基于抑郁症PFC-NAc-VTA神经环路研究疏肝和胃汤的抗抑郁作用机制

发布时间：2018-05-06 16:05

本文选题：抑郁症 + PFC-NAc-VTA神经环路　；参考：《湖北中医药大学》2017年博士论文

【摘要】：目的抑郁症逐渐成为现代社会的常见及高发疾病,给社会和患者的生活都带来了巨大的压力,但抑郁症发病机制复杂,明确抑郁症的发病机制,对于抑郁症的诊治至关重要。本研究以中医“肝郁”与抑郁症的相似性为切入点,基于抑郁模型PFC-NAc-VTA神经环路的病理生理变化,旨在阐释中医“肝郁”发生的中枢机制,并通过观察疏肝和胃汤对抑郁模型PFC-NAc-VTA神经环路的影响作用研究其抗抑郁机制,为深入研究中医药治疗抑郁症提供理论和实验依据。方法1.理论研究:采用文献分析法,归纳古今医家对抑郁症相关病证的临床辨治规律,并梳理《伤寒杂病论》中抑郁症相关病证的辨治规律,分析中医肝郁与抑郁症的相关性;通过整理PFC-NAc-VTA神经环路与抑郁症发病的联系,分析中医肝郁的中枢机制与PFC-NAc-VTA神经环路的相关性。2.实验研究:(1)模型制作及评价:采用慢性不可预知性应激结合孤养法造模4w,造模完成后给予三种不同浓度疏肝和胃汤及氟西汀干预7d,并运用旷场实验、强迫游泳实验进行行为学评价。(2)神经环路相关指标的检测:采用尼氏染色法观察抑郁模型大鼠PFC脑区切片的病理改变,并统计PFC区正常神经元的数目;采用高效液相法分别检测抑郁模型大鼠PFC、NAc及VTA三个脑区中DA、GABA、Glu含量;采用RT-PCR检测抑郁模型大鼠NAc区PSD95m RNA、NR2B m RNA的相对表达量;采用western blot检测抑郁模型大鼠NAc区PSD95、NR2B的相对表达量。结果1.理论研究:古代医家认为,抑郁症与情志之郁证关系密切,可兼夹诸郁,又涉及脏腑功能紊乱及气血津液的失调。现代临床辨证规律研究显示,情志失调为抑郁症的主要病因,肝失疏泄为抑郁症病机核心,以影响及心、脾、胃等多脏腑为抑郁症的传变特点,以气血津液阴阳失调为抑郁症的病理变化特点,治法以疏肝解郁,调畅气机为主。现代医学认为抑郁症的发病机制与PFC-NAc-VTA神经环路的功能失调相关,因此可以从PFC-NAc-VTA神经环路为切入点探讨“肝郁”的中枢机制。2.实验研究:实验一:疏肝和胃汤对抑郁模型大鼠行为学的影响(1)模型组旷场实验静止时间延长,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤各剂量组及氟西汀组静止时间缩短,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤高剂量组与氟西汀组比较,差异无统计学意义(P0.05)。(2)模型组旷场实验总运动距离缩短,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤各剂量组及氟西汀组总运动距离增加,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤中剂量组总运动距离较氟西汀组长,差异有统计学意义(P0.01),高剂量组与氟西汀组比较,差异无统计学意义(P0.05)。(3)模型组旷场实验中央区域穿越格数减少,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤中、高剂量组及氟西汀组中央区域穿越格数增多,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤中、高剂量组与氟西汀组比较,差异无统计学意义(P0.05)。(4)模型组强迫游泳实验不动时间延长,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤各剂量组及氟西汀组不动时间缩短,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤中、高剂量组与氟西汀组比较,差异无统计学意义(P0.05)。实验二:疏肝和胃汤对抑郁模型大鼠PFC区病理变化的影响(1)光镜下尼氏小体呈深蓝色或紫蓝色,多为块状或细颗粒状,分布于核周围或树突内,尼氏体靠近胞体周围处较为明显。空白组PFC区神经元数目较多,尼氏小体颜色较深,分布于细胞核周围。神经元细胞形态饱满,结构完整,边缘清晰;模型组PFC区神经元数目相对较少,尼氏小体颜色较淡,分布疏松,细胞形态不规则,结构不完整,尼氏小体数目减少或消失,部分细胞核偏向一侧,细胞肿大,出现空泡区;经过治疗药物干预后,各治疗组尼氏小体数目较模型组增多但少于空白组,较模型组尼氏小体颜色更深,结构较完整、清晰。(2)模型组PFC区正常神经细胞数量减少,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤各剂量组及氟西汀组PFC区正常神经细胞数量增加,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤中、高剂量组与氟西汀组比较,差异无统计学意义(P0.05)。实验三:疏肝和胃汤对抑郁模型大鼠PFC-NAc-VTA神经环路中DA、GABA、Glu含量的影响(1)PFC区中,模型组DA含量降低,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤中、高剂量组及氟西汀组DA含量升高,与模型组比较差异有统计学意义(P0.05或P0.01);其中,疏肝和胃汤高剂量组与氟西汀组比较,差异无统计学意义(P0.05)。NAc区中,模型组DA含量降低,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤各剂量组及氟西汀组DA含量升高,与模型组比较差异有统计学意义(P0.05或P0.01);其中,疏肝和胃汤中剂量组与氟西汀组比较,差异无统计学意义(P0.05)。VTA区中,模型组DA含量降低,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤低、中剂量组及氟西汀组DA含量升高,与模型组比较差异有统计学意义(P0.05或P0.01);其中,疏肝和胃汤低、中剂量组DA含量较氟西汀组低,差异有统计学意义(P0.01)。(2)PFC区中,模型组Glu含量升高,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤各剂量组及氟西汀组Glu含量降低,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤中剂量组Glu含量较氟西汀组低,差异有统计学意义(P0.05),低、高剂量组与氟西汀组比较,差异无统计学意义(P0.05)。NAc区中,模型组Glu含量降低,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤中剂量组及氟西汀组Glu含量升高,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤中剂量组与氟西汀组比较,差异无统计学意义(P0.05)。VTA区中,模型组Glu含量升高,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤各剂量组及氟西汀组Glu含量降低,与模型组比较差异有统计学意义(P0.05或P0.01);其中,疏肝和胃汤各剂量组与氟西汀组比较,差异无统计学意义(P0.05)。(3)PFC区中,模型组GABA含量降低,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤中剂量组及氟西汀组GABA含量升高,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤中剂量组与氟西汀组比较,差异无统计学意义(P0.05)。NAc区中,模型组GABA含量升高,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤中、高剂量组GABA含量降低,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤中、高剂量组与氟西汀组比较,差异有统计学意义(P0.01)。VTA区中,模型组GABA含量升高,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤中剂量组及氟西汀组GABA含量降低,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤中剂量组与氟西汀组比较,差异无统计学意义(P0.05)。实验四:疏肝和胃汤对抑郁模型大鼠NAc区PSD95 m RNA和NR2B m RNA表达的影响(1)模型组NAc区PSD95 m RNA的相对表达量降低,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤各剂量组及氟西汀组NAc区PSD95 m RNA的相对表达量升高,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤中、高剂量组PSD95 m RNA的相对表达量高于氟西汀组,差异有统计学意义(P0.01);低剂量组与氟西汀组比较,差异无统计学意义(P0.05)。(2)模型组NAc区NR2B m RNA的相对表达量降低,与空白组比较差异有统计学意义(P0.05);疏肝和胃汤各剂量组及氟西汀组NAc区NR2B m RNA的相对表达量升高,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤各剂量组NAc区NR2B m RNA的相对表达量高于氟西汀组,差异有统计学意义(P0.01)。实验五:疏肝和胃汤对抑郁模型大鼠NAc区PSD95和NR2B表达的影响(1)模型组NAc区PSD95相对表达量降低,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤各剂量组及氟西汀组PSD95相对表达量升高,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤中、高剂量组PSD95相对表达量高于氟西汀组,差异有统计学意义(P0.01),低剂量组与氟西汀组比较,差异无统计学意义(P0.05)。(2)模型组NAc区NR2B相对表达量降低,与空白组比较差异有统计学意义(P0.01);疏肝和胃汤各剂量组及氟西汀组NR2B相对表达量升高,与模型组比较差异有统计学意义(P0.01);其中,疏肝和胃汤低、中剂量组NR2B相对表达量高于氟西汀组,差异有统计学意义(P0.01)。结论1.古代医家所论之“郁”涉及范围较广,包括五运六气之郁、脏腑之郁及情志之郁等,至明清以后专论郁证,才与现代抑郁之“郁”联系紧密。2.近十年来抑郁症的中医病因及证候分布规律研究显示,情志失调为抑郁症的主要病因,肝失疏泄为其病机核心,以影响及心、脾、胃等多脏腑为其传变特点,以气血津液阴阳失调为其病理变化特点,治法以疏肝解郁,调畅气机为主。3.慢性应激引起的PFC区神经元病理改变,PFC-NAc-VTA神经环路DA含量的降低、Glu含量的升高、GABA含量的相应改变,以及NAc区突触相关蛋白PSD95、NR2B及其m RNA表达的下降,可能是抑郁症的发病机制。4.疏肝和胃汤能够改善抑郁模型大鼠的抑郁状态,并且能减轻抑郁模型大鼠PFC区神经元的受损程度,调节PFC-NAc-VTA神经环路中DA、Glu、GABA含量的异常上升或下降,且能够逆转模型大鼠NAc区突触相关蛋白PSD95、NR2B及其m RNA的异常下调,提示疏肝和胃汤可能是通过对PFC-NAc-VTA神经环路中上述指标的调节发挥抗抑郁作用的。5.中医“肝郁”发生的中枢机制可能与PFC区神经元病理变化,PFC-NAc-VTA神经环路中DA、Glu、GABA的异常表达以及NAc区突触相关蛋白PSD95、NR2B及其m RNA的异常下调有关。6.研究结果提示,疏肝和胃汤发挥抗抑郁作用的潜在机制之一,可能是通过调节PFC-NAc-VTA神经环路中的多个靶点实现的。
[Abstract]:Objective depression has gradually become a common and high incidence disease in modern society, which brings great pressure to the life of society and patients. However, the pathogenesis of depression is complicated. The pathogenesis of depression is very important for the diagnosis and treatment of depression. This study is based on the similarity of "liver depression" with depression, based on depression and depression. The pathophysiological changes of the model PFC-NAc-VTA nerve loop are designed to explain the central mechanism of "liver depression" in Chinese medicine, and to study the antidepressant mechanism of the depression model PFC-NAc-VTA neural loop by observing the effect of Shugan and stomach soup on the depression model, and to provide theoretical and experimental basis for the in-depth study of Chinese medicine for the treatment of depression. Method 1. theory study: Using the method of literature analysis, this paper summarizes the law of the clinical differentiation and treatment of depression related syndromes by ancient and modern doctors, and combs the law of differentiation and treatment of the syndrome of depression related to depression in the theory of typhoid and miscellaneous diseases, analyzes the correlation between the liver depression and depression of traditional Chinese medicine, and analyzes the central mechanism and PFC-N of the liver depression of traditional Chinese medicine by sorting out the connection between the PFC-NAc-VTA nerve loop and the depressive disorder. The correlation.2. experimental study of Ac-VTA neural loop: (1) model making and evaluation: using chronic unpredictable stress combined with soliton method to create a model of 4W, after the completion of the model, three different concentrations of liver and stomach soup and fluoxetine intervention were given, and the field experiment was used to evaluate the behavior of the forced swimming test. (2) the examination of the related indexes of the nerve loop. Test: the pathological changes in the PFC brain section of the depressive model rats were observed by Nissl's staining, and the number of normal neurons in the PFC region was measured. The content of DA, GABA and Glu in the three brain regions of the depressive model rats were detected by high performance liquid phase method, and the relative expression of PSD95m RNA in the NAc region of the depressive model rats was detected by RT-PCR. Western blot was used to detect the relative expression of PSD95 and NR2B in the NAc area of the depression model rats. Results 1. theoretical study: ancient doctors believed that depression was closely related to emotional depression syndrome, and it could be combined with depression and imbalance of viscera function and Qi and blood body fluid. Modern clinical syndrome differentiation study showed that emotional disorder was the main disease of depression. As the core of the depression, liver loss and catharsis are the core of the depression, and the multiple Zang Fu organs such as the heart, the spleen and the stomach are the characteristics of the depressive disorder, and the pathological changes of depression are characterized by the imbalance of Qi and blood Yin and Yin and Yin and Yang, and the treatment method is mainly to remove the stagnation of the liver and regulate the Qi, and the pathogenesis of depression is related to the dysfunction of the PFC-NAc-VTA nerve loop. The experimental study on the central mechanism of "liver depression" can be studied from the PFC-NAc-VTA neural loop as the breakthrough point. Experiment 1: the effect of Shugan Hewei decoction on the behavior of the depression model rats (1) the time of the static time of the model group was prolonged, and the difference was statistically significant (P0.01), the dosage group of Shugan Hewei decoction and the static fluoxetine group were static. There was significant difference between the model group and the model group (P0.01), and there was no significant difference between the high dose group of Shugan Hewei decoction and the fluoxetine group (P0.05). (2) the total movement distance of the model group was shorter than that in the blank group (P0.01); the total dose group of Shugan Hewei decoction and the general transport of fluoxetine group were compared with that of the blank group. The difference of dynamic distance between the model group and the model group was statistically significant (P0.01), and the total motion distance of the dose group in the liver and stomach soup was more significant than that of the fluoxetine group (P0.01). There was no significant difference between the high dose group and the fluoxetine group (P0.05). (3) the number of crossing lattices in the central area of the model group was reduced and the blank group was in the blank group. The difference was statistically significant (P0.01); in the high dose group and the fluoxetine group, the number of crossing lattices increased in the high dose group and the fluoxetine group, and there was a significant difference between the model group and the model group (P0.01). There was no significant difference between the high dose group and the fluoxetine group (P0.05). (4) the time of the forced swimming test in the model group was not significant. The difference has statistical significance (P0.01) compared with the blank group, and the time of each dose group and fluoxetine group in the Shugan Hewei decoction group and the fluoxetine group were shorter than the model group (P0.01). The difference between the high dose group and the fluoxetine group was not statistically significant (P0.05). Experiment two: the depression of liver and stomach soup to the depression model. The effect of pathological changes in PFC area of type rats (1) the Nissl body was dark blue or purple blue under light microscope, mostly massive or fine grainy, distributed around the nucleus or dendrite, Nissl body was near the cell body. The number of neurons in the blank group PFC area was more, the Nissl body color was deep and distributed around the nucleus. Full, complete structure, clear edge; the number of neurons in the model group PFC is relatively small, Nissl body color is light, loose, irregular cell morphology, incomplete structure, the number of Nissl corpuscles decreasing or disappearing, partial nucleus to one side, cell enlargement, empty bubble area; after treatment of drug after the prognosis, the number of Nissl corpuscles in the treatment group More than the model group but less than the blank group, Nissl body color was deeper and more complete and clear than the model group. (2) the number of normal nerve cells in the PFC area of the model group decreased, and the difference was statistically significant (P0.01). The number of normal nerve cells in each dose group of Shugan Hewei decoction and the PFC area of fluoxetine group was increased, and the difference was compared with the model group. There was statistical significance (P0.01); among them, there was no significant difference between the high dose group and the fluoxetine group (P0.05). Experiment three: the effect of Shugan Hewei decoction on the content of DA, GABA and Glu in the PFC-NAc-VTA nerve loop of the depression model rats (1) the DA content in the model group decreased, and the difference was statistically significant (P0.) compared with the blank group (P0.) 01): the content of DA in the high dose group and the fluoxetine group increased in the high dose group and the fluoxetine group. The difference was statistically significant (P0.05 or P0.01) in the model group (P0.05 or P0.01), and there was no significant difference between the high dose group and the fluoxetine group (P0.05) in.NAc area, and the content of the model group decreased, and the difference was statistically significant (P0.01). The DA content in each dose group of liver and stomach soup and fluoxetine group was higher than that in the model group (P0.05 or P0.01), and there was no significant difference in the dose group of the liver and stomach soup with the fluoxetine group (P0.05) in.VTA area (P0.05), the model group had a lower DA content, and the difference was statistically significant (P0.01) compared with the blank group (P0.01); the liver and stomach soup had a significant difference (P0.01). The DA content in the middle dose group and the fluoxetine group was higher than that in the model group (P0.05 or P0.01), in which the liver and stomach soup were low and the medium dose group DA content was lower than that of the fluoxetine group (P0.01). (2) in the PFC region, the Glu content in the model group increased, and the difference was statistically significant (P0.01) compared with the blank group (P0.01); and the liver soothing and the liver of the group were statistically significant (P0.01). The contents of Glu in each dose group and fluoxetine group were lower than those in the model group (P0.01), and the Glu content in the dose group of the liver and stomach soup was lower than that in the fluoxetine group (P0.05), and the difference was lower in the high dose group than in the fluoxetine group (P0.05).NAc region, and the Glu content in the model group decreased. Compared with the blank group, the difference was statistically significant (P0.01). The content of Glu in the dose group and the fluoxetine group in the Shugan Hewei decoction and the fluoxetine group had a significant difference (P0.01), and the difference between the dose group of the liver and the stomach soup was not statistically significant (P0.05).VTA region, and the Glu content in the model group was higher than that in the blank group, and compared with the blank group. The difference was statistically significant (P0.01); the content of Glu in each dose group of Shugan Hewei decoction and fluoxetine group decreased significantly (P0.05 or P0.01) compared with the model group (P0.05 or P0.01), and there was no significant difference between each dose group of Shugan Hewei decoction and fluoxetine group (P0.05). (3) in the PFC region, the content of GABA in the model group decreased and the difference was different from that of the blank group. There was statistical significance (P0.01); the content of GABA in the dose group and the fluoxetine group in the Shugan Hewei decoction group was higher than that in the model group (P0.01). There was no significant difference in the dose group between the soothing liver and the stomach soup and the fluoxetine group (P0.05) in.NAc region, and the GABA content in the model group increased, and the difference was statistically significant compared with the blank group. P0.01; in the liver and stomach soup, the content of GABA in the high dose group decreased, and the difference between the model group and the model group was statistically significant (P0.01). In the liver and stomach soup, the high dose group and the fluoxetine group were compared with the fluoxetine group, the difference was statistically significant (P0.01) in the.VTA region, the GABA content in the model group increased, and the difference was statistically significant (P0.01) compared with the blank group (P0.01); the liver and the stomach were significantly different from the blank group. The content of GABA in the medium dose group and the fluoxetine group decreased, and there was a significant difference between the model group and the model group (P0.01). There was no significant difference between the dose group in the Shugan Hewei soup and the fluoxetine group (P0.05). Experiment four: the effect of Shugan Hewei decoction on the expression of PSD95 m RNA and NR2B m RNA in the NAc region of the depressive model rats (1) NAc District PSD95 The relative expression of M RNA was lower than that in the blank group (P0.01), and the relative expression of PSD95 m RNA in each dose group of Shugan Hewei decoction and the PSD95 m RNA in fluoxetine group was higher than that in the model group (P0.01), and the relative expression of PSD95 m RNA in the high dose group was higher than that of fluoxetine in the liver and stomach soup. The difference was statistically significant (P0.01), and there was no significant difference between the low dose group and the fluoxetine group (P0.05). (2) the relative expression of NR2B m RNA in the NAc area of the model group was reduced, and the difference was statistically significant (P0.05), and the relative expression of the NR2B m RNA in the each dose group of Shugan Hewei decoction and the NAc region of the fluoxetine group increased, and the model was higher than that of the model group. The comparative difference between the group and the group was statistically significant (P0.01), and the relative expression of NR2B m RNA in the NAc region of the Shugan Hewei decoction group was higher than that of the fluoxetine group, the difference was statistically significant (P0.01). Experiment five: the effect of Shugan Hewei decoction on the PSD95 and NR2B expression in the NAc region of the depression model rats (1) the PSD95 relative expression in the NAc area of the model group was lower than that in the blank group. The relative expression of PSD95 in each dose group of Shugan Hewei decoction and fluoxetine group was higher than that in the model group (P0.01), and the relative expression of PSD95 in the high dose group was higher than that in the fluoxetine group (P0.01), and the difference was statistically significant (P0.01), and the low dose group was compared with the fluoxetine group. The difference was not statistically significant (P0.05). (2) the relative expression of NR2B in the NAc area of the model group was reduced, and the difference was statistically significant (P0.01), and the relative expression of NR2B in each dose group of Shugan Hewei decoction and the group of fluoxetine was higher than that in the model group (P0.01), of which the liver and stomach soup were low and the medium dose group NR2B was relatively expressed. The difference was significantly higher than that of fluoxetine group (P0.01). Conclusion 1. ancient doctors had a wide range of depression, including five transport and six qi stagnation, viscera depression and emotional depression. After the Ming and Qing Dynasties, the depression of depression was closely related to the "depression" of modern depression, which was closely related to the etiology and syndrome distribution of depression in the recent ten years. The study shows that emotional disorder is the main cause of depression, liver loss and catharsis are the core of its pathogenesis, and many viscera with influence and heart, spleen, stomach and other viscera are the pathological changes. The pathological changes of Qi and blood body fluid Yin and Yang disorders are its pathological changes. The treatment method is to dredge the liver to solve depression and regulate the pathological changes of the neuron in the PFC area caused by the.3. chronic stress, and the PFC-NAc-VTA nerve. The decrease of DA content, the increase of Glu content, the corresponding changes in the content of GABA, and the decrease of the expression of PSD95, NR2B and m RNA in the NAc region, may be the pathogenesis of depression, which may improve the depression state of the depression model rats, and reduce the damage of the neurons in the PFC area of the depression model rats. The abnormal increase or decline of DA, Glu, GABA content in the PFC-NAc-VTA nerve loop can reverse the abnormal downregulation of PSD95, NR2B and m RNA in the NAc region of the model rats, suggesting that the liver and stomach soup may be the.5. Chinese medicine "liver depression" that can play an antidepressant role in the PFC-NAc-VTA neural loop. The central mechanism may be related to the pathological changes of PFC neurons, abnormal expression of DA, Glu, GABA in PFC-NAc-VTA nerve circuit and synapse in NAc region.

【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R277.7

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