对抑郁大鼠脑组织单胺类神经递质及其代谢产物的影响[J].吉林大学学报(医学版),2007,33(4)：704-707.
[5]Smith LJ, HendersonJ A, AbellCW, et al. Effects of ovarian steroidsand raloxifene on proteins that synthesiz, transport, and degrade serotonin in the rapheregion of macaques[J]. Neuropsycho pharmacology,2004,29(11):2035-2045.
[6]BetheaCL, MirkesSJ, Michelson D. Effects of oral estrogen, raloxifene and arzoxifene on gene expression in serotonin neurons of macaques[J]. Psychoneuroendocrinology,2002,27(4):431-445.
[7]GundlahC, LuNZ.BetheaCL. Ovarian steroid regulation of monoamineoxidase-A and B mRNAs in the macaque dorsalraphe and hypothalamic nucle[J]. Psychopharmacology,2002,160(3):271-282.
[8]Rivera HM, Santollo J, Nikonova LV, et al. Estradiol increases the anorexia associated with increased 5-HT(2C) receptor activation in ovariectomized rats[J].Physiol Behav,2012,105(2):188-94.
[9]Chavez C, Hollaus M, Scarr E, et al. The effect of estrogen on dopamine and serotonin receptor and transporter levels in the brain:an autoradiography study[J].Brain Res,2010,19(1321):51-59.
[10]Hiroi R, Neumaier JF. Estrogen decreases 5-HT1B autoreceptor mRNA in selective subregion of rat dorsal raphe nucleus:inverse association between gene expression and anxiety behavior in the open field[J]. Neuroscience,2009,158(2):456-64.
[11]Mcewen B. Estrogen actions throughout the brain[J]. Recent Prog HormRes,2002,57(1):357-384.
[12]张吉强,姚青,蔡文琴.卵巢切除对小鼠基底前脑内雌激素α受体表达的影响[J].第三军医大学学报,2003,25(3)：261-263.
[13]GundlahC, LuNZ, MirkesSJ, et al. Estrogen receptor beta (ER(3) mRNA and protein in serotonin neurons of macaques[J].Brain ResMol Brain Res,2001,91 (1-2):14-22.
[14]Walf AA, Frye CA. Administration of estrogen receptor beta-specific selective estrogen receptor modulators to the hippocampus decrease anxiety and depressive behavior of ovariectomized rats[J].Pharmacol Biochem Behav,2007,86(2):407-414.
[15]Walf AA, Koonce CJ, Frye CA. Estradiol or diarylpropionitrile decrease anxiety-like behavior of wildtype, but not estrogen receptor beta knockout mice [J].Behavioral Neuroscience,2008,122(5): 974-981.
[16]Serova LI, Harris HA, Maharjan S, et al. Modulation of responses to stress by estradiol benzoate and selective estrogen receptor agonists[J]. J Endocrinol,2010,205(3):253-262.
[17]Le Saux M, Di Paolo T. Influence of oestrogenic compounds on monoamine transporters in rat striatum[J]. J Neuroendocrinol,2006,18(1):25-32.
[18]A1-Sweidi S, Morissette M, Di Paolo T. Effect of oestrogen receptors on brain NMDA receptors of 1-methy1-4-pheny1-1,2,3,6-tetrahydropyridine mice[J].2012,24(11):1375-1385.
[19]Maharjan S, Serova L, Sabban EL.Transcriptional regulation of tyrosine hydroxylase by estrogen: opposite effects with estrogen receptors alpha and beta and interactions with cyclic AMP[J].J Neurochem,2005,93(6):1502-1514.
[20]Soare.Soares for the treatment of depressive disorders in perimenopausal women:a double-blind, randomized, placebo controlled trial[J]. ArchGen Psychiatry,2001,58:529-534.
[21]罗阳,何国平.性激素及神经递质与产后抑郁症关系的Meta分析[J].中国妇幼保健,2007,22(8)：1020-1023.
[22]罗元恺.妇女更年期综合征的中医治疗[J].新中医,1992,24(1)：16-17.
[23]叶燕萍.更年期综合征辨证分型的研究[J].新中医,2000,32(9)：36-37.
[24]李汉明.辨证分型治疗更年期综合征75例[J].湖南中医学院学报,1995,15(2)：311.
[25]孙静.胥受天.从肝论治更年期综合征经验[J].江西中医药,2003,34(1)：9-10.
[26]袁立霞,刘刚.从肝论治妇女更年期综合征[J].山东中医杂志,2011,26(7)：439-440.
[27]张丽丽,姚秀英.绝经前后诸症从肝论治58例[J].新中医,2007,39(3)：65-66.
[28]杜巧琳,王小云.舒肝胶囊治疗更年期妇女抑郁症36例临床观察[J].新中医,2008,40(10)：61-62.
[29]唐凤.丹栀逍遥散临床应用[J].辽宁中医杂志,2006,33(10)：1344.
[30]谈勇,许小风,卢苏.绝经前后诸证心肾病机探析[J].南京中医药大学报,2001,17(6)：340.
[31]池雷.宁心敛阴汤治疗更年期综合征20例[J].河南中医,2009,29(12)：1201.
[32]李力,任婕,杜彩风,等.更年期综合征中医证候及证候要素分布特点的文献分析[J].中华中医药杂志,2008,23(3)：194-197.
[33]陈家旭,万霞,胡立胜.围绝经期综合征辨证分型的文献研究[J].中华中医药杂志,2006,21(11)：649-651.
[34]申春悌,张华强,朱雄华,等.400例更年期综合征临床证候辨证标准现场调查分析[J].中国中西医结合杂志,2004,24(6)：517-520.
[35]辛意,王天芳,杜彩凤,等.基于经验辨证的更年期综合征围绝经期和绝经后期常见证候及证候要素分布[J].中西医结合学报,2009,7(6)：522-526.
[36]汪涛,秦锋.逍遥散对产后抑郁大鼠海马中单胺类神经递质含量的影响[J].中西医结合学报,2010,8(11)：1075-1079.
[37]严亨秀,任昉,顾健.柴胡疏肝散对实验性肝郁证大鼠的影响[J].中药药理与临床,2006,22(6)：5-6.
[38]王桐生,谢鸣,张艳霞,等.肝郁模型大鼠行为学与脑单胺递质的变化及柴胡的干预作用[J].中华中医药杂志,2008,23(10)：934-937.