逍遥散加减方对高催乳激素血症肝郁脾虚证模型大鼠下丘脑多巴胺能神经元细胞凋亡及内质网应激的影响

Effects of Modified Xiaoyao Powder(逍遥散加减方)on Hypothalamic Dopaminergic Neurons Apoptosis and Endoplasmic Reticulum Stress in Hyperprolactinemia Model Rats with Liver Constraint and Spleen Deficiency Syndrome

目的探讨逍遥散加减方治疗高催乳激素血症(HPRL)肝郁脾虚证的可能作用机制。方法126只SD大鼠随机分成空白组,模型组,甲磺酸溴隐亭组,逍遥散加减方高、中、低剂量组,每组21只。除空白组外,其他各组均采用甲氧氯普胺腹腔注射联合慢性复合应激法制作HPRL肝郁脾虚证病证结合大鼠模型,总造模周期21天。实验第22天开始空白组与模型组给予4 ml生理盐水灌胃,甲磺酸溴隐亭组给予甲磺酸溴隐亭片0.001 g/(kg·d)灌胃,逍遥散加减方高、中、低剂量组分别给予逍遥散加减方60、30、15 g/(kg·d)灌胃,各组大鼠均灌胃共14天。给药结束次日称量大鼠体质量后,采用免疫荧光法测定下丘脑酪氨酸羟化酶(TH)阳性细胞数;采用ELISA法检测血清催乳素(PRL)含量;采用蛋白免疫印记法检测下丘脑的活化型半胱氨酸蛋白酶3(Cleaved-Caspase3)、C/EBP同源蛋白(CHOP)蛋白含量;原代培养多巴胺能神经元1周后,采用流式细胞仪检测多巴胺能神经元细胞凋亡。结果与空白组比较,模型组大鼠体质量、TH阳性细胞数降低,PRL含量及大鼠下丘脑组织CHOP、Cleaved-Caspase3蛋白表达量显著升高,多巴胺能神经元细胞凋亡率亦升高(P<0.01)。与模型组比较,甲磺酸溴隐亭组和逍遥散加减方高、中、低剂量组血清PRL含量显著降低,TH阳性细胞数升高;甲磺酸溴隐亭组及逍遥散加减方高剂量组CHOP蛋白表达降低,甲磺酸溴隐亭组和逍遥散加减方各剂量组Cleaved-Caspase3蛋白表达亦降低;甲磺酸溴隐亭组多巴胺能神经元细胞凋亡率显著升高,而逍遥散加减方各剂量组细胞凋亡率降低(P<0.05或P<0.01)。与逍遥散加减方中剂量组比较,逍遥散加减方高、低剂量组血清PRL含量显著升高(P<0.05或P<0.01)。与甲磺酸溴隐亭组和逍遥散加减方高剂量组比较,逍遥散加减方中、低剂量组CHOP蛋白表达升高,逍遥散加减方低剂量组Cleaved-Caspase3蛋白降低(P<0.01)。结论逍遥散加减方可能通过抑制CHOP介导的内质网应激相关凋亡通路减轻HPRL大鼠模型的多巴胺能神经元损伤。

Objective To study the possible mechanism of Modified Xiaoyao Powder(逍遥散加减方,MXP)in the treatment of hyperprolactinemia(HPRL)with liver constraint and spleen deficiency syndrome.Methods A total of 126 SD rats were randomly divided into blank group,model group,bromocriptine mesylate group,high-,medium-and low-dose MXP groups,with 21 rats in each group.Except for the blank group,all other groups used intraperitoneal injection of metoclopramide plus chronic compound stress method to develop the HPRL model with liver constraint and spleen deficiency syndrome,taking a total modeling period of 21 days.Since the 22nd day of the experiment,the blank group and the model group were given intragastric administration of 4 ml of normal saline,and the bromocriptine mesylate group was given 0.001 g/(kg·d)of bromocriptine mesylate tablets by gavage;MXP at the dosage of 60,30,and 15 g/(kg·d)was administered by gavage in the high-,medium-and low-dose group,respectively.The administration period was 14 days for all groups.After weighing the rats on the next day of last administration,the number of hypothalamic tyrosine hydroxylase(TH)positive cells was determined by immunofluorescence;the serum prolactin(PRL)content was detected by ELISA;the protein content of activated cysteine protease 3(Cleaved-Caspase3)and C/EBP homologous protein(CHOP)in the hypothalamus was detected by Western blotting.The apoptosis of dopaminergic neurons was detected by flow cytometry after primary culture for 1 week.Results Compared to those in the blank group,the body weight and the number of TH positive cells in the model group decreased,while the content of PRL,the expression of CHOP and Cleaved-Caspase3 proteins in the hypothalamus tissue increased significantly,as well as the apoptosis rate of dopaminergic neurons(P<0.01).Compared to those in the model group,the serum PRL content significantly decreased while the number of TH positive cells increased in the bromocriptine mesylate group and high-,medium-,and low-dose MXP groups;the expression of CHOP protein decreased in the bromocriptine mesylate group and the high-dose MXP group;the expression of Cleaved-Caspase3 protein in the bromocriptine mesylate group and MXP all dose groups also decreased;the apoptosis rate of dopaminergic neurons in the bromocriptine mesylate group increased significantly,while that of MXP all dose groups decreased(P<0.05 or P<0.01).Compared to those in the medium-dose MXP group,the serum PRL level in the high-and low-dose groups significantly increased(P<0.05 or P<0.01).Compared to those in the bromocriptine mesylate group and the high-dose MXP group,the expression of CHOP protein in the medium-and low-dose groups increased,and the Cleaved-Caspase3 protein in the low-dose group decreased(P<0.01).Conclusion MXP may alleviate dopaminergic neuron damage in HPRL rat model by inhibiting CHOP-mediated endoplasmic reticulum stress-related apoptosis pathway.