| Depression is one kind of frequent mental disorder and the lifetime prevalence is up to 15 ~20 %. The high disease incidence and harm induced by it have gotten more and more attention in medical science realm. Antidepressant drugs have potential market demand. The manufacture and exploitation of new anti-depression drugs in traditional Chinese medicine are still in the first step, the research needs to be improved, but the potentiality of market exploitation is enormous.Thymoleptics based on MA strategy emerge effective hysteresis in clinic and almost 30% patients with major depression have no reaction to any Thymoleptics. Because the pathological factors of depression are complex, there exist many potential targets of anti-depression. Many evidence of animal experiments and clinical researches indicate that we should explore new anti-depression drugs and promote antidepressant effects surpassing MA synapse strategy.Also, the adaptive changes of thymoleptics based on MA stratege to NMDAR in definite encephalic region, like downregulating NMDAR function and emerging effects resemble to NMDAR antagonist, may be the essence of their antidepressant effects. Many animal experiments and clinical research es indicate that NMDAR antagonists have antidepressant effects. Rivalry of NMDAR function can improve the symptoms of severe patients and the effects are better than thymoleptics. So we conclude that the antidepressant strategy of regulating NMDAR may be more prior than that of traditional MA. The big problem of NMDAR antagonists is the side effect leading to mental disease. There are several controlling sites in NMDAR complex and the heterology of NMDAR provide some selective drug targets. The antagonist focused on them can prevent the excitable toxicity but will not block all NMDAR, avoiding the emergence of side effects.To sum up, turning the aberrant glu function to normal is an important feature of anti-depression effects, which is fulfilled by acting directly on the glutamatergic synapses targets or on others neurotransmitter system like 5-HT,NE,GABA to remotely affect glutamatergic system.Our working team introduced the hypothesis for the first time in 1999: there eixist some concrete central nerve mechanisms in addition to the whole regulation of the " liver" function(liver controlling dispersion, adjusting emotion) in TCM on sympathesis-adrenal medulla and the hypothalamus-pituitary gland-internet system. Meanwhile there exists unity between part and whole in mechanism of action. We take Jia Wei Si Ni San(JWSNS) as a representative regulating liver formula to regulate the psychological stress reaction and have done some relative researches, which have tentatively confirmed our investing hypothesis.Taking the depression induced by psychological stress as the cutting-point of ourresearch, integrating the latest pathomechanism research reviews of depression and our previous working foundation, we further prefer a hypothesis: the central nerve mechanism of the "liver" controlling dispersion and adjusting emotion to anti-depression is acted by down-regulating the NMDAR function and their excitable toxicity, protecting the hippocampus neurons by revealing the stress damage and regulating HPA axis.Taking the depression caused by psychological stress as research object, combining the mechanism of action of the regulating "liver" formula, according to the related theory of formula-syndroms-effects-zangfu in TCM, the research further reveals the essence of "liver" function and scientific connotation of "liver" theory, riches and innovates the theory of state of viscera and emotion pathopoiesis of TCM, provides scientific foundation for regulating "liver" formula to prevent and cure psychological stress diseases. Meanwhile it also provides ideas and technology warnings for investigating different ZangFu function and pathopoiesis mechanism of etiological factors.The achievements of the item will provide guiding and theory base for preventing and curing emotion diseases. Our country lacks of competition ability in antidepressant drugs market for a long time, and many medical businesses have taken more attentions on developing TCM to exert anti-depression effects based on our country' market style. The manufacture and exploitation of new drug for anti-depression in traditional Chinese medicine are still in the first step, the research depth and width are still need to be improved, but the potentiality of market exploitation is enormous. This achievement will be helpful for further clarifying the etiopathogenesis of depression and developing new antidepressant drugs of TCM with independent intellectual property rights, which will elevate competition ability and promote the national economical development. Part one research reportsSection one Effects of regulating "liver" formula-JWSNS on weights and sucrose preference of CMUS model.Establish the modified chronic mild unpredictable stess model(CMUS) according to Willner P. Wistar rats, grade SPF, male, 180 - 220g in weight. Offered by animals' centre in Guangzhou university of TCM. Model rats were placed in one independent room and accepted different stess stimulus within 21 days. The stressors consist of restraint for lh, 45° cage tilt for 7h, soiled cage (250 ml water in sawdust bedding) for 17h, new invader(two cages combined) for 23h, persistent illumination for 36h, empty bottle for lh, food and water deprivation (23h each time). All rats were divided into 4 groups according to the weight and base Sucrose Consumptions: Normal group, without any stimulus;CMUS group, applied stimulus without formula;JWSNS group, offered 2ml JWSNS concentration every day at AM 8: 30;MK801 group, offered MK8010.2mg/Kg/d with intraperitoneal injection. Normal group and Model group were given 2ml distilled water. The normalgroup was placed in different room compared with others.Composition and dosage of JWSNS: Bupleurum Root 15 g> White Peony Root 15 g^ Bitter Orange 6 gs Wolfberry Fruit 15 g> Cape jasmine Fruit 5 g^ Rehmannia Root 18 gN Concha haliotidis 30 g. The formula are bought from the medical material company of Guang Zhou and appraised as pure crude formula by the drug department. Take the traditional Chinese medicine into coarse powder and soaked in eight multiple warm water for 0.5h, simmering decoction for 4h after boiled. Attention: even mixed and filtered with three-tier gauze;the 2nd and 3rd decotation with 6 multiple water for 2h. merged the thrice physic liquor and concentrated to 1. 69g/ml and preserved it in refrigerator at 4 centigrade after being cooled.After food and water deprivation for 23h later, the Sucrose Consumptions test was taken at PM 15: 00-16: 00 on the last day of the procedure of 21 days CMUS. In the mean time, the weights were tested. All the data were expressed with X+S and analyzed by t-test and ANOVA with SPSS statistics software.There' s no difference between different groups in weight and sucrose preference (p>0.05) before stress procedure. After 21 days of model-making, the weight and the added-weight of model group, JWSNS group and MK801 group are lower than normal group(p<0.01), while there' s no difference among the 3 groups. This indicates that CMUS can decrease the weights of rats, while JWSNS and MK801 have no effects on that. The sucrose preference of model group is significantly lower than that of normal group (p<0.01) , while the other 3 groups have no difference. This indicates that CMUS can decrease the sucrose preference, while JWSNS and MK801 can reverse the tendency deduced by CMUS.Through the sucrose preference test on CMUS model, we can find that MK801 has effects of anti-depression. MK801 is one kind of nonselective NMD A receptor antagonists, which can traverse the blood brain barrier. The result thoroughly demonstrates that the excessive activation of NMDAR may be one of the pathomechanism of depression induced by chronic stress and down-regulating the NMDAR function may be an important strategy to treat depression.We concluded that formula of regulating "liver" therapy can regulate the stress reaction based on our previous research . This research result further demonstrates that JWSNS has the effects of anti-depression, which is in coincidence with the clinical reports. But it still needs to be tested whether JWSNS exerts anti-depression effects by acting on NMDAR. Section two The protective effects of regulating "liver" formula on hippocampusneuron damage of CMUS ratsGroups, formula and medication are the same as section one. The apoptosis rates of hippocampus neurons are detected with flow cytometry, the TTC dyeing of hippocampus brain slices ex vivo is tested with chromatometry.The results indicate that there exists enormous neurons apoptosis in model rats. Compared with the model group, there is little apoptosis cells in normal group (P<0.01) , which indicate that the 21 days' CMUS model can evoke hippocampus neurons apoptosis.From the results of brain slice TTC dyeing, we can find that OD values and damage rates of hippocampus slices in model group are obviously lower than normal group (p<0.01), while JWSNS group and MK801 group can increase the OD values (p<0.05,p<0.01) and decrease the damage rates (p<0.05,p<0.01).The results of TTC dyeing show that 20% serum containing effective components of JWSNS can increase the OD value of brain slices damaged by high density Glu(200umol/L) (p<0.01) and decrease the damage rates (p<0.01),while giving 200umol/L Glu solo to brain slices, the OD values are obviously lower than normal group(p<0.01) and hippocampus damage rates are obviously higher(p<0.01).From the tests of hippocampus neurons apoptosis rates and TTC dyeing, we confirm that there truly exist hippocampus damage and apoptosis in CMUS model rats. MK801 can totally reverse the tendency, which fully illustrate that NMDAR play an important role in hippocampus damage during stress and interfering the NMDAR function is the main treating strategy in preventing and curing hippocampus damage induced by depression.It is sure that JWSNS has some protective effects on CMUS model rats(p<0.05), but not as good as MK801. We can consider that JWSNS also could reverse the excitable toxicity induced by high concentration of Glu. But further study still needs to interpret how JWSNS regulate NMDAR to protect hippocampus neurons and anti-depression.We can consider that the protective effects of JWSNS on hippocampus neurons are parts of its pharmacological foundation of its antidepressant effects based on the effects of MK801 ,JWSNS and the pathomechanism of hippocampus neurons degenerative disease and neurons aregeneration.Section 3 Effects of JWSNS on hippocampus NMDAR channels of CMUS model ratsGroups, formula and medication are the same as section one. The single channel current report and analysis of NMDAR in rats hippocampus: attach on cell patch clamp. We apply positive ion imaging system to detect the Ca + concentration in single cell of hippocampusneurons.The results indicate that the opening probability of NMDAR channels in model group are higer than those of normal group (p<0.05), while JWSNS and MK801 can significantly decrease the opening probability of NMDAR channels in stressed rats (p<0.05, p<0.01). There' s no statistical significance among these four groups in the average opening time of NMDAR channels in hippocampus (p>0.05). Ca concentration of model group is higher than that of normal group (p<0.01), while JWSNS and MK801 can obviously decrease the concentration (p<0.01).From our research, we can find that the opening probability of single NMDAR channel is significantly higher in CMUS model rats compared with normal rats, although there' s no statistically significance in the average opening time, but the data of CMUS model rats are still higher than normal rats, which fully indicate that the single NMDAR is hyperfunction in hippocampus of CMUS model rats. Bidder, etc found that the opening probability of NMDAR might be relative to their C- terminal phosphorylation. So we think that i s the reason why the ratios in chronic stress depression are higher. To sum up, the degree of C- terminal phosphorylation of NMDA subtypes becomes higher and the activation of NMDAR signal transduction pathway in CMUS model rats can initiate a series of events like the emergence of oxygen free radical, downregulation of BDNF, resulting in the damage or death of hippocampus neurons.The intracellular free Ca2+ concentration can also reflect the active state of NMDAR . There is fine goodness of fit between the results of electrophysiological characteristics of NMDAR channel and Ca2 + concentration of hippocampus neurons in model group.Some researches also indicate that the changes of active state of NMDAR in hippocampus are the key mediate factors of behavior dysfunction after stress. And the probable mechanism is that continuously activating the NMDAR causes damage to hippocampus pyramid neurons, inducing depression. Different types of stress lead to the changes of the NMDAR active by effecting their synapse structures and function plasticity in hippocampus and amygdala , which are closely relative to behaviors. Hyperactivity of NMDAR leads to the atrophy of hippocampus, which is relative to depression.But JWSNS and MK801 can both suppress the hyperactivity of single NMDAR channel and decrease intracellular Ca2 + concentration, which indicate that during chronic stress depression , hippocampus NMDAR are hyperfunction and regulating "liver" formula can decrease their activement, preventing the increasing of the phosphorylation. of their subtypes. Regulating "liver" formula regulate excitable toxicity signal transduction mainly by changing hippocampus NMDAR channels" dynamics features, which relates to its protective effects on hippocampus neurons and anti-depression. Combined with results inthe second part that JWSNS can prevent hippocampus neurons from damage by high concentration of Glu ,we tentatively conclude that JWSNS protect hippocampus neurons of chronic stressed depression individuals from damage probably by regulating NMDAR function., which may be one of its direct effect mechanisms, but its concrete effect targets should be further studied.Section 4 Effects of JWSNS on the expression of NMDAR subunits of CMUS model ratsApply chronic mild unpredictable stress model, and the stressors are in the list: electric attack(voltage 30V, electric current intensity 1.0 mA, 1 stimulus each 1 min and last for 10s, 30 min), swimming in 4°C water(5 min each time), clipping the tail (1 min), food or water deprivation (24h each time), and restraint(2h each time), all methods above were arranged randomly during 21 days, one method every day, then the materials were drew out on the 22nd day.Groups, formula and medication are the same as section one. Detecting method of NR1, NR2A, NR2B: immunohistochemistry method.The positive staining neurons of NR1, NR2A, NR2B distribute in many realms of hippocampus, which looks buffy, round or oval .Compared with the color in endochylema, it is more distinctive in cellular membrane, the cell bodies are bigger,. The positive staining neurons of NR1, NR2A, NR2B of model group are more than those of normal group (PO.01), while JWSNS can significantly decrease the numbers (P<0.01,P<0.05).From the result we can find that the expression of NR1 and NR2B increased in model group(P<0.01). The expression of NR2A increased also, but there is no statistical significance. The results indicates that the NMDAR increased in stress induced depression and the expression disorder of NMDAR subunits would induce activement changes of receptor compounds, which were the chief factors in determining the affinity of NMDAR to its ligands. The expression changes of NMDAR subunits will affect the channel activities .which are in accordance with the results of section three. Regulating liver formula-JWSNS can obviously decrease the expression of NR1, NR2A, NR2B (P<0. 01), which are in coincidence with the reports that thymoleptics can decrease the expression of NMDAR subunits and make NMDA adaptable to the change. This may have close relations with the effects of JWSNS which can change hippocampus NMDAR channel activities and decrease the intracellularCa2+ concentration.We just tested the total amount of NMDAR (activated and unactivated) in hippocampus, so the result can only provide references to the NMDAR subunits ' expressive changes under activated state of stress induced depression. From the results, the expression of NR2B increased significantly while NR2A had no change in model rats, which partly indicated that mainly those NMDAR located outside synapses increased in stress induced depression, which are the reasoncausing excitable toxicity to hippocampus neurons and Ca overloading and neuron apoptosis. Recipes of regulating liver therapy-JWSNS has effects on both NR2A and NR2B, which has no specificity to any of the subunits.Part 3 Summary discussion and conclusionSection 1 Summary Discussion 1 Behavior effects of anti-depression by Regulating "liver" formula-JWSNS.Central NMDAR are the key substance of the brain functions like emotion, learning and memory and participate in many pathological process of neurons and emotion diseases. NMDAR is very sensitive to some psychological medicines. Kalivas found that NMDAR expressed in cerebral cortex, hippocampus, corpora striatum, amygdala, etc in high-density by radioligand binding method, while those realms are considered to be related to recognition and emotional function.In constraint and forced swimming stress, we use C-fos as probe and find hippocampus neurons were largely activated, while the activation could be depressed by NMDAR antagonist, which indicate that Glu-neurotransmitters have taken part in it. The increase of Glu in hippocampus caused by constraint and forced swimming stress emerged in the first several minutes and the persistence time are longer than that of stress.The activity and quantity of NMDAR in brain after stress is closely related to irritability behavioral disturbance. Before stress, perfusing NMDAR block agent into the brain can decrease irritability anxiety and depression of animals, which hint NMDAR may have important function in behavior effect of brain mechanism induced by stress. So NMDAR have important function in depression induced by chronic stress, and excessive activation of NMDAR is related to depressed emotions of animals. But this research find regulating "liver" formula-JWSNS has function of resisting to depression, and improve "anhedonia" behaviors of depressed animals, which may be mediated by adjusting the function of NMDAR.(2) Regulating "liver" formula-JWSNS can resist hippocampus neurons excitable toxicity and protect hippocampus neurons.One mechanism of hippocampus damage induced by stress is Glu induced by GC and the excitability of NMDAR that Ca +depended on. Many research certified that stress and the increasing GC can result in the advance of Glu concentration outside hippocampus cells. Glu make use of NMDAR of cell envelope to promote Ca + inflowing, and increase the level of Ca + in cell, which activate related enzymes that Ca + depended on or regulated, dissolve cystoskeleton, decompose some protein, changesome gene expression, and result in the death or deletion of nerve cells. So exitotoxicity of Glu is fundamentally mediated by NMDAR, and the main function of NMDAR in depression is that exitotoxicity caused by excessive activation which result in the easy damage or death of center system, especially to hippocampus neurons.The research find that hippocampus neurons manifested obvious damage, overexciability of NMDAR and increasing nomadic Ca2+ concentration. But not only regulating "liver" formula-JWSNS can degrade the function of NMDAR, step down Ca2+ concentration, resist damage of hippocampus neurons, but also it can obviously resist exitotoxicity function in cell model of hippocampus neurons damaged by high concentration Glu. That is to say adjusting NMDAR function is an important mechanism of JWSNS' anti-damage effects of hippocampus neurons of depression. (3) The way of regulating " liver" formula-JWSNS adjusting NMDAR channelRegulating " liver" formula-JWSNS can degrade opening probability of NMDAR channels of depression, reduce opening time, decrease function of NMDAR channel, and drawdown calcium signal mediated by NMDAR channels, thereby regulating "liver" formula-JWSNS can resist apoptosis of hippocampus neurons and protect hippocampus neurons. (4) Regulating "liver" formula-JWSNS can regulate HPA axis" excitability of stress induced depressionTraditional opinions consider that the excitability of HPA axis is related to hyperthalamus, pituitary, adrenal. General opinions now consider hyperthalamus and hippocampus are the two main brain realms of regulating the excitability of HPA axis. Hyperthalamus control the excitability of HPA axis under normal condition while hippocampus control that under stress condition.The influence of NMDAR on HPA axis: NMDAR can promote the gene expression and release of CRH to activate HPA axis by acting on hyperthalamus and other realm like hippocampus. In addition, NMDAR can depress the negative feedback of HPA axis induced by stress by regulating the expression of GR. So NMDAR can activate HPA axis directly or by depressing the negative feedback of HPA axis under stress condition. To sum up, the excessive activation of HPA axis depends on the activated degree of NMDAR channels of hippocampus, while depressing the activation of NMDAR can suppress the overexcitation of HPA axis.Our previous researches found that JWSNS can decrease the CORT in blood plasma and ACTH, CRH in hypothalamus and blood plasma to suppress the accentuation of HPA induced by chronic psychological stress. Combined with this result, we can conclue that JWSNS could fulfill its effects of depressing the accentuation of HPA induced by chronic psychological stress by downregulating the NMDAR function.The analysis of anti- depression using the theory of the liver controlling dispersion.Our previous research suggest that there is central nerve mechanism for the theory of liver controlling dispersion and adjusting emotion, which has been proved tentatively. The central nerve mechanism of So called "dispersion" is relative to regulating hypothalamus-pituitary-adrenal axis, maybe it is relative to adjusting the expression of various neurotransmitters and their synthetases, neuron peptides, hormones, CAMP system, immediate early genes and fos, which shows the virtue of many levels and targets. The brain areas Include amygdala hippocampus, ect.Taking the depression induced by psychological stress as the cutting-point of our research, the results further prove that the regulatinging mechanism on hippocampus of liver controlling dispersion .which is down regulating EAA receptors, protecting hippocampus neurons, ensuring the controlling function of hippocampus on HPA axis, so that the body can shut off the stress reaction normally, preventing HPA axis from excessive activation.Section 2 conclusions1 Regulating "liver" formula-JWSNS can mediate antidepressant effects by regulating NMDAR functions2 Regulating "liver" formula-JWSNS can resist to hippocampus neurons excitable toxicity and protect hippocampus neurons.3 Regulating "liver" formula-JWSNS downregulate the overexciability of NMDAR of chronic stress depression mainly by adjusting the NMDAR channel dynamics characteristics of hippocampus neurons and the degree of subunit phosphorylation and so on.4 Regulating "liver" formula-JWSNS can regulate HPA axis function of stressed depression by adjusting NMDAR function5 Regulating mechanism on hippocampus of liver controlling dispersion is that down regulating EAA receptors, protecting hippocampus neurons, ensuring the controlling function of hippocampus on HPA axis, so that the body can shut off the stress reaction normally, preventing HPA axis from excessive activation.
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