| Objective:We chose this topic,which is based on the theoretical understanding that "liver controlling conveyance and dispersion",and used depression-like emotions with typical changes of liver qi as the entry point to explore how the maternal,which have abnormal dispersion of liver,affecting offspring’s emotions,behaviors and cognitive performance under the theoretical interpretation of Chinese medicine.Meanwhile,deepen the theoretical understanding of "liver controlling conveyance and dispersion and regulating emotions".Pre-pregnancy chronic mild stress(CMS)was used to simulate the stress in modern society,through CORT-BDNF-ERK1/2 signal cascade hippocampal damage to discuss the mechanism of the mood,behavior,and neurodevelopment change of the male offspring,interpretation of the maternal suffered from negative life event influence on offspring,found out the regularity and essence of the emotion and behavior change and the high prevalence of depressive-like affective disorder in offspring.New concepts and hypotheses are expected to propose as while as provide a new perspective for the related research and guide the development of new drugs for offspring depression susceptibility to achieve breakthroughs.Methods:1.Pre-pregnancy depression induced by CMS stress in female rats(stagnation of liver qi)was intervened by Shuyu capsule and Fluoxetine,then we assessed the syndrome manifestations,in order to make sure that the reliability and validity of the evoked target emotions,to verify the reliability of the animal model as well as drug effectiveness.2.Select male offspring for a series of behavioral tests related to emotion and learning and memory like sucrose preference test(SPT),open field test(OFT),forced swimming test(FST),and Morris water maze(MWM)in order to know how the Pre-pregnancy CMS on maternal effected the behavioral phenotypic of offspring.Serum CORT content of male offspring rats in each group was detected by ELISA.The protein contents of BDNF,ERK1/2,p-ERK1/2,CREB and p-CREB in hippocampus were detected by WB.The expression of BDNF m RNA,ERK1 m RNA,ERK2 m RNA and CREB m RNA in hippocampus were detected by RT-PCR.To investigate the effect of CMS stress on CORT content and BDNF-ERK1/2signaling pathway,and the key micro mechanism of Shu Yu capsule intervention on emotion and learning and memory-related behavior of male offspring from female rats with stagnation of liver qi before pregnancy.3.Based on the results of the above signal path changes,we integrated application of adrenalectomy,exogenous CORT import and BDNF microperfusion to detect the depression-like behavior in offspring.ELISA was used to detect the serum CORT content.The protein contents of BDNF,ERK1/2,p-ERK1/2,CREB and p-CREB in hippocampus were all detected by WB.The expression of BDNF m RNA,ERK1 m RNA,ERK2 m RNA and CREB m RNA in hippocampus were detected by RT-PCR.These methods were used to clarify the mechanism of hippocampal injury,and CORT,BDNF-ERK1/2 in offspring depression-like behavior.Results:1.Pre-pregnancy CMS induced stagnation of liver qi depression in female rats.The results of behavioral tests showed that: the growth rate of body weight,the preference coefficient of sucrose water in SPT,and the distance travelled in the central area and the number of entries,the residence time in the central area in OFT were significantly lower in the model group(p<0.0001,p<0.05,p<0.01,p<0.01,p<0.05).After the intervention with Fluoxetine and Shu Yu capsule,the above abnormal changes were effectively alleviated.Molecular biological tests showed that,during pregnancy,the CORT level was significantly higher in the model group(p<0.05).After treated with Fluoxetine and Shu Yu capsule,the above abnormal changes were effectively alleviated.2.The results of behavioral evaluation and mechanism exploration experiment of male offspring rats born from maternal with pre-pregnancy liver qi stagnation.The results of behavioral tests showed that: the coefficient of sucrose preference,the distance,the number of entries and the residence time in the central area of the open field test were significantly reduced in the model group(p<0.05,p<0.05,p<0.05,p<0.05);The escape latency was prolonged during the exploration period in MWM test(p<0.05),and the distance,time,and the number of platform crossing were reduced(p<0.05,p<0.01,p<0.05);Both the number and the time of suspension immobility was increased(p<0.05,p<0.01),along with reduced latency(p<0.05).Both depression-like behaviors and cognitive impairment were improved after prophylactic administration of Shu Yu capsules and Fluoxetine.Molecular biological tests showed that the corticosterone content was increased in the offspring of CMS model group(p<0.05);The content of hippocampal BDNF,p-ERK1/2,and p-CREB protein in the offspring was decreased(p<0.05,p<0.05,p<0.05).The expression of hippocampal BDNF m RNA,ERK1 m RNA,ERK2 m RNA and CREB m RNA in the offspring were significantly decreased,(p<0.01,p<0.05,p<0.01,p<0.05).After the intervention of Shuyu capsule and fluoxetine,the content of CORT in male offspring decreased,and the expression of BDNF-ERK1/2 protein and m RNA showed different degrees of increase.3.To validate the relationship between abnormal male offspring behavior and BDNF-ERK1/2 pathway and hippocampal damage.After adrenalectomy operation,compared with the sham group,the ADX+CORT100mg group showed a significant decrease in the sucrose preference coefficient(p<0.05);the distance,the number of entries,and the residence time in the central area have decreased(p<0.01,p<0.05,p<0.05);In the FST,an increase was reported in suspension immobility time and immobility number(p<0.01,p<0.01),and a reduced latency period(p<0.01).In Y maze,the rats in ADX and ADX+CORT 100 mg groups showed learning and memory dysfunction,the total arm entry times,rotation times and the scores of spontaneous rotation were significantly reduced(p<0.05,p<0.05,p<0.05).Molecular biological tests showed that compared with the Sham group,serum CORT levels were reduced in the ADX group(p<0.05);hippocampal BDNF and p-ERK1/2 protein levels were increased(p<0.05,p<0.05);BDNF m RNA,ERK1,ERK2 m RNA expression was increased(p<0.05,p<0.01,p<0.05).Compared with the ADX group,the ADX+CORT100mg group had increased CORT content(p<0.01);hippocampal BDNF,p-ERK1/2and p-CREB content were decreased(p<0.05,p<0.01,p<0.01);BDNF m RNA,ERK1 m RNA expression was decreased(p<0.05,p<0.05).After BDNF microinjection,the results of behavioral tests showed that in the CORT model,there was a significant increase in body weight and the sucrose preference coefficient compared to the saline control group,and the travelled distance,the number of entries and the time spent in the central area in the OFT(p<0.05,p<0.01,p<0.05,p<0.05,p<0.05);In the forced swimming test,decreased forced swimming suspension immobility time and number of immobility(p<0.01,p<0.0001)and prolonged latency period with significant differences(p<0.05)were seen.During the exploration period,time in target quadrant and number of platform crossings were both increased(p<0.05,p<0.05),with escape latency shortened(p<0.05).After BDNF treatment,ELISA results showed that: CORT content decreased significantly(p<0.01);WB assay results showed that: BDNF,p-ERK1/2,p-CREB protein content increased(p<0.05,p<0.05,p<0.05);RT-PCR results showed that BDNF m RNA,ERK1 m RNA,ERK2 m RNA,CREB m RNA expression were significantly increased(p<0.01,p<0.01,p<0.05,p<0.05).Conclusion:1.The maternal generation of liver qi stagnation can have a negative effect on female fetal pregnancy and the growth and development of offspring through the functional connection between liver and kidney,liver and the Chong,Ren,Du,dai pulse.2.An effective rat model of depression with liver-qi stagnation syndrome can be prepared by chronic mild stress paradigm.The increase of maternal CORT content is a risk factor for fetal growth environment in maternal uterus during pregnancy.3.High-level CORT of offspring selectively acts on hippocampus,adversely affect emotions and learning memory functions through downregulation of the BDNF-ERK1/2 signaling pathway.Shu Yu capsule can reduce the influence of intrauterine adverse environment on the development of offspring by correcting the abnormality of glucocorticoid level and the pathway.4.By inhibiting CORT overproduction or exogenous supplementation of BDNF can effectively increase BDNF-ERK1/2 expression and reduce hippocampal damage. |
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