白金胶囊抗抑郁作用及作用机制研究

【作者】 张荣；

【导师】 孙建宁；

【作者基本信息】 北京中医药大学 ， 中药药理学， 2017， 硕士

【摘要】 抑郁症是一种情感性精神障碍症,临床表现为情绪低落、消极、悲观、内疾、注意力不集中、对事物失去兴趣、睡眠和饮食障碍等。抑郁症发病机制复杂,患者多出现单胺类神经递质含量不足或功能低下、下丘脑-垂体-肾上腺轴功能失调、神经营养因子受损和炎症反应等。基于上述发现,目前临床上首选用于治疗抑郁症的药物多为神经递质再摄取抑制剂、5-HT受体抑制剂等的化学药。研究开发用于治疗抑郁症的中药复方药物成为热点。白金胶囊原方为临床经验方,具有良好的抗抑郁作用。本研究样品为其现代新制剂白金胶囊,对该制剂的抗抑郁作用及初步作用机制进行研究。研究采用慢性不可预知应激和嗅球切除两种抑郁模型,给药后监测实验动物旷场活动、自主活动、糖水消耗等行为学,确认白金胶囊的抗抑郁作用。并通过测定实验动物脑内神经递质,脑内5-HT受体以及血清中CORT、CRH、ACTH、TNF-α、IL-6、IL-1β的含量,对白金胶囊抗抑郁作用机制进行初探。1白金胶囊对慢性不可预知应激致抑郁症模型大鼠的影响目的:研究白金胶囊对慢性不可预知温和应激结合孤养致抑郁模型大鼠抗抑郁作用,并对其抗抑郁作用机制进行初探。方法:112只SD大鼠,雌雄各半,适应性饲养1周,期间通过体重、糖水实验及旷场实验,选择得分相近的96只大鼠(雌/雄各半),随机分为正常对照组、抑郁症模型组、白金胶囊12.6 g/kg剂量组、4.2g/kg剂量组、1.4g/kg剂量组及盐酸氟西汀分散片3.5 mg/kg剂量组,每组12只。分组后连续7周给予慢性应激刺激,通过体重、糖水消耗实验及旷场实验观察是否造模成功。造模成功后,继续给予应激刺激并给予药物治疗4周,期间通过监测体重、自主活动次数、糖水消耗实验以及旷场实验观察治疗情况。给药结束后,采用HPLC-ECD法对实验大鼠脑皮层、海马组织中单胺类神经递质进行定量分析,放免法对实验大鼠血清中CORT、ACTH、CRH及炎性细胞因子进行含量测定,对白金胶囊的抗抑郁作用机制进行初探。结果:与正常对照组相比,模型动物旷场运动距离、垂直运动次数以及自主活动次数明显减少(P<0.01),糖水消耗百分比显著降低(P<0.01),体重明显降低(P<0.01)。与模型组相比,白金胶囊中、低剂量组可显著增加大鼠自主活动次数(P<0.05),各剂量组可显著升高模型大鼠糖水消耗百分比(P<0.05,P<0.01),还可显著增加水平运动距离和垂直运动次数(P<0.01)。表明白金胶囊具有抗抑郁作用。抗抑郁作用机制的研究结果显示:与正常对照组相比,模型动物脑皮层组织中DA、DOPAC、NE、5-HT的含量有下降的趋势,5-HIAA有升高趋势,而皮层中DA的代谢产物HVA含量明显升高(P<0.05),(DOPAC+HVA)/DA及5-HIAA/5-HT的代谢率显著升高(P<0.05);海马组织中5-HT及其代谢产物5-HIAA、HVA的含量显著降低(P<0.05,P<0.01),NE的含量有升高趋势,5-HIAA/5-HT代谢率比值明显增加(P<0.05);血清CRH、TNF-α、IL-6的含量明显升高(P<0.05)。给药4周后,白金胶囊中、低剂量组可显著降低模型动物脑皮层HVA的含量并有升高5-HT、DA的趋势,还可显著降低模型动物脑皮层(DOPAC+HVA)/DA及5-HIAA/5-HT的代谢率(P<0.05,P<0.01),其中中剂量组可显著升高NE的含量(P<0.05);白金胶囊高、中剂量组可显著降低模型大鼠血清CRH的含量(P<0.05,P<0.01),高剂量组可显著降低血清中TNF-α、IL-1β的含量(P<0.05),中剂量组有降低血清IL-1β含量的趋势。结论:白金胶囊对慢性不可预知应激致抑郁症模型大鼠具有抗抑郁作用,其作用机制可能与其增加脑皮层单胺类神经递质5-HT、DA的含量,抑制HPA轴兴奋性及降低炎性细胞因子水平有关。2白金胶囊对嗅球切除致抑郁症模型大鼠的影响目的:研究白金胶囊对慢性不可预知应激致抑郁大鼠的抗抑郁作用,并对其抗抑郁作用机制进行初探。方法:96只SD大鼠,雌雄各半,适应性饲养1周,随机分成假手术组(n=16)和手术组(n=80),手术组用10%水合氯醛腹腔注射麻醉,毁损大鼠双侧嗅球,假手术组与手术组平行操作,但不毁损嗅球,术后用青霉素防感染,恢复2周。将手术组随机分为抑郁症模型组、白金胶囊12.6 g/kg剂量组、4.2 g/kg剂量组、1.4g/kg剂量组及盐酸氟西汀分散片3.5 mg/kg剂量组,每组12只,连续给药4周。给药第4周对各组大鼠分别进行体重及自主活动的测定、新奇抑制摄食实验以及旷场实验行为学检测。给药结束后,分别采用LC-MS/MS测定大鼠脑皮层中单胺类神经递质和氨基酸的含量,放免法测定大鼠血清中CORT、ACTH、CRH及促炎性细胞因子的含量,RT-PCR法测定大鼠脑皮层5-HT1A/2A及mGlu受体表达来进一步探讨其抗抑郁作用机制。结果:与正常对照组相比,模型动物自主活动明显增加(P<0.01),摄食潜伏期显著延长(P<0.01),水平运动距离和垂直运动次数明显增加(P<0.05)。与模型组相比,白金胶囊各剂量组可显著缩短模型动物摄食潜伏期(P<0.05);高剂量组可显著缩短模型动物水平运动距离并减少垂直运动次数(P<0.05),其余组有减少的趋势;中剂量组可显著减少模型动物自主活动(P<0.05),其余组有减少的趋势。表明白金胶囊具有抗抑郁作用。抗抑郁作用机制的研究结果显示:与正常对照组相比,模型动物脑皮层中5-HT、Orn的含量明显降低(P<(0.05),Glu、Asp、5-HIAA、DOPAC和HVA的含量以及(DOPAC+HVA)/DA的代谢率明显升高(P<0.05),DA的含量有降低的趋势,5-HIAA/5-HT的代谢率有升高的趋势;模型动物脑皮层mGlu mRNA以及5-HT2A mRNA 表达上调,5-HT1A mRNA表达下调(P<0.05,P<0.01);血清中 CORT、TNF-α、IL-6以及IL-1β的含量明显升高(P<0.05)。给药4周后,白金胶囊高、低剂量组可显著升高模型动物脑皮层中5-HT、DA的含量(P<0.05,P<0.01);各剂量组可显著降低脑皮层Glu、Asp的含量并升高Orn的含量,上调5-HT1A mRNA表达(P<0.05,P<0.01);高、中剂量组可显著下调模型动物脑皮层5-HT2A mRNA表达并降低模型动物血清CORT的含量(P<0.05,P<0.01);高剂量组还可显著降低动物血清中TNF-α、IL-6的含量(P<0.01),中剂量组有降低动物血清TNF-α、IL-6含量的趋势。结论:白金胶囊对嗅球切除致抑郁症模型大鼠具有抗抑郁的作用,其作用机制可能与升高脑皮层单胺类神经递质5-HT、DA的含量、抑制兴奋性氨基酸、调节脑内5-HT受体表达、抑制HPA轴兴奋性以及降低炎性细胞因子水平有关。小结1本课题建立慢性不可预知应激刺激(CUMS)结合孤养模型和嗅球切除(OBX)模型,这两种动物模型可成功表现出快感缺失、自主活动改变、对外界新鲜环境探知欲望改变等抑郁症状,表明这两种抑郁模型复制成功。2白金胶囊给药4周后,可缓解抑郁模型动物表现出的快感缺失、自主活动改变、对新奇环境的探索能力加强以及摄食能力改变的症状,表明其具有抗抑郁作用。3白金胶囊抗抑郁作用机制可能与升高脑内单胺类神经递质含量、抑制兴奋性氨基酸、调节5-HT受体表达、抑制HPA轴功能过度兴奋以及抑制炎性细胞因子水平有关。更多还原

【Abstract】 Depression is a kind of affective disorder,with the clinical feature of negative,pessimistic,inattention,lose interest in things,sleep and eating disorders and so on.The pathogenesis of depression is complex,while,most of patients show symptoms in molecular biology as hypofunction or insufficient of monoamine neurotransmitter,dysfunction of hypothalamic-pituitary-adrenal,impairment of neurotrophic factors and inflammatory reaction.Based on these symptoms,most of the drugs used for the treatment of depression are chemical agents such as the neurotransmitter reuptake inhibitors,5-HT receptor inhibitor and so on.Therefore,it is significant to explore a effective and harmfulless medicine for the treatment of depression.Baijin capsule which showed well therapeutic effect on depression was originally derived from formula of clinical experence.However,the pharmacodynamics study on Baijin capsule have not been carried out,and the pathogenesis of depression of Baijin capsule is ambiguous.Thus,in order to verify the antidepressant-like activity and clarify the antidepressant mechanism,studies on the rat depression model caused by chronic unpredictable mild stress and the rat depression model caused by olfactory bulbectomy were carried out from the perspective of monoamine neurotransmitter,HPAA and proinflammatory cytokine.1 Effect of Baijin capsule on rat depression model caused by chronic unpredictable mild stressObjective:Investigate the anti-depression effect of Baijin capsule on depressed rat,and clarify the antidepressant mechanism.Methods:96 SD rats,selected from 112 rats according to their comprehensive score of body weight,sugar water experiment and open field test,were divided into 6 groups:control group,model group,fluoxetine group,Baijin capsule group with high dosage,middle dosage,and low dosage.Body weight,sweet water wastage and open field test were used to evaluate the established depression model after being given chronic unpredictable mild stress for 7 weeks continuously.Then give corresponding medicines by oral administration for 4 weeks continuously,after establishing the model successfully.And body weight,autonomic activity times,sweet water wastage and open field test were detected to evaluate the anti-depression effect of Baijin capsule.After then,monoamine neurotransmitter in the cerebral cortex,and hippocampus of the rats were determined by HPLC,and CORT,ACTH,CRH and proinflammatory factor in the serum of the test rats were determined by radioimmunoassay to explore the possible the antidepressant mechanism of Baijin capsule.Results:Compared with the rat in control group,the body weight,autonomic activity times,sweet water wastage,horizontal movement distance and the number of vertical movement of the rat were all decreased obviously(P<0.01).Compared with the model rat,autonomic activity times of the rat in middle and low dose group of Baijin capsule were increased(P<0.05);sweet water wastage,horizontal movement distance and the number of vertical movement of the rat in Baijin capsule group were all increased(P<0.05,P<0.01),which mean that Baijin capsule possess anti-depression effect on the depression rat.Compared with the control group,the concentrate of DA,DOPAC,NE,and 5-HT in the cerebral cortex in model rat had the tendency to reduce.While metabolic rates of(DOPAC+HVA)/DA,and 5-HIAA/5-HT in the cerebral cortex were elevated(P<0.05).The concentrateions of 5-HT,5-HIAA,and HVA in hippocampus of the model rat were decreased(P<0.05,P<0.01),and the metabolic rate of 5-HIAA/5-HT in the hippocampus was increased(P<0.05).The CRH,TNF-α,IL-6 in the serum were changed drastically(P<0.05).However,compared with the model,Baijin capsule could inhibit the change of the monoamine neurotransmitter,CRH,TNF-α and IL-6 of the rat in the model group(P<0.05,P<0.01).Conclusion:Baijin capsule possess the effect of anti-depression,and the anti-depression effect might be related to the regulation effect of Baijin capsule on the content of monoamine neurotransmitter in the cerebral cortex and on the content of CRH,TNF-α and IL-6 in the rat plasma.2 Effect of Baijin capsule on rat depression model caused by olfactory bulbectomyObjective:Investigate the anti-depression effect of Baijin capsule on depressed rat,and clarify the antidepressant mechanism.Methods:96 SD rats were divided into sham-operated group(16)and operating group(80).Then the olfactory bulb of the rat in the operating group were destroyed,and penicillin was used for avoiding infection.After establishing the model successfully,the 80 rats in the operating group were divided into 5 groups:model group,fluoxetine group,Baijin capsule group with high dosage,middle dosage,and low dosage.The body weight and autonomic activity times were determined to evaluate the established model.And then the body weight,autonomic activity times,novelty-suppressed feeding,and open field test were carried out to investigate the anti-depression effect of Baijin capsule,after giving corresponding medicines by oral administration for 4 weeks continuously.In order to study on the anti-depression mechanism of Baijin capsule,the neurotransmitter in the cerebral cortex of the rat in the Baijin capsule group were determined by LC-MS/MS,and the the CORT,ACTH,CRH and proinflammatory factor were determined by radioimmunoassay.At the same time,Real-time PCR were used to determine the expression of 5-HT1A/2A and mGlu receptors.Results:Compared with the rat in the control group,autonomic activity times,horizontal movement distance,and the number of vertical motion of the rat in the model rat were increased(P<0.05,P<0.01).Feeding latency in the model rat was evidently extended(P<0.01).Compared with the rat in the model group,autonomic activity times,horizontal movement distance,and the number of vertical motion of the rat in the Baijin capsule group were decreased dramatically(P<0.05).Compared with the rat in the control group,the content of 5-HT,DA and Orn in cerebral cortex were decreased apparently(P<0.05),the expression of 5-HT1A receptor was also down-regulate(P<0.01);the content of 5-HIAA,DOPAC,HVA,Glu,and ASP were increased obviously(P<0.05),and the value of(DOPAC+HVA)/DA was also increased distinctly(P<0.05).The content of CORT,TNF-α,IL-6 and IL-1β in rat plasma were increased(P<0.05).The expression of 5-HT2A and mGlu receptors were up-regulate(P<0.05).Compared with the rat in the model group,the the content of 5-HT,DA and Orn and the expression of 5-HT1AR in cerebral cortex of the rat in the Baijin capsule group were increased dramatically,while the content of Glu,and ASP and the expression of 5-HT2AR were decreased,and the value of(DOPAC+HVA)/DA and 5-HIAA/5-HT were decreased(P<0.05,P<0.01).The content of CORT,TNF-α and IL-6 in rat plasma were decreased(P<0.05,P<0.01).Conclusion:Baijin capsule possess the effect of anti-depression,and the anti-depression effect might be related to the regulation effect of Baijin capsule on the content of monoamine neurotransmitter and its receptors,Amino acid in the cerebral cortex and on the content of CRH,TNF-α and IL-6 in the rat plasma.Summary1 This study adopts the depression rat model with chronic unpredictable mild stress or the model of olfactory bulb.These two kinds of animal models has been successfully showed the loss of sensation,autonomic activity change,and the change of the detection desire of the outside world fresh environment and feeding,which could show that models had been established successfully.2 Baijin capsule can alleviate depression animal model which showed a lack of pleasure,autonomous activity change,the change of the ability to explore new environment and feeding capacity,which can show the antidepressant effect of Baijin capsule.3 The antidepressant mechanism of Baijin capsule may be related to regulating the content of monoamines neurotransmitter,and amino acid and regulating its receptor expression in the cerebral cortex,regulation and control function of HPA axis and promoting inflammatory cytokine levels in the serum.更多还原