产后抑郁大鼠行为学与激素水平、单胺递质变化及补益心脾法干预机制研究
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摘要
目的：以产后抑郁模型动物为研究载体,采用具有补益心脾作用的参芪解郁方进行干预,从行为学、脑组织形态学、神经内分泌系统相关激素与受体、单胺递质及其代谢产物的变化等方面,探讨产后抑郁症(PPD)神经生物学发病机制以及中医补益心脾法的干预作用,明确参芪解郁方治疗PPD的作用途径,进一步完善补益心脾法在PPD治疗方面的理论学说。
方法：选用雌性SD大鼠,随机分为正常、假手术、模型、中药、西药组,每组均设处理后1、2、4、6w四个不同观察时点。模型、中药、西药组经去势手术后连续雌孕激素注射制备PPD模型,后分别予双蒸水、参芪解郁方、氟西汀灌胃干预。正常组给予等容积双蒸水,假手术组行假手术后予双蒸水灌胃。所有动物分别于1、2、4、6w不同时点予行为学测试。测试完毕后,行相关指标检测：①通过HE染色观察大鼠脑前额皮质、海马、下丘脑神经细胞损伤情况；②采用酶联免疫法和放射免疫法检测血清HPA、HPG轴激素水平；③采用免疫组化法测定脑前额皮质、海马、下丘脑ERα、β表达；④采用高效液相法测定脑前额皮质、海马单胺递质及其代谢产物含量。
结果：
1.一般情况观察
1.1外观与活动状态：模型大鼠精神、活动状态较正常、假手术组差,表现为皮毛晦暗,毛发梳理、寻觅探查等动作明显减少,自由活动度降低,于造模后1、2w时显著,4、6w时虽有好转,但仍较正常大鼠状态差。经药物干预后,中、西药组上述抑郁征象较模型组改善,6w时可基本恢复正常状态。
1.2体重：模型组各时点均较正常、假手术组降低(P<0.01,P<0.05)。中药组2、4、6w及西药组各时点均较模型组升高(P<0.01,P<0.05)。模型、中药、西药组体重均呈增加趋势,但模型组较中、西药组增长幅度明显缓慢。
2.行为学观察
2.1蔗糖水消耗：模型组各时点较正常、假手术组降低(P<0.01,P<0.05)。西药组2w及中、西药组4、6w较模型组升高(P<0.01)。模型组显示为逐渐下降规律,中、西药组则表现逐渐上升趋势。
2.2 OFT：模型组各时点水平与垂直得分均较正常、假手术组降低(P<0.01,P<0.05)；中、西药组除1w外余各时点水平得分与中药组1w及中、西药组2、4w垂直得分均较模型组升高(P<0.01,P<0.05)。模型、中药、西药组水平得分及西药组垂直得分均呈升高趋势,模型、中药组垂直得分显示降低后又逐渐升高规律。
2.3 FST：模型组各时点不动时间较正常、假手术组延长(P<0.01,P<0.05),各时点挣扎时间与2、4、6w游泳时间均较以上两组缩短(P<0.01)。中、西药组各时点不动时间较模型组缩短(P<0.01,P<0.05),二者各时点挣扎时间及2、4、6w游泳时间较模型组延长(P<0.01,P<0.05)。模型组不动时间呈增加后又减少趋势,挣扎时间呈稍减少后逐渐增加,此后又迅速减少趋势,游泳时间呈降低后又逐渐升高趋势；中、西药组不动时间呈逐渐减少趋势,挣扎时间表现为逐渐增加后又降低,游泳时间则表现逐渐升高趋势。
3.脑组织病理结构观察
正常、假手术组前额皮质、海马细胞数量多,排列整齐,胞浆饱满,核仁清晰；下丘脑细胞排列规律,形态正常。模型组上述三个部位病理损伤1、2w尤为显著,4、6w无明显恢复。前额皮质、海马细胞数量明显减少或呈萎缩状,间隙增大,排列疏松紊乱,细胞淡染,着色不匀,海马部分细胞亦出现空泡化及核固缩；下丘脑细胞肿胀明显,着色不均,部分细胞出现核固缩。中、西药组病理损伤1w较模型组有所减轻,2w改善明显,4、6w状况恢复基本接近正常水平。
4.神经内分泌系统相关激素测定
4.1 HPA轴激素：模型组各时点CRH、ACTH、Cor均较正常、假手术组升高(P<0.01,P<0.05)。西药组4、6w的CRH,除中、西药组1w及西药组4w外二者余各时点ACTH,中、西药组2、4、6w的Cor均较模型组降低(P<0.01,P<0.05)。中、西药组ACTH呈逐渐减少趋势；模型、中药、西药组Cor呈升高后又逐渐降低趋势,而模型组升高显著减低缓慢。
4.2 HPG轴激素：模型组各时点GnRH、FSH,1w的LH,2、4、6w的E2均较正常、假手术组降低(P<0.01,P<0.05)；2、4、6w的LH,1w的E2,各时点P均较以上两组升高(P<0.01,P<0.05)。西药组各时点GnRH、E2及2、4、6w的FSH、1w的LH,中药组2、4、6w的GnRH、FSH及各时点E2均较模型组升高(P<0.01,P<0.05)；中、西药组2、4、6w的LH及各时点P均较模型组降低(P<0.01,P<0.05)。模型、中药、西药组GnRH、FSH、LH均呈逐渐升高趋势,E2、P呈明显下降趋势,上述指标经中、西药物干预后可基本恢复正常水平。
5.ER表达变化
5.1 ERα：模型组2、4、6w前额皮质、海马、下丘脑表达较正常、假手术组减少(P<0.01,P<0.05),1w海马、下丘脑表达较以上两组增加(P<0.01,P<0.05)。中药组4、6w前额皮质,西药组2、4、6w前额皮质、下丘脑,中、西药组2、4、6w海马,中药组各时点下丘脑表达均较模型组增加(P<0.01,P<0.05)。模型、中药、西药组三个部位表达均呈下降趋势,且以模型组下降更为明显。
5.2 ERβ：模型组1w前额皮质,1、2w海马、下丘脑表达均较正常、假手术组增加(P<0.01,P<0.05),4、6w前额皮质、下丘脑及6w海马表达均较以上两组明显减少(P<0.01)。中、西药组6w前额皮质及4、6w海马,中药组4、6w及西药组2、4、6w下丘脑表达均较模型组增加(P<0.01,P<0.05)。各组三个部位表达变化与ERα相似。
6.单胺递质及其代谢产物检测
6.1单胺递质：模型组各时点前额皮质、海马5-HT、NE、DA均较正常、假手术组减少(P<0.01,P<0.05)。中药组2、4、6w及西药组各时点前额皮质,中、西药组各时点海马5-HT、NE均较模型组增加(P<0.01,P<0.05),中药组4、6w及西药组2、4、6w前额皮质,中药组1、6w及西药组1w海马DA均较模型组增加(P<0.01,P<0.05)。模型组前额皮质、海马5-HT,前额皮质NE、DA均呈逐渐下降趋势,海马NE呈明显下降后又轻度升高趋势,海马DA则表现为升高后逐渐下降规律。中药组前额皮质及中、西药组海马5-HT,西药组前额皮质及中药组海马NE均呈逐渐升高趋势；中药组前额皮质NE表现为稍下降后又逐渐上升规律；中、西药组海马DA则表现为下降趋势。
6.2单胺递质代谢产物：模型组1w前额皮质、海马5-HIAA较正常、假手术组增加(P<0.01,P<0.05),余各时点两部位5-HIAA及各时点两部位DOPAC含量均较以上两组减少(P<0.01,P<0.05)。中、西药组1w前额皮质、海马5-HIAA较模型组减少(P<0.01,P<0.05)；二者4、6w前额皮质5-HIAA,2、4、6w海马5-HIAA及前额皮质DOPAC均较模型组增加(P<0.01,P<0.05)。模型组前额皮质、海马5-HIAA均呈下降趋势,前额叶皮质DOPAC呈逐渐下降后又升高趋势。中、西药组前额皮质、海马5-HIAA则呈明显减少后又逐渐增加趋势；中药组海马DOPAC表现为升高后又逐渐下降规律。
结论：
1.采用去势后连续激素注射并突然停撤方法建立PPD动物模型,模型大鼠行为学方面可出现活动能力减弱、探索能力降低、面对绝望环境挣扎逃脱反应下降等改变,脑组织形态学方面亦有相应结构损伤发生。该模型在病因学上较好地模拟了PPD发病状态,且重复性好,可为深入探索PPD发病机制及新药研发等提供良好的研究平台。
2.具有补益心脾作用的参芪解郁方对PPD模型大鼠抑郁行为有明显调节作用,同时可促进脑组织病理损伤的恢复。与对照药物SSRIs氟西汀相比,二者在大鼠抑郁行为及脑组织病理损伤方面疗效相当,此为中医补益心脾法治疗PPD提供了理论依据,同时也为中药参芪解郁方的药效学研究奠定基础。
3.建立的PPD模型大鼠血清HPA、HPG轴激素平衡紊乱,与E特异性结合ERα、β功能低下,脑内单胺递质及其代谢产物含量降低,上述系统的改变可能是PPD疾病发生发展的重要神经生物学物质基础。
4.中药参芪解郁方对模型大鼠神经内分泌系统相关激素水平、脑内ER表达及单胺递质与代谢产物含量具有明显改善作用,对上述各独立系统的平衡调节以及对各系统间的整体稳态调控可能是其效应发挥的主要途径。
Objective:In this study, Shenqi Jieyu formula with the effect of tonifying heart and spleen was used to treat postpartum depression (PPD) model animals, and changes in behavior, pathomorphology of brain, related hormones and receptors in neuroendocrine system, monoamine neurotransmitters and their metabolites were tested. It was expected to explore the pathogenesis of neurobiology of PPD and intervening mechanism of Chinese medical therapeutic method of tonifying heart and spleen. And it was also looked forward to explicit the action pathway of Shenqi Jieyu formula and further improve the theory of tonifying heart and spleen treatment for PPD.
Methods:Female SD rats were randomly divided into groups of normal (N), sham operation (SO), model (M), Chinese Medicine (CM), western medicine (WM). All the rats were observed at 1,2,4,6w respectively after preparation. M, CM and WM received continuous estrogen and progesterone injection after castration, and there was no intervention for N and sham operation for SO. Then rats were given intragastric administration of distilled water for N, SO and M, Shenqi Jieyu formula for CM, fluoxetine for WM. All of the rats were observed with behaviors tests containing sucrose water consumption (SWC), open-field test (OFT) and forced swimming test (FST) at 1,2,4,6w respectively. After such tests, correlated indexes were detected as follow:①observing nerve cell damages in prefrontal cortex, hippocampus, hypothalamus in rats by hematoxylin and eosin stain;②checking serum hormone levels of HPA and HPG axis by ELISA and RIA;③measuring expressions of ER alpha and beta in prefrontal cortex, hippocampus, hypothalamus in brain through immunohistochemistry;④surveying contents of monoamine neurotransmitters and their metabolites in prefrontal cortex, hippocampus with HPLC.
Results:
1. Observation of general conditions
1.1 Appearance and active state:The spirit and active state of M were worse than N and SO. M manifested such symptoms as dull fur, significantly reduced action of hair combing, less explorement and decreased free movement. These symptoms above worst manifested at 1, 2w and partly improved at 4,6w. However, the main status of M was still worse than N at 6w. After drug intervention, the depressive signs above in CM and WM were improved and nearly returned to normal status at 6w.
1.2 Weight:Weight of rats in M at each time point were lower than N and SO (P<0.01, P<0.05). Weight in CM at 2,4,6w and in WM at each time point were higher than M (P<0.01, P<0.05). The condition of rats' weight in M, CM and WM all showed an increasing trend, however, the growing speed of weight in M was obviously slower than CM and WM.
2. Observation of praxiology
2.1 SWC:SWC in M was lower than N and SO (P<0.01, P<0.05). SWC in WM at 2w and in CM, WM at 4,6w were higher than M (P<0.01). The volume of SWC in M displayed a gradually declined regularity, however, a gradual upward trend in CM and WM.
2.2 OFT:Horizontal and vertical scores in M at each time point were lower than N and SO (P<0.01, P<0.05). Horizontal scores in CM, WM at 2,4,6w as well as vertical scores in CM at 1w and in CM, WM at 2,4w were higher than M (P<0.01, P<0.05). Horizontal scores in M, CM, WM and vertical scores in WM all showed a rising trend, while vertical scores in M and CM manifested a formerly declined and then gradually increased trend.
2.3 FST:The immobility time in M at each time point were longer than N and SO (P<0.01, P<0.05), struggling time in M at each time point as well as swimming time at 2,4, 6w were shorter than two groups above (P<0.01). Compared with M, the immobility time in CM and WM at each time point reduced (P<0.01, P<0.05), both groups' struggling time at each time point as well as swimming time at 2,4,6w increased (P<0.01, P<0.05). There was a formerly increased and then reduced trend for immobility time in M, and a decreased tendency forward, by following, with gradually increased then rapidly grew downwards pattern for struggling time, meanwhile, a decreased and then gradually increased trend for swimming time. In CM and WM, the regularity manifested gradually decreased for immobility time, gradually increased and then decreased for struggling time, gradually increased for swimming time.
3. Observation for pathological structure of brain
Cell counts of prefrontal cortex and hippocampus in N and SO were abundant, with well-arranged order, well-stacked hyalomitome and distincted nucleolus. Cells in hypothalamus lined in regularity, with normal shape as well. The pathological changes at three places above in M damaged extremely at 1,2w, with unobvious recovery at 4,6w. Cell counts of prefrontal cortex and hippocampus reduced remarkably and some of these were atrophy, with large gap, arranged in loose disorder, cell lightly stained and asymmetrically colored, additionally, cells in hippocampus also partly appeared vacuolization and pyknosis. Cells in hypothalamus swelled obviously, with nonuniformly colored, some of cells also appeared pyknosis. Compared with M, the damaging status of CM and WM at 1w relieved slightly, ameliorated obviously at 2w, with closely recuperation to normal status at 4,6w.
4. Tests for related hormones of neuroendocrine system
4.1 Hormones of HPA axis:Compared with N and SO, CRH, ACTH and Cor in M at each time point were higher (P<0.01, P<0.05). CRH in WM at 4,6w as well as ACTH in CM at 2,4,6w and in WM at 2,6w, additionally, Cor in CM, WM at 2,4,6w were all lower than M (P<0.01, P<0.05). ACTH in CM and WM displayed a gradually decreased tendency. In addition, Cor in M, CM and WM showed a gradually raised and then downgraded trend, however, Cor in M manifested the regularity of rising obviously and decreasing slowly.
4.2 Hormones of HPG axis:M's GnRH, FSH at each time point as well as LH at 1w, E2 at 2,4,6w were all lower than N and SO (P<0.01, P<0.05), while LH at 2,4,6w as well as E2 at 1w, P at each time point were higher than two groups above (P<0.01, P<0.05). Furthermore, WM's GnRH, E2 at each time point and FSH at 2,4,6w, LH at 1w as well as CM's GnRH, FSH at 2,4,6w, E2 at each time point were all higher than M (P<0.01, P<0.05), while LH at 2, 4.6w and P at each time point in CM and WM were totally lower than M (P<0.01, P<0.05). GnRH, FSH and LH in M, CM and WM all showed the gradually increased trend, while E2 and P appeared obviously decreased regularity. These hormones mentioned above could basically recover to normal level through drug intervention.
5. Variation of ER expressions
5.1 ER alpha:Expressions in prefrontal cortex, hippocampus and hypothalamus in M at 2,4,6w were less than N and SO (P<0.01, P<0.05), while more than the two groups above at 1w (P<0.01, P<0.05). Compared with M, expressions in prefrontal cortex in CM at 4,6w, prefrontal cortex and hypothalamus in WM at 2,4,6w, hippocampus in CM, WM at 2,4,6w, hypothalamus in CM at each time point all increased (P<0.01, P<0.05). Expressions of the three locations above in M, CM and WM all showed a downward tendency, furthermore, with a remarkable downtrend in M.
5.2 ER beta:Both expressions in prefrontal cortex at 1w and in hippocampus, hypothalamus at 1,2w in M were all more than N and SO (P<0.01, P<0.05), however, there had a significant reduction in prefrontal cortex and hypothalamus at 4,6w as well as in hippocasmpus at 6w (P<0.01). Compared with M, expressions in prefrontal cortex at 6w and hippocampus at 4,6w in CM, WM as well as hypothalamus at 4,6w in CM and 2,4,6w in WM all increased (P<0.01, P<0.05). Changes for expressions of ER beta at three locations above in each group made a similar regularity with ER alpha.
6. Survey of monoamine neurotransmitters and their metabolites
6.1 Monoamine neurotransmitters:Contents of 5-HT, NE, DA in prefrontal cortex and hippocampus in M at each time point were all lower than N and SO (P<0.01, P<0.05). Compared with M, contents of 5-HT, NE in prefrontal cortex in CM at 2,4,6w and WM at each time point as well as in hippocampus in CM, WM at each time point all increased (P<0.01, P<0.05), with the same situation for contents of DA in prefrontal cortex in CM at 4, 6w, WM at 2,4,6w as well as in hippocampus in CM at 1,6w, WM at 1w (P<0.01, P<0.05). 5-HT in prefrontal cortex, hippocampus as well as NE, DA in prefrontal cortex in M all showed a downward trend, while NE in hippocampus displayed a significantly decreased and then slightly increased regularity, in addition, DA in hippocampus manifested the opposite tendency, making a status of increasing by degrees and then falling down.5-HT in prefrontal cortex in CM and in hippocampus in CM, WM as well as NE in prefrontal cortex in WM and in hippocampus in CM all manifested a gradually increased trend, while NE in prefrontal cortex in CM displayed a formerly slightly descended then increased situation, furthermore, DA in hippocampus in CM and WM showed a trend of decreasing.
6.2 Metabolites of monoamine neurotransmitters:Contents of 5-HIAA in prefrontal cortex, hippocampus in M at 1w were more than N and SO (P<0.O1, P<0.05), while 5-HIAA for other time points and DOPAC at each time point in both two places were lower than the two groups above (P<0.01, P<0.05). Compared with M, contents of 5-HIAA in prefrontal cortex, hippocampus in CM and WM at 1w reduced (P<0.01, P<0.05), however, with a reversed condition for 5-HIAA in prefrontal cortex at 4,6w as well as both 5-HIAA in hippocampus and DOPAC in prefrontal cortex at 2,4,6w (P<0.01, P<0.05).5-HIAA in prefrontal cortex, hippocampus in M all displayed a decreased trend, while with a first gradually descended and then increased regularity for DOPAC in prefrontal cortex. Additionally,5-HIAA in prefrontal cortex and hippocampus in CM, WM showed an obviously grew downwards then gradually increased situation, whereas DOPAC in hippocampus in CM illustrated an opposite trend of formerly increasing and then descending.
Conclusions:
1. Through suddenly hormones withdraw after continuous injection of estrogen and progesterone to build PPD animal model, PPD rats manifested a series of behavioral changes: active and exploring movements attenuated, interests reduced, the response of struggle to escape out of the despair environment declined. Furthermore, the histomorphology of brain also had the corresponding structural damages. This model, which possesed a well repetitiveness, successfully simulated the status of PPD in etiology, meanwhile, it might also provide a good platform for further exploration for pathogenesis as well as new drug discovery in PPD.
2. Shenqi Jieyu formula with the effect of tonifying heart and spleen could not only obviously improve depressive behaviors in PPD rats, but also promote the recovery of pathological injury of brain tissue. Compared with the control drug SSRIs fluoxetine, both interventions had coincidental curative effects for depressive behaviors and pathological harm of brain in model rats, which might provide a theoretical groundwork for tonifying heart and spleen treatment for PPD and also establish the basis for pharmacodynamics study with Shenqi Jieyu formula.
3. There existed balanced disorders in serum hormones of HP A and HPG axises, as well as dysfunction in E-specific combinationed ER alpha and beta, furthermore, content reduction of monoamine neurotransmitters and their metabolites in brain in PPD model rats. These alteration mentioned above in each system might be the important neurobiological material base during the occurrence and development in PPD.
4. Shenqi Jieyu formula could remarkably improve related hormones'level of the neuroendocrine system and promote ER expressions in brain, additionally, increase the contents of monoamine neurotransmitters and their metabolites. Adjusting each of these independent systems as well as regulating various systems as a whole steady state may the major route for therapeutic effects educing and also play a very important role in the whole process.
方法：选用雌性SD大鼠,随机分为正常、假手术、模型、中药、西药组,每组均设处理后1、2、4、6w四个不同观察时点。模型、中药、西药组经去势手术后连续雌孕激素注射制备PPD模型,后分别予双蒸水、参芪解郁方、氟西汀灌胃干预。正常组给予等容积双蒸水,假手术组行假手术后予双蒸水灌胃。所有动物分别于1、2、4、6w不同时点予行为学测试。测试完毕后,行相关指标检测：①通过HE染色观察大鼠脑前额皮质、海马、下丘脑神经细胞损伤情况；②采用酶联免疫法和放射免疫法检测血清HPA、HPG轴激素水平；③采用免疫组化法测定脑前额皮质、海马、下丘脑ERα、β表达；④采用高效液相法测定脑前额皮质、海马单胺递质及其代谢产物含量。
结果：
1.一般情况观察
1.1外观与活动状态：模型大鼠精神、活动状态较正常、假手术组差,表现为皮毛晦暗,毛发梳理、寻觅探查等动作明显减少,自由活动度降低,于造模后1、2w时显著,4、6w时虽有好转,但仍较正常大鼠状态差。经药物干预后,中、西药组上述抑郁征象较模型组改善,6w时可基本恢复正常状态。
1.2体重：模型组各时点均较正常、假手术组降低(P<0.01,P<0.05)。中药组2、4、6w及西药组各时点均较模型组升高(P<0.01,P<0.05)。模型、中药、西药组体重均呈增加趋势,但模型组较中、西药组增长幅度明显缓慢。
2.行为学观察
2.1蔗糖水消耗：模型组各时点较正常、假手术组降低(P<0.01,P<0.05)。西药组2w及中、西药组4、6w较模型组升高(P<0.01)。模型组显示为逐渐下降规律,中、西药组则表现逐渐上升趋势。
2.2 OFT：模型组各时点水平与垂直得分均较正常、假手术组降低(P<0.01,P<0.05)；中、西药组除1w外余各时点水平得分与中药组1w及中、西药组2、4w垂直得分均较模型组升高(P<0.01,P<0.05)。模型、中药、西药组水平得分及西药组垂直得分均呈升高趋势,模型、中药组垂直得分显示降低后又逐渐升高规律。
2.3 FST：模型组各时点不动时间较正常、假手术组延长(P<0.01,P<0.05),各时点挣扎时间与2、4、6w游泳时间均较以上两组缩短(P<0.01)。中、西药组各时点不动时间较模型组缩短(P<0.01,P<0.05),二者各时点挣扎时间及2、4、6w游泳时间较模型组延长(P<0.01,P<0.05)。模型组不动时间呈增加后又减少趋势,挣扎时间呈稍减少后逐渐增加,此后又迅速减少趋势,游泳时间呈降低后又逐渐升高趋势；中、西药组不动时间呈逐渐减少趋势,挣扎时间表现为逐渐增加后又降低,游泳时间则表现逐渐升高趋势。
3.脑组织病理结构观察
正常、假手术组前额皮质、海马细胞数量多,排列整齐,胞浆饱满,核仁清晰；下丘脑细胞排列规律,形态正常。模型组上述三个部位病理损伤1、2w尤为显著,4、6w无明显恢复。前额皮质、海马细胞数量明显减少或呈萎缩状,间隙增大,排列疏松紊乱,细胞淡染,着色不匀,海马部分细胞亦出现空泡化及核固缩；下丘脑细胞肿胀明显,着色不均,部分细胞出现核固缩。中、西药组病理损伤1w较模型组有所减轻,2w改善明显,4、6w状况恢复基本接近正常水平。
4.神经内分泌系统相关激素测定
4.1 HPA轴激素：模型组各时点CRH、ACTH、Cor均较正常、假手术组升高(P<0.01,P<0.05)。西药组4、6w的CRH,除中、西药组1w及西药组4w外二者余各时点ACTH,中、西药组2、4、6w的Cor均较模型组降低(P<0.01,P<0.05)。中、西药组ACTH呈逐渐减少趋势；模型、中药、西药组Cor呈升高后又逐渐降低趋势,而模型组升高显著减低缓慢。
4.2 HPG轴激素：模型组各时点GnRH、FSH,1w的LH,2、4、6w的E2均较正常、假手术组降低(P<0.01,P<0.05)；2、4、6w的LH,1w的E2,各时点P均较以上两组升高(P<0.01,P<0.05)。西药组各时点GnRH、E2及2、4、6w的FSH、1w的LH,中药组2、4、6w的GnRH、FSH及各时点E2均较模型组升高(P<0.01,P<0.05)；中、西药组2、4、6w的LH及各时点P均较模型组降低(P<0.01,P<0.05)。模型、中药、西药组GnRH、FSH、LH均呈逐渐升高趋势,E2、P呈明显下降趋势,上述指标经中、西药物干预后可基本恢复正常水平。
5.ER表达变化
5.1 ERα：模型组2、4、6w前额皮质、海马、下丘脑表达较正常、假手术组减少(P<0.01,P<0.05),1w海马、下丘脑表达较以上两组增加(P<0.01,P<0.05)。中药组4、6w前额皮质,西药组2、4、6w前额皮质、下丘脑,中、西药组2、4、6w海马,中药组各时点下丘脑表达均较模型组增加(P<0.01,P<0.05)。模型、中药、西药组三个部位表达均呈下降趋势,且以模型组下降更为明显。
5.2 ERβ：模型组1w前额皮质,1、2w海马、下丘脑表达均较正常、假手术组增加(P<0.01,P<0.05),4、6w前额皮质、下丘脑及6w海马表达均较以上两组明显减少(P<0.01)。中、西药组6w前额皮质及4、6w海马,中药组4、6w及西药组2、4、6w下丘脑表达均较模型组增加(P<0.01,P<0.05)。各组三个部位表达变化与ERα相似。
6.单胺递质及其代谢产物检测
6.1单胺递质：模型组各时点前额皮质、海马5-HT、NE、DA均较正常、假手术组减少(P<0.01,P<0.05)。中药组2、4、6w及西药组各时点前额皮质,中、西药组各时点海马5-HT、NE均较模型组增加(P<0.01,P<0.05),中药组4、6w及西药组2、4、6w前额皮质,中药组1、6w及西药组1w海马DA均较模型组增加(P<0.01,P<0.05)。模型组前额皮质、海马5-HT,前额皮质NE、DA均呈逐渐下降趋势,海马NE呈明显下降后又轻度升高趋势,海马DA则表现为升高后逐渐下降规律。中药组前额皮质及中、西药组海马5-HT,西药组前额皮质及中药组海马NE均呈逐渐升高趋势；中药组前额皮质NE表现为稍下降后又逐渐上升规律；中、西药组海马DA则表现为下降趋势。
6.2单胺递质代谢产物：模型组1w前额皮质、海马5-HIAA较正常、假手术组增加(P<0.01,P<0.05),余各时点两部位5-HIAA及各时点两部位DOPAC含量均较以上两组减少(P<0.01,P<0.05)。中、西药组1w前额皮质、海马5-HIAA较模型组减少(P<0.01,P<0.05)；二者4、6w前额皮质5-HIAA,2、4、6w海马5-HIAA及前额皮质DOPAC均较模型组增加(P<0.01,P<0.05)。模型组前额皮质、海马5-HIAA均呈下降趋势,前额叶皮质DOPAC呈逐渐下降后又升高趋势。中、西药组前额皮质、海马5-HIAA则呈明显减少后又逐渐增加趋势；中药组海马DOPAC表现为升高后又逐渐下降规律。
结论：
1.采用去势后连续激素注射并突然停撤方法建立PPD动物模型,模型大鼠行为学方面可出现活动能力减弱、探索能力降低、面对绝望环境挣扎逃脱反应下降等改变,脑组织形态学方面亦有相应结构损伤发生。该模型在病因学上较好地模拟了PPD发病状态,且重复性好,可为深入探索PPD发病机制及新药研发等提供良好的研究平台。
2.具有补益心脾作用的参芪解郁方对PPD模型大鼠抑郁行为有明显调节作用,同时可促进脑组织病理损伤的恢复。与对照药物SSRIs氟西汀相比,二者在大鼠抑郁行为及脑组织病理损伤方面疗效相当,此为中医补益心脾法治疗PPD提供了理论依据,同时也为中药参芪解郁方的药效学研究奠定基础。
3.建立的PPD模型大鼠血清HPA、HPG轴激素平衡紊乱,与E特异性结合ERα、β功能低下,脑内单胺递质及其代谢产物含量降低,上述系统的改变可能是PPD疾病发生发展的重要神经生物学物质基础。
4.中药参芪解郁方对模型大鼠神经内分泌系统相关激素水平、脑内ER表达及单胺递质与代谢产物含量具有明显改善作用,对上述各独立系统的平衡调节以及对各系统间的整体稳态调控可能是其效应发挥的主要途径。
Objective:In this study, Shenqi Jieyu formula with the effect of tonifying heart and spleen was used to treat postpartum depression (PPD) model animals, and changes in behavior, pathomorphology of brain, related hormones and receptors in neuroendocrine system, monoamine neurotransmitters and their metabolites were tested. It was expected to explore the pathogenesis of neurobiology of PPD and intervening mechanism of Chinese medical therapeutic method of tonifying heart and spleen. And it was also looked forward to explicit the action pathway of Shenqi Jieyu formula and further improve the theory of tonifying heart and spleen treatment for PPD.
Methods:Female SD rats were randomly divided into groups of normal (N), sham operation (SO), model (M), Chinese Medicine (CM), western medicine (WM). All the rats were observed at 1,2,4,6w respectively after preparation. M, CM and WM received continuous estrogen and progesterone injection after castration, and there was no intervention for N and sham operation for SO. Then rats were given intragastric administration of distilled water for N, SO and M, Shenqi Jieyu formula for CM, fluoxetine for WM. All of the rats were observed with behaviors tests containing sucrose water consumption (SWC), open-field test (OFT) and forced swimming test (FST) at 1,2,4,6w respectively. After such tests, correlated indexes were detected as follow:①observing nerve cell damages in prefrontal cortex, hippocampus, hypothalamus in rats by hematoxylin and eosin stain;②checking serum hormone levels of HPA and HPG axis by ELISA and RIA;③measuring expressions of ER alpha and beta in prefrontal cortex, hippocampus, hypothalamus in brain through immunohistochemistry;④surveying contents of monoamine neurotransmitters and their metabolites in prefrontal cortex, hippocampus with HPLC.
Results:
1. Observation of general conditions
1.1 Appearance and active state:The spirit and active state of M were worse than N and SO. M manifested such symptoms as dull fur, significantly reduced action of hair combing, less explorement and decreased free movement. These symptoms above worst manifested at 1, 2w and partly improved at 4,6w. However, the main status of M was still worse than N at 6w. After drug intervention, the depressive signs above in CM and WM were improved and nearly returned to normal status at 6w.
1.2 Weight:Weight of rats in M at each time point were lower than N and SO (P<0.01, P<0.05). Weight in CM at 2,4,6w and in WM at each time point were higher than M (P<0.01, P<0.05). The condition of rats' weight in M, CM and WM all showed an increasing trend, however, the growing speed of weight in M was obviously slower than CM and WM.
2. Observation of praxiology
2.1 SWC:SWC in M was lower than N and SO (P<0.01, P<0.05). SWC in WM at 2w and in CM, WM at 4,6w were higher than M (P<0.01). The volume of SWC in M displayed a gradually declined regularity, however, a gradual upward trend in CM and WM.
2.2 OFT:Horizontal and vertical scores in M at each time point were lower than N and SO (P<0.01, P<0.05). Horizontal scores in CM, WM at 2,4,6w as well as vertical scores in CM at 1w and in CM, WM at 2,4w were higher than M (P<0.01, P<0.05). Horizontal scores in M, CM, WM and vertical scores in WM all showed a rising trend, while vertical scores in M and CM manifested a formerly declined and then gradually increased trend.
2.3 FST:The immobility time in M at each time point were longer than N and SO (P<0.01, P<0.05), struggling time in M at each time point as well as swimming time at 2,4, 6w were shorter than two groups above (P<0.01). Compared with M, the immobility time in CM and WM at each time point reduced (P<0.01, P<0.05), both groups' struggling time at each time point as well as swimming time at 2,4,6w increased (P<0.01, P<0.05). There was a formerly increased and then reduced trend for immobility time in M, and a decreased tendency forward, by following, with gradually increased then rapidly grew downwards pattern for struggling time, meanwhile, a decreased and then gradually increased trend for swimming time. In CM and WM, the regularity manifested gradually decreased for immobility time, gradually increased and then decreased for struggling time, gradually increased for swimming time.
3. Observation for pathological structure of brain
Cell counts of prefrontal cortex and hippocampus in N and SO were abundant, with well-arranged order, well-stacked hyalomitome and distincted nucleolus. Cells in hypothalamus lined in regularity, with normal shape as well. The pathological changes at three places above in M damaged extremely at 1,2w, with unobvious recovery at 4,6w. Cell counts of prefrontal cortex and hippocampus reduced remarkably and some of these were atrophy, with large gap, arranged in loose disorder, cell lightly stained and asymmetrically colored, additionally, cells in hippocampus also partly appeared vacuolization and pyknosis. Cells in hypothalamus swelled obviously, with nonuniformly colored, some of cells also appeared pyknosis. Compared with M, the damaging status of CM and WM at 1w relieved slightly, ameliorated obviously at 2w, with closely recuperation to normal status at 4,6w.
4. Tests for related hormones of neuroendocrine system
4.1 Hormones of HPA axis:Compared with N and SO, CRH, ACTH and Cor in M at each time point were higher (P<0.01, P<0.05). CRH in WM at 4,6w as well as ACTH in CM at 2,4,6w and in WM at 2,6w, additionally, Cor in CM, WM at 2,4,6w were all lower than M (P<0.01, P<0.05). ACTH in CM and WM displayed a gradually decreased tendency. In addition, Cor in M, CM and WM showed a gradually raised and then downgraded trend, however, Cor in M manifested the regularity of rising obviously and decreasing slowly.
4.2 Hormones of HPG axis:M's GnRH, FSH at each time point as well as LH at 1w, E2 at 2,4,6w were all lower than N and SO (P<0.01, P<0.05), while LH at 2,4,6w as well as E2 at 1w, P at each time point were higher than two groups above (P<0.01, P<0.05). Furthermore, WM's GnRH, E2 at each time point and FSH at 2,4,6w, LH at 1w as well as CM's GnRH, FSH at 2,4,6w, E2 at each time point were all higher than M (P<0.01, P<0.05), while LH at 2, 4.6w and P at each time point in CM and WM were totally lower than M (P<0.01, P<0.05). GnRH, FSH and LH in M, CM and WM all showed the gradually increased trend, while E2 and P appeared obviously decreased regularity. These hormones mentioned above could basically recover to normal level through drug intervention.
5. Variation of ER expressions
5.1 ER alpha:Expressions in prefrontal cortex, hippocampus and hypothalamus in M at 2,4,6w were less than N and SO (P<0.01, P<0.05), while more than the two groups above at 1w (P<0.01, P<0.05). Compared with M, expressions in prefrontal cortex in CM at 4,6w, prefrontal cortex and hypothalamus in WM at 2,4,6w, hippocampus in CM, WM at 2,4,6w, hypothalamus in CM at each time point all increased (P<0.01, P<0.05). Expressions of the three locations above in M, CM and WM all showed a downward tendency, furthermore, with a remarkable downtrend in M.
5.2 ER beta:Both expressions in prefrontal cortex at 1w and in hippocampus, hypothalamus at 1,2w in M were all more than N and SO (P<0.01, P<0.05), however, there had a significant reduction in prefrontal cortex and hypothalamus at 4,6w as well as in hippocasmpus at 6w (P<0.01). Compared with M, expressions in prefrontal cortex at 6w and hippocampus at 4,6w in CM, WM as well as hypothalamus at 4,6w in CM and 2,4,6w in WM all increased (P<0.01, P<0.05). Changes for expressions of ER beta at three locations above in each group made a similar regularity with ER alpha.
6. Survey of monoamine neurotransmitters and their metabolites
6.1 Monoamine neurotransmitters:Contents of 5-HT, NE, DA in prefrontal cortex and hippocampus in M at each time point were all lower than N and SO (P<0.01, P<0.05). Compared with M, contents of 5-HT, NE in prefrontal cortex in CM at 2,4,6w and WM at each time point as well as in hippocampus in CM, WM at each time point all increased (P<0.01, P<0.05), with the same situation for contents of DA in prefrontal cortex in CM at 4, 6w, WM at 2,4,6w as well as in hippocampus in CM at 1,6w, WM at 1w (P<0.01, P<0.05). 5-HT in prefrontal cortex, hippocampus as well as NE, DA in prefrontal cortex in M all showed a downward trend, while NE in hippocampus displayed a significantly decreased and then slightly increased regularity, in addition, DA in hippocampus manifested the opposite tendency, making a status of increasing by degrees and then falling down.5-HT in prefrontal cortex in CM and in hippocampus in CM, WM as well as NE in prefrontal cortex in WM and in hippocampus in CM all manifested a gradually increased trend, while NE in prefrontal cortex in CM displayed a formerly slightly descended then increased situation, furthermore, DA in hippocampus in CM and WM showed a trend of decreasing.
6.2 Metabolites of monoamine neurotransmitters:Contents of 5-HIAA in prefrontal cortex, hippocampus in M at 1w were more than N and SO (P<0.O1, P<0.05), while 5-HIAA for other time points and DOPAC at each time point in both two places were lower than the two groups above (P<0.01, P<0.05). Compared with M, contents of 5-HIAA in prefrontal cortex, hippocampus in CM and WM at 1w reduced (P<0.01, P<0.05), however, with a reversed condition for 5-HIAA in prefrontal cortex at 4,6w as well as both 5-HIAA in hippocampus and DOPAC in prefrontal cortex at 2,4,6w (P<0.01, P<0.05).5-HIAA in prefrontal cortex, hippocampus in M all displayed a decreased trend, while with a first gradually descended and then increased regularity for DOPAC in prefrontal cortex. Additionally,5-HIAA in prefrontal cortex and hippocampus in CM, WM showed an obviously grew downwards then gradually increased situation, whereas DOPAC in hippocampus in CM illustrated an opposite trend of formerly increasing and then descending.
Conclusions:
1. Through suddenly hormones withdraw after continuous injection of estrogen and progesterone to build PPD animal model, PPD rats manifested a series of behavioral changes: active and exploring movements attenuated, interests reduced, the response of struggle to escape out of the despair environment declined. Furthermore, the histomorphology of brain also had the corresponding structural damages. This model, which possesed a well repetitiveness, successfully simulated the status of PPD in etiology, meanwhile, it might also provide a good platform for further exploration for pathogenesis as well as new drug discovery in PPD.
2. Shenqi Jieyu formula with the effect of tonifying heart and spleen could not only obviously improve depressive behaviors in PPD rats, but also promote the recovery of pathological injury of brain tissue. Compared with the control drug SSRIs fluoxetine, both interventions had coincidental curative effects for depressive behaviors and pathological harm of brain in model rats, which might provide a theoretical groundwork for tonifying heart and spleen treatment for PPD and also establish the basis for pharmacodynamics study with Shenqi Jieyu formula.
3. There existed balanced disorders in serum hormones of HP A and HPG axises, as well as dysfunction in E-specific combinationed ER alpha and beta, furthermore, content reduction of monoamine neurotransmitters and their metabolites in brain in PPD model rats. These alteration mentioned above in each system might be the important neurobiological material base during the occurrence and development in PPD.
4. Shenqi Jieyu formula could remarkably improve related hormones'level of the neuroendocrine system and promote ER expressions in brain, additionally, increase the contents of monoamine neurotransmitters and their metabolites. Adjusting each of these independent systems as well as regulating various systems as a whole steady state may the major route for therapeutic effects educing and also play a very important role in the whole process.
引文
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