| Research purpose and senseMany clinical and experimental studies confirmed that chronic stress play an important role in occurrence and development of many diseases, but the exact mechanism has not yet been fully explained. Thus currently, the treatment measures of anti-stress have many defects, such as uncertain or a single target, obvious side effects, and so on that greatly limited the clinical application of these drugs. Xiao Yao San as a Chinese herbal formula is an official prescription promulgated in the Pharmacopoeia of the Imperial Medical Bureau in the Song Dynasty, and it has been commonly used in the treatment of depression related diseases until today. Nearly a thousand years of clinical practice proved that it has the effects of coursing the liver and resolving depression, fortifying the spleen and supplementing the blood. Modern clinical and experimental studies have confirmed that the mental stress-induced learning and memory decline, depression, anxiety and other symptoms can be improved or eliminated by treating with Xiao Yao San, it has exact effects of sedation, analgesia, anticonvulsant, anti-anxiety, and anti-depression. In this study, hypothesis on mechanism of anti-chronic stress of Xiao Yao San has been proposed on the basis of analysis based on related research. And around this hypothesis, a series of experiments was conducted to investigate the possible central mechanism of the anti-chronic stress of Xiao Yao San, in the process of that the cultured rat hippocampal neurons were used as model.Research methodsTo establish a primary culture method of hippocampal neurons of new born rats, prepare serum of rats suffered with chronic stress, serum of chronic stress rats treated with Xiao Yao San, and the serum of normal control group, and use dizocilpine as a tool for drug, then treated each group with different factors according to the experimental design, and study the effects of the serum of chronic stress rat model and the serum treated with Xiao Yao San on the cell viability, expression of NMDA receptor subunits'mRNA, intracellular free Ca2+ concentration and the expression of intracellular glucocorticoid receptor (iGR) in the cultured hippocampal neurons of rats.1 Hippocampal neurons of newborn rats within 24 hours were isolated by the method of trypsin digestion combining with that of mechanical dispersion, which were planted with medium containing 10% fetal bovine serum, and maintained feeding with serum-free medium. Identification of neurons and its purity were conducted by neural microtubule-associated protein (Map-2).2 30 rats were randomly divided into 3 groups:Normal control group (saline 2ml/d, ig, not to be stimulated by stress), chronic stress model group (saline 2ml/d, ig, and 1 hour after, each to be stimulated by stress), chronic stress model treated with Xiao Yao San group (Xiao Yao San decoction 2ml/d, ig, and 1 hour after, each to be stimulated by stress). The multi-stress model by Cart was improved and used in this experiment. Stressors used in this experiment include electric shock through feet (30V,0. 1Hz, lsec stimuli and lsec interval each time,30times/d), ice water swimming (4℃or so,5min), prohibition cf water (24h), fasting (24h), binding (4h). Each stressor was assigned randomly everyday, the same stressor in adjacent days and regularity of the stimulaticn or adaptability of the rats must be avoided. Modeling was lasted for 21 days. Blood of each group were collected by artery puncture through abdominal aorta after administration in 1 to 2 hours on the last day of modeling, then separated serum by centrifugation at 3000 rpm, serum of each group were mixed respectively, and inactivated in water bath at 56℃for 30min, then filtrated bacteria by using filter of 0.22μm under sterile conditions. Packed serum of each group into lml per tube and marked clearly and then stored at-20℃in refrigerator.3 Hippocampal neurons were divided into 7 groups after cultured for 8 days: (1)control group (normal cells, no treatment), (2)normal serum control group, (3)normal serum+glutamate group, (4)chronic stress model serum+glutamate group, (5)chronic stress model serum+glutamate+MK-801 group, (6)Xiao Yao San treatment serum+glutamate group, (7)Xiao Yao San treatment serum+ glutamate+MK-801 group. Each group was pretreated with serum or together with MK-801 for 20min, then after that added glutamate for the following 30min. Cell viability of each group was detected by MTT assay. The methods of Taqman fluorescence probe real-time quantitative PCR and relative quantification were used to determine the expression of NMDAR subunits mRNA. Intracellular free Ca2+ concentration in cultured hippocampal neurons in the simulated micro-environment of chronic stress and after intervention with the serum treated with Xiao Yao San were detected by confocal laser microscope at the same period of time. The methods of Western blot and relative quantification was used to determine the protein expression of iGR in the cultured rat hippocampal nerve cells after being conducted by the serum of chronic stress rat model and the serum treated with Xiao Yao San.Results1 The structures of nerve cells were clear and integral, and the purity of the neurons is up to 85.71% after cultured for 8 days.2 Hippocampal neurons viability of the normal serum+glutamate group and the chronic stress model serum+glutamate group were relatively low, and there were no significant differences in other groups. Hippocampal neurons viability in each group was approximately the same, which can meet the needs of follow-up assay.3 Neurons viability was affected obviously, NR1, NR2A, NR2B mRNA were all increased in different degrees, concentration of free Ca2+ intracellular increased significantly, and the protein expression of iGR was decreased significantly when micro-environment of chronic stress simulated by glutamate and serum of rat model with chronic stress was effected on the cultured hippocampal neurons.4 When cultured hippocampal neurons were prepared with MK-801, viability of the cultured nerve cells could be improved markedly, NR2A and NR2B mRNA were significantly decreased, concentration of free Ca2+ intracellular in cultured rat hippocampal neurons was decreased, but MK-801 has failed to improve the situation of increased Ca2+ influx and the downregulation of iGR expression.5 Neurons viability showed a trend of increasing, the expression of NR2A mRNA was upregulated significantly but that of NR2B mRNA was downregulated and NR1mRNA expression was not significantly decreased, concentration of free Ca2+ intracellular hadn't increased significantly and chronic stress-induced downregulation of iGR could be against significantly though there still were differences between the group with control group. When serum of chronic stress treated with Xiao Yao San was effected on the neurons.6 When MK-801 and serum of chronic stress treated with Xiao Yao San were used together, hippocampal neurons viability and expression of NR2A and NR2B mRNA were closer to normal, and glutamate had failed to lead to Ca2+ overload, chronic stress-induced downregulation of iGR could be significant(?)y antagonized and there was no significant difference between the group with control group. Conclusion1 No need to strictly control the digestion time and used the Ara-C to purify cells, neurons with good condition and high purity were obtained.2 The state of chronic stess micro-environment could be simulated by using serum of chronic stress rat model together with glutamate on the cultured hippocampal neurons, which caused the similar pathological results with that of chronic stress.3 Imbalance state of NR subunits under chronic stress could be corrected, and the normal ratio of NR2A AND NR2B could be maintained by using serum of chronic stress treated with Xiao Yao San, and it had effects of inhibiting Ca2+ overload and antagonizing chronic stress-induced downregulation of iGR in hippocampal neurons.4 Ca2+ overload and downregulation of iGR induced by chronic stress could not be completely blocked by using MK-801, there might be some other signaling pathways other than that of Glu-NR that interacted in the processes. Besides the effect of inhibition the activation of NR caused by MK-801, serum of chronic stress treated with Xiao Yao San played a role in neurons stability maintenance, and protected neurons against injury induced by chronic stress when MK-801 and serum of chronic stress treated with Xiao Yao San were used together. It may work through a variety of signaling pathways including Glu-NR-Ca2+-iGR to maintain the steady-state of the neurons, and then to protect neurons from the neurotoxic effects of excitatory.SummaryMicro-environment of chronic stress around neurons in vivo could be simulated by serum of chronic stress rats together with glutamate, which could cause excessive activation of NR and calcium overload intracellular, subsequently reduced synthesis of iGR, and ultimately led to damage of hippocampal neurons and pathological results similar to chronic stress. Xiao Yao San treatment serum could correct the imbalance state of the NR subunits mRNA expression, maintain the normal proportion of NR2A and NR2B and calcium homeostasis intracellular, and keep iGR from decreased expression caused by chronic stress. It played a role in maintaining stability of hippocampal nerve cells, and protected neurons against the effects of chronic stress. Application of MK-801 made it clear that Xiao Yao San may work through multiple signaling pathways including that of Glu-NR-Ca2+-cAMP-iGR. |
PDF Full Text Request