经前舒颗粒对PMS肝气郁证模型大鼠不同脑区雌激素受体表达的影响
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摘要
目的:制备经前期综合征(premenstrual syndrome,PMS)肝气郁证大鼠模型,研究该模型的评价指标;检测不同脑区内雌激素受体的表达,探讨PMS肝气郁证发病的微观机制,并探究中药复方制剂经前舒颗粒对PMS肝气郁证的干预作用。
方法:采用束缚法、居住-入侵法制备PMS肝气郁证大鼠模型,以调肝方药经前舒颗粒进行药物干预。采用细胞涂片法观察大鼠阴道细胞形态,确定动情周期:接收期与非接收期。以旷场实验和悬尾实验两种行为学方法评价模型。造模后,将各组大鼠断头处死,迅速取出额叶皮层、海马、下丘脑、边缘叶这四个脑区。提取脑组织的总蛋白。用蛋白质印迹技术(western blot,WB)检测各脑区雌激素受体α与β(ERα、ERβ)两种受体在不同脑区的表达水平。
结果:与空白组相比,居住-入侵造模组(居住组)及居住-入侵造模给药组(居住给药组)大鼠体重变化显著(P<0.05,P<0.01)。行为学检测表明:在接收期,与空白组大鼠相比,束缚组和居住组大鼠悬尾不动时间显著增加(P<0.01);而与两造模组相比,相应的两造模给药组大鼠悬尾不动时间均显著减少(P<0.01)。在非接受期,与空白组大鼠相比,束缚组和居住组大鼠旷场实验的水平得分、垂直得分及总分均显著减少,但悬尾不动时间显著增加(P<0.01);与两造模组相比,相应的两造模给药组大鼠旷场水平得分、垂直得分及总分均显著增加,但悬尾不动时间均显著减少(P<0.01)。蛋白印迹实验表明,与空白组大鼠相比,束缚组大鼠额叶皮层、海马、下丘脑、边缘叶ERα表达显著升高(P<0.01);居住组大鼠边缘叶与下丘脑ERα表达显著升高(P<0.01),皮层表达量亦显著升高(P<0.05);束缚组与居住组各指标均无显著性差异;与模型组比较,给药组各脑区的ERα表达水平不同程度的降低。与空白组大鼠相比,束缚组大鼠额叶大脑皮层、海马、下丘脑、边缘叶ERβ表达显著升高(P<0.01);居住组大鼠皮层与海马ERβ表达显著升高(P<0.01),在下丘脑、边缘叶的表达亦升高(P<0.05);束缚组与居住组各指标均无差异;与模型组比较,给药组各脑区的ERβ表达水平不同程度的降低。
结论:用居住-入侵法可有效制备PMS肝气郁证大鼠模型,旷场实验可有效评价该模型,悬尾实验不适宜用来评价PMS肝气郁证大鼠模型。大鼠不同脑区的ERα与ERβ表达与PMS肝气郁证关系密切,额叶皮层、海马、下丘脑、边缘叶中的ERα与ERβ表达变化可能是导致PMS肝气郁证的重要病因之一。中药经前舒颗粒对该病证模型大鼠有不同程度的调节作用。
Objectives:Our aims of this study are to introduce model rats with Premenstrual Syndrome liver-qi depression, research evaluation method of the model rats; explore expression levels of estrogen receptor in different brain regions; seek for parts of the mechanisms of Premenstrual Syndrome liver-qi depression; and study the effect on Jingqianshu granule acting on the liver-qi depression.
Methods : Model rats with PMS liver-qi depression were introduced by bondaging the limbs and using the method of resident-intruder respectively, and some of them were treated with Jingqianshu granule. In the process, we observed the vagina cell shape to make sure the estrum of rats: receptive phases and non-receptive phases, and then estimated model rats by open-field test and tailing suspension test. After introducing model, we killed the rats by decapitation and dissected frontal lobe cortex, hippocampus, hypothalamus and limbic lobe from the brains. Total proteins were extracted from these brain tissues. After that, expression levels of estrogen receptorα&β(ERα, ERβ) were determined in the encephalic regions with western blot method.
Results : Compared with the blank group, the body weight of the resident-intruder group (resident group) and the resident-intruder medication administration group (resident-medication administration group) increased significantly (P<0.05,P<0.01). In the tailing suspension test, it showed that in receptive phases, compared with the blank group, the scores of the bondage group increased significantly(P<0.01); While compared with two model groups, the scores of two model- medication administration groups decreased significantly(P<0.01). In non-receptive phases, compared with the blank group, the crossing score, the rearing score and the total score of the bondage group and the resident group in open-field decreased significantly, but scores of tailing suspension test increased significantly(P<0.01); Compared with two model groups, the rearing score and the total score of two model- medication administration groups in open-field increased significantly, but the scores of tailing suspension test decreased significantly(P<0.01). According to the results of western blot , it showed that compared with the blank group , the ERαexpression levels of frontal lobe cortex, hippocampus, hypothalamus and limbic lobe of the bondage group increased significantly(P<0.01); the ERαexpression levels of hippocampus and hypothalamus of the resident group increased significantly(P<0.01), that of frontal lobe cdortex increased, too(P<0.05); however, there was insignificant deviation in every index of the bondage group and resident group. Compared with the model groups, the ERαexpression levels of the medication administration groups decreased with different degrees. Compared with the blank group, the ERβexpression levels of hippocampus, hypothalamus and limbic lobe of the bondage group increased significantly(P<0.01); the ERβexpression levels of frontal lobe cortex and hippocampus of the resident group increased significantly(P<0.01), those of hypothalamus and limbic lobe increased, too(P<0.05); however, there was insignificant deviation in every index of the bondage group and resident group. Compared with the model groups, the ERβexpression levels of the medication administration groups decreased with different degrees.
Conclusions:The model rats with PMS liver-qi depression can be introduced by the method of resident-intruder, the model rats can be estimated by open-field experiment, but the tailing suspension test is not fit to estimate the model rats. There is a intimate relationship between the expressions of the ERαand ERβin different brain regions and the PMS liver-qi depression. The syndrome can be cured by the Jingqianshu granule. The changes of the expression levels of ERαand ERβin frontal lobe cortex, hippocampus, hypothalamus and limbic lobe may be one of the reasons that can cause PMS liver-qi depression.
方法:采用束缚法、居住-入侵法制备PMS肝气郁证大鼠模型,以调肝方药经前舒颗粒进行药物干预。采用细胞涂片法观察大鼠阴道细胞形态,确定动情周期:接收期与非接收期。以旷场实验和悬尾实验两种行为学方法评价模型。造模后,将各组大鼠断头处死,迅速取出额叶皮层、海马、下丘脑、边缘叶这四个脑区。提取脑组织的总蛋白。用蛋白质印迹技术(western blot,WB)检测各脑区雌激素受体α与β(ERα、ERβ)两种受体在不同脑区的表达水平。
结果:与空白组相比,居住-入侵造模组(居住组)及居住-入侵造模给药组(居住给药组)大鼠体重变化显著(P<0.05,P<0.01)。行为学检测表明:在接收期,与空白组大鼠相比,束缚组和居住组大鼠悬尾不动时间显著增加(P<0.01);而与两造模组相比,相应的两造模给药组大鼠悬尾不动时间均显著减少(P<0.01)。在非接受期,与空白组大鼠相比,束缚组和居住组大鼠旷场实验的水平得分、垂直得分及总分均显著减少,但悬尾不动时间显著增加(P<0.01);与两造模组相比,相应的两造模给药组大鼠旷场水平得分、垂直得分及总分均显著增加,但悬尾不动时间均显著减少(P<0.01)。蛋白印迹实验表明,与空白组大鼠相比,束缚组大鼠额叶皮层、海马、下丘脑、边缘叶ERα表达显著升高(P<0.01);居住组大鼠边缘叶与下丘脑ERα表达显著升高(P<0.01),皮层表达量亦显著升高(P<0.05);束缚组与居住组各指标均无显著性差异;与模型组比较,给药组各脑区的ERα表达水平不同程度的降低。与空白组大鼠相比,束缚组大鼠额叶大脑皮层、海马、下丘脑、边缘叶ERβ表达显著升高(P<0.01);居住组大鼠皮层与海马ERβ表达显著升高(P<0.01),在下丘脑、边缘叶的表达亦升高(P<0.05);束缚组与居住组各指标均无差异;与模型组比较,给药组各脑区的ERβ表达水平不同程度的降低。
结论:用居住-入侵法可有效制备PMS肝气郁证大鼠模型,旷场实验可有效评价该模型,悬尾实验不适宜用来评价PMS肝气郁证大鼠模型。大鼠不同脑区的ERα与ERβ表达与PMS肝气郁证关系密切,额叶皮层、海马、下丘脑、边缘叶中的ERα与ERβ表达变化可能是导致PMS肝气郁证的重要病因之一。中药经前舒颗粒对该病证模型大鼠有不同程度的调节作用。
Objectives:Our aims of this study are to introduce model rats with Premenstrual Syndrome liver-qi depression, research evaluation method of the model rats; explore expression levels of estrogen receptor in different brain regions; seek for parts of the mechanisms of Premenstrual Syndrome liver-qi depression; and study the effect on Jingqianshu granule acting on the liver-qi depression.
Methods : Model rats with PMS liver-qi depression were introduced by bondaging the limbs and using the method of resident-intruder respectively, and some of them were treated with Jingqianshu granule. In the process, we observed the vagina cell shape to make sure the estrum of rats: receptive phases and non-receptive phases, and then estimated model rats by open-field test and tailing suspension test. After introducing model, we killed the rats by decapitation and dissected frontal lobe cortex, hippocampus, hypothalamus and limbic lobe from the brains. Total proteins were extracted from these brain tissues. After that, expression levels of estrogen receptorα&β(ERα, ERβ) were determined in the encephalic regions with western blot method.
Results : Compared with the blank group, the body weight of the resident-intruder group (resident group) and the resident-intruder medication administration group (resident-medication administration group) increased significantly (P<0.05,P<0.01). In the tailing suspension test, it showed that in receptive phases, compared with the blank group, the scores of the bondage group increased significantly(P<0.01); While compared with two model groups, the scores of two model- medication administration groups decreased significantly(P<0.01). In non-receptive phases, compared with the blank group, the crossing score, the rearing score and the total score of the bondage group and the resident group in open-field decreased significantly, but scores of tailing suspension test increased significantly(P<0.01); Compared with two model groups, the rearing score and the total score of two model- medication administration groups in open-field increased significantly, but the scores of tailing suspension test decreased significantly(P<0.01). According to the results of western blot , it showed that compared with the blank group , the ERαexpression levels of frontal lobe cortex, hippocampus, hypothalamus and limbic lobe of the bondage group increased significantly(P<0.01); the ERαexpression levels of hippocampus and hypothalamus of the resident group increased significantly(P<0.01), that of frontal lobe cdortex increased, too(P<0.05); however, there was insignificant deviation in every index of the bondage group and resident group. Compared with the model groups, the ERαexpression levels of the medication administration groups decreased with different degrees. Compared with the blank group, the ERβexpression levels of hippocampus, hypothalamus and limbic lobe of the bondage group increased significantly(P<0.01); the ERβexpression levels of frontal lobe cortex and hippocampus of the resident group increased significantly(P<0.01), those of hypothalamus and limbic lobe increased, too(P<0.05); however, there was insignificant deviation in every index of the bondage group and resident group. Compared with the model groups, the ERβexpression levels of the medication administration groups decreased with different degrees.
Conclusions:The model rats with PMS liver-qi depression can be introduced by the method of resident-intruder, the model rats can be estimated by open-field experiment, but the tailing suspension test is not fit to estimate the model rats. There is a intimate relationship between the expressions of the ERαand ERβin different brain regions and the PMS liver-qi depression. The syndrome can be cured by the Jingqianshu granule. The changes of the expression levels of ERαand ERβin frontal lobe cortex, hippocampus, hypothalamus and limbic lobe may be one of the reasons that can cause PMS liver-qi depression.
引文
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