电针对慢性应激模型大鼠HPA轴-Glu-NMDA受体-NO路径调节作用机理的研究

【作者】 史榕荇；

【导师】 图娅；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2007， 博士

【摘要】 抑郁症是一种常见的精神疾病,其终生患病率高达15 %-20%。抑郁症居高不下的发病率以及造成的危害,已经越来越引起医药卫生界的重视。近年来,治疗抑郁症的新药层出不穷,但由于抑郁症的发病机制很复杂,抗抑郁剂在临床上出现疗效滞后现象,并且大约有30%左右的重度抑郁症人群对目前的各型抗抑郁剂没有反应,而且无法摆脱毒、副作用大,患者依从性差等问题。行之有效的治疗抑郁症的方法有着急迫而且巨大的需求。针刺疗法源于中医学理论,立足于整体观念辨证论治,对机体产生广泛的良性调节作用。近年来针灸治疗抑郁症越来越多的应用于临床,产生了较好的临床疗效,且未发现不良反应。针刺作为替代疗法以其特有的优势受到人们的广泛关注,但针灸抗抑郁疗法的研发才刚刚起步,其产生疗效的机制有待深入阐明,研究的深度和广度都有待进一步提高。我们的研究以HPA轴-Glu-NMDA受体-NO路径为研究的切入点,选择慢性应激刺激诱发的抑郁模型大鼠作为研究对象。本文选择临床上治疗抑郁症行之有效的穴位解释治疗机理,使基础实验研究更贴近临床,以便服务于临床。通过研究电针对慢性应激模型细胞因子、激素和相关受体等的影响,探讨电针抗抑郁治疗的作用机制。通过引入NMDA受体拮抗剂MK-801,从该路径内多个环节相互促进与制约的复杂关系中,探讨电针对NMDA受体通道的作用途径。从而研究电针抗抑郁作用的可能机制。研究方法及结论:1.采用7种不可预知的应激原结合孤养方法建立大鼠慢性应激模型。结论:此次实验研究我们选择的慢性应激结合孤养模型,是在CUMS模型和孤养模型上的发展,此模型效果稳定,可重复性高,接近抑郁症的发病特点,是研究抑郁较好、较有效的模型,目前被广泛采用的抑郁症动物模型。2.通过开野试验和游泳试验,研究慢性应激模型大鼠的行为学变化,从而检验造模成功与否及电针对慢性应激模型大鼠行为学的影响。结论:①造模后大鼠的水平运动发生了显著改变,模型组大鼠的活动度明显降低,给予电针治疗的慢性应激大鼠活动性有所改善,在水平运动次数上与模型组大鼠比较有显著性差异（P<0.01）;造模后大鼠的垂直运动明显减少,模型组大鼠的活动度明显降低,给予电针治疗的慢性应激大鼠探究活动有所改善,在垂直运动次数上与模型组大鼠比较有显著性差异（P<0.05）;说明电针可以改善抑郁模型大鼠活动性及探究兴趣,疗效与MK-801相当。②造模后大鼠强迫游泳不动时间明显增加,模型组大鼠的活动度明显降低（P<0.01）,给予电针治疗的慢性应激大鼠的强迫游泳不动时间得到改善,与模型组大鼠比较有显著性差异（P<0.05）;MK-801对应激导致的大鼠活动性降低也有明显改善作用（P<0.05）。提示电针可以改善抑郁模型大鼠活动性,而这种改变的原因可能与动物与恶劣环境抗争的意识得到增强有关。3.运用放免法检测大鼠下丘脑CRF,垂体ACTH、肾上腺CORT、血清CORT。判断慢性应激大鼠是否存在HPA轴亢进及电针对HPA轴相关细胞因子及激素的影响。结论:慢性应激大鼠下丘脑CRF过度分泌（P<0.05）。检测模型组垂体的ACTH的分泌有增多的趋势,但相对于空白组并不显著,仅有增高的趋势。模型组肾上腺、血清中CORT与空白组相比显著增加（P<0.05）。说明下丘脑CRF作用于垂体的ACTH含量的增加最终导致了肾上腺和血液中CORT的升高。慢性应激状态下大鼠出现HPA轴亢进,电针可以降低CRF、CORT,进而对亢进的HPA轴产生良性调整作用。4.运用放免法检测大鼠海马CORT,运用比色法检测大鼠海马内Glu含量,判断慢性应激大鼠是否影响海马内兴奋性氨基酸含量及电针对海马内相关激素及神经递质的影响。结论:经长期慢性应激刺激后,大鼠海马内CORT显著升高（P<0.05）,谷氨酸含量明显升高（P<0.05）。电针可以明显降低大鼠海马内CORT、Glu的含量（P<0.05）,有效纠正海马所处的应激状态,降低由HPA轴亢进而导致的对海马的兴奋性毒性作用。5.运用免疫组化法检测大鼠海马内NMDA受体含量,判断慢性应激大鼠是否存在NMDA受体含量的改变,探讨电针对海马内NMDA受体干预作用可能存在的机制。结论:慢性应激条件下,大鼠海马内NMDA受体含量显著增加,模型组明显高于正常组（P<0.05）;电针可明显降低慢性应激模型大鼠海马内NMDA受体含量（P<0.05）;经MK-801干预后,大鼠海马内NMDA受体含量亦明显降低（P<0.05）。电针可通过调节海马神经元NMDA受体的含量,抑制慢性应激性抑郁症NMDA受体的过度激活控制NMDA受体通道的开放,减轻细胞毒性作用。6.运用荧光定量PCR法检测大鼠海马内NR2A、NR2BmRNA含量,判断慢性应激大鼠是否存在NMDAR受体含量改变,研究在HPA轴亢进状态下,上位中枢海马可能存在的分子病理学改变,分析电针如何通过干预海马内NMDAR受体而对海马内神经递质通路产生调整作用。结论:模型组与空白组比较,NR2AmRNA含量显著增加（P<0.01）,电针干预使慢性应激状态下大鼠海马NR2AmRNA的表达有显著下调（P<0.01）,MK-801（P<0.01）可明显下调海马内NR2AmRNA表达。模型组与空白组比较,NR2BmRNA含量显著增加（P<0.01）,电针干预使慢性应激状态下大鼠海马NR2BmRNA的表达有显著下调（P<0.01）,MK-801可明显下调海马内NR2BmRNA表达（P<0.01）。电针通过调节海马神经元NR2A、NR2BmRNA的过量表达,控制NMDA受体通道的开放,从而减轻抑郁症海马的细胞毒性作用。7.运用放免法检测大鼠海马内iNOS、NO₃^-含量,判断慢性应激大鼠是否存在一氧化氮含量的改变及电针改善海马内一氧化氮的毒性作用。结论:接受慢性应激刺激后,模型组大鼠海马组织iNOS和NO含量显著升高,模型组与正常组比较有极显著差异（P<0.01）;电针组与模型组比较有显著差异（P<0.01）,海马iNOS和NO含量明显下调（P<0.01）;电针组与MK-801组无显著差异。电针通过调节海马神经元NR2A、NR2BmRNA的过量表达,控制NMDA受体通道的开放,抑制iNOS的激活,从而减轻NO对抑郁症海马的细胞毒性作用。本课题研究表明,电针可以通过调整NMDA受体通道,调节HPA轴-Glu-NMDA受体-NO路径,进而改善慢性应激模型大鼠行为。具有一定的实验依据,值得深入研究,并加以开发利用。更多还原

【Abstract】 Depression is one kind of frequent mental disorder and the lifetime prevalence is up to15 -20%.T’he high disease incidence and harm induced by it have gotten more and more attention in medical science realm.In recently years,many types of anti-depression medications came to the market. However,their effectiveness is limited to controlling certain symptoms. And almost 30% patients with major depression have no reaction to any medicine.Other than the unwanted side-effects such as increased toxin in blood stream, the patients often exhibit behavioral problems such resistance to following orders.An effective therapeutic method is being demanded urgently.Acupuncture,one of the major parts of Traditional Chinese Medicine,has a universal function which can balance the situation of the people. Moreover, it has the prevalence of no side effects and has been acceptted widely in clinical practice. It has been laid more and more focus throughout all over the world.But the research is just on the early period ,and the mechanism of the electroacupucnture (EA) effects depression is still need further exploration.This research emphases on the HPA axis-Glu-NMDA receptor-NO path. We choose the long-time unpredicted chronic stress model that can stimulate the predisposing cause of the depression. we use the frequent most of the acupoints and try to provide basic theory for clinical practice.Analysis the effects of EA on the hormones and related receptors in model rats,we try to explain the mechanism of how EA effects depression.We fetch in MK-801 in our reseach, explore this path from diffirent points and try to explain the mechanism.Discuss if the EA effects NMDA receptor pathway is the mechanism of acupuncture.Methods and conclusion:1.Seven kinds of chronic stress are used unpredictable.Conclusion:the model which we selected is a feasible model,and has been widely used in medical research field.2.We studied the depression level of the model rats by open-field and swim experiment. Evaluate if we made a successful long-time chronic stress model. Analysis EA effects model rats` behavior.Conclusion:⑴By open-field experiment, we observed the rearing times and the crossing times of the model rats. The results show that the rearing times and the crossing times of the rats in the EA group significantly increased compared with the rats of the model group (P<0.01). This results indicate that the EA therapy can improve the activities and the explorative interest of the model rats.⑵We observed the swim of the model rats. The result shows that the stillness time during swim of rats in the EA group decreased significantly compared with the rats of the model group (P<0.05). And the stillness time of the rats in the EA group approximate to that of the MK-801 group. These results indicate that the EA therapy can increase the sensibility of the model rats.3. Analysis the CRF in hypothalamus,ACTH in pituitary gland,CORT in adrenal gland by RIA method.Conclusion:CRF and CORT increased greatly in model group (P<0.05).EA decreased the CRF and CORT level significantly and compared with MK-801group has no difference.4.Analysis the CORT and Glu level in hippocampus by RIA method.Conclusion: CORT and Glu level in hippocampus increased greatly in model group (P<0.05).EA decreased the CORT and Glu level in hippocampus significantly and compared with MK-801 group has no difference.5. Analysis the NMDA receptor level in CA3 immuno histochemical study.Conclusion: the NMDA receptor level in hippocampus increased greatly in model group (P<0.05).EA decreased the NMDA receptor level in hippocampus significantly and compared with MK-801 group has no difference.6. Analysis the NR2A,NR2BmRNA level in hippocampus by Real-time Quantitative ReverseTranscription-polymerase Chain Reaction method.Conclusion:the NR2A,NR2BmRNA level in hippocampus increased greatly in model group (P<0.01). EA decreased NR2A,NR2BmRNA level in hippocampus significantly (P<0.01). And compared with MK-801 group has no difference.7.Analysis the iNOS level by RIA methord, NO3 - level by colorimetry method.Conclusion:the iNOS and NO3– level in hippocampus increased significantly (P<0.01). EA decreased the iNOS and NO3– level in hippocampus significantly (P<0.01). And EA compared with MK-801group has no difference.更多还原