Effect Of Liver Qi Stagnation Before Pregnancy On The Expression Of BDNF And NMDA Receptor Of Female Rats’ Male Offspring And Regulated Action Of Chaihu Shugan Powder

Research backgroundThe theory of TCM thinks that kidney governing reproduction.However,the experimental and clinical studies show that the theory of liver governs reproduction has been widely accepted.Liver controlling dispersion,which can make essential qi distribute,and it also can store blood and regulate the volume of blood.According to the theory of"essence and blood sharing with the same origin",if liver blood is enough,the essence of reproductive can be transformed.If the function of liver controlling dispersion is normal,qi movement is smoothness,qi and blood is harmonizes,Chong and Ren channels is coordination,so it can make man’s sperm strong and a woman’menstruation normal.Liver channel surrounds the genitals,which directly contacts with reproduction.Therefore,there is the theory of liver governs reproduction.The experimental results show that the stress in different stages of the mother is not conducive to the development of the nervous system of the offspring,which may cause the abnormal behavior of the offspring to some extent.Most of the previous studies on prenatal stress before and after the domestic and foreign literatures have focused on the effects of post pregnancy stress on offspring,but few studies have been done on the effects of prenatal stress on offspring.The syndrome of liver depression is often caused by chronic restraint stress,so it is of great significance to study the effect of depression before pregnancy on the development of offspring.Therefore,based on the theory of"liver governs reproduction",the model of maternal with liver depression syndrome before pregnancy was modified by chronic restraint stress method,then mated with normal male rats and produced offspring rats as the research object of this subject.The study trys to study hippocampal neurons morphology and the change of the number of the male offsprings,analyze whether its mechanism has a relationship with abnormalities of hippocampal brain-derived neurotrophic factor（Brain-Derived neurotrophic,factor,BDNF）and glutamic acid N-methyl-D-aspartate（N-methyl-D-aspartate,NMDA）expression of 2A and 2B receptor of male offspring rats.After modeling,Chaihu Shugan Powder was used to the female rats in order to know whether it have effect on the expressions of BDNF,NR_2A and NR_2B of offspring male rats in the hippocampus.This subject is the following research which effect of liver depression on reproductive function,to further research on the expression of BDNF and NMDA in hippocampus and neural development of survival offspring rats,to provide a scientific basis and new ideas for eugenics.Methods18 female Wistar rats were randomly divided into normal group,model group and Chaihu Shugan Powder group by Excel,with weight of 240±20g and 75-80 days of age,each group was 12 rats.The weight of 9 male Wistar rats was 220±20g,and age was85-90 days.After 5 days of adaptive feeding,the female rat models of liver depression were modified by chronic restraint stress.9 male Wistar rats were fed normally.The macroscopic characteristics of rats were observed at zeroth,7,14 and 21 days of the experiment,and the weight of the rats was weighed.The behavior of the rats was recorded by the elevated plus maze test on the twenty-first day of the experiment.After 21days of chronic restraint stress and after the assessment is completed,the female rats and male rats were cage feeding together in the same cage according to 2:1.Examing vaginal smear of female rats under microscope at 8:00 everyday,and making fine bolt can be seen for pregnant signs.Pregnancy female rats were reared in single cage until to dilivery.21days later,male offspring were divided into model offspring group,normal offspring group and Chaihu Shugan Powder offspring group（Chinese medicine group）according to their parents.The expression of BDNF,NR_2A and NR_2B protein in hippocampus of male offspring rats were determined by immunohistochemical method;the expression of BDNF,NR2A and NR2B mRNA of hippocampal in male offspring rats were detected by qRT-PCR;neurons in cell morphology and abnormal changes of male offspring rats were observed by HE.ResultModeling and evaluation of female rats with liver depression after 3 weeks,compared with the normal group rats,rats in model group showed that yellow fur and dull,listlessness and burnout,less hiding in the corner,their eyes had abnormal secretions,auricle color fades,drinking and eating were reduced,loose stool,the liver depression symptoms was obvious.While the mother state of Chaihu Shuga Powder group was better than in model group,the appearance and performance.were close to the normal group.2 immunohistochemical resultsIn hippocampus CA₃ region,compared with the male offspring of normal control group,the the expression of BDNF proteion of model offspring group male rats was decreased（P<0.01）,the expression of NR_2B receptor of model offspring group male rats was decreased（P<0.01）,there was statistical differences,and the expression of NR_2A receptor was decreased,but it has no significant difference;compared with the male offspring of model offspring group,the expression of NR_2A and NR_2B receptors protein were increased,and it had significant differences（P<0.05）,but there were no significant differences between the expression of BDNF protein.In hippocampus DG region,compared with the male offspring of normal control group,the expression of BDNF and NR_2Awere decreased（P<0.01）,and there was statistical differences,and the expression of NR_2B was also decreased but there was no statistical difference;compared with the male offspring of model group,the expression of NR_2A proteion was increased（P<0.01）,the expression of NR_2B proteion was also increased（P<0.05）,and they all have significant difference,while there was no significant diffetence in the expression of BDNF.3 qRT-PCR experimental resultsCompared with the normal offspring group,the expression of BDNF mRNA in the hippocampus of male rats in model offspring group was reduced（P<0.01）;the expression of NR_2B mRNA in the hippocampus of male rats in model offspring group was also reduced（P<0.05）,the expression of NR_2A mRNA was decreased,but there was no significant difference;compared with the modle offspring group,the expression of BDNF mRNA in the hippocampus of male rats in TCM offspring group was increased（P<0.01）,and the the expression of NR_2B mRNA was also increased（P<0.05）,the expression of NR_2A was also increased,but it had no significant difference.4.HE staining resultsCompared with the normal offspring group,the number of damage neurons in hippocampus CA₃ and DG regions of male rat offspring in model offspring group was increased,and the number of synapses was decreased.The nerve cells were arranged in disorder and sparsly,and appeared karyopyknosis;there was no significant difference between model offspring group and male rats of TCM offspring group in the number of synapse and nerve cells in hippocampus CA₃ and DG regions.Conclusion1.21 days of chronic restraint stress could reproduce a comparatively ideal of model rats with liver qi stagnation,and Chaihu Shugan Powder could alleviate symptoms of maternal with liver qi stagnation syndrome.2.Female rats with liver qi stagnation before pregnancy could reduce the expression of BDNF and NR_2B receptor protein in hippocampus CA₃ region of male offspring rats,reduce the expression of BDNF and NR_2A receptor protein in hippocampus DG region of male offspring rats,and it also could down-regulate the expression of BDNF and NR_2B mRNA and injury hippocampus neuron,these phenomena may affect the learning and memory function of male offspring rats.3 Chaihu Shugan Powder has certain curative effect on liver qi stagnation syndrome,which may indirectly effect of maternal before pregnancy with liver qi stagnation syndrome to male offspring rats through the matrix and reversing the expression of between NR_2A and NR_2B receptor protein of male offspring rat in hippocampal CA₃ and DG regions.