| 1 ObjectiveThere are some corresponding laws between prescriptions and syndromes,and the selectivity of syndromes in the study of traditional Chinese medicine(TCM)prescriptions should be taken into account.TCM syndromes usually have the characteristics of cross-system and dynamic evolution.Therefore,in the replication of syndrome models,we should pay attention to cross-system multi-index comprehensive evaluation.There are differences in the same syndromes among the different sexes,however,the systematic exploration and evaluation of the syndrome models based on gender differences is still rare.In this study,based on the perspective of liver depression syndrome and gonadal axis in females,the connotation of nervous-endocrine theory of the female rat model induced by restraint stress were researched.In addition,based on the theory of correlation between prescription and syndrome,the regulating effect of Chaihu Shugan Powder,a classic formula which used to treat the syndrome of liver depression,were observed,thus to provide a good understanding of the modern biological connotation of correlation between the method of dispersing stagnated liver qi and liver depression syndrome.The paper includes two parts:literature review and experimental study.The literature review mainly focuses on the relationship between liver and reproductive system,liver depression syndrome and female reproductive disease.The concept of neuroendocrine disease is proposed in this paper.The experimental study explored the relationship between TCM syndromes and neuroendocrine function of the rats with syndrome of liver depression induced by restraint stress and the effects of Chaihu Shugan Powder.2 MethodsRats were randomly divided into normal control group,syndrome of liver depression group(model group),Chaihu Shugan Powder high dose group(H group),and Chaihu Shugan Powder low dose group(L group),with 10 rats in each group.The model was established by chronic restraint stress for 4 weeks,and the normal control group was not treated.After modeling for 14 days,the rats in the L group and H group were given the Chaihu Shugan Powder for the dose of 1.26g/kg and 0.31g/kg(equivalent to 2 and 0.5 times of human consumption)once a day for 2 weeks.The rats in the model group and the normal control group were given the same amount of distilled water.On the 27th day,the vaginal smears were observed in each group,and the animals were sacrificed after abdominal anesthesia,the sample of serum and plasma were collected from the abdominal aorta,the hypothalamus,pituitary,bilateral ovarian and other organizations were separated and fixed in 10%neutral formalin or liquid nitrogen frozen in the-80℃.At last,the following indicators were observed:1)Before and after the experiment,the vaginal smears of rats were observed.2)During the modeling,the body weight and appearance(state,activity,emotion,sleep,food intake,and hair color)of the rats were observed.The sucrose preference test(SPT)was carried out at the end of the second and fourth weeks,the open field test(OFT)was taken at the end of fourth week.3)The indicators in the hypothalamic-pituitary-adrenal/thyroid/gonadal axis were determined by radioimmunoassay or enzyme-linked assay.All data were analysed using SPSS 20.0 software and presented as the means ± standard deviation of the means.Statistical significance was assessed using one-way analysis of variance(ANOVA)followed by SNK for multiple comparisons.The non-normal distribution data were analyzed by nonparametric statistical analysis.Count data is expressed as a percentage,and the difference between groups is Fisher’s exact test.All charts are made using GraphPad Prism 6 software.3 Results(1)Syndrome indicators:① general appearance:compared with the normal group,the rats in model group showed a state of hyperactivity,irritability and dry stool from the first week,and became get together,less action,slow action,reduce diet,dry stool,and dry hair from the second week.The body weight were reduced to varying degrees during the 2-4 weeks(p<0.05 or p<0.01).The model rats showed an addition sucrose preference at the 2th week and a reduction sucrose preference at the 4th week of the SPT(p<0.01).At the 4th week,the model rats displayed a reduction time spent,central zone frequency and upright frequency of the OFT(p<0.01).Compared with the model group,the activity,sleep and stool of rats in H group were moderately regulated,the body weight was significantly increased at the 3rd week(p<0.05 or p<0.01),and showed an addition sucrose preference at the 4th week(p<0.01),and displayed an addition time spent,central zone frequency and upright frequency of the OFT,(p<0.01).There was no significant change in the above-mentioned indexes of L group(p>0.05).Compared with the L group,the above indexes in the H group had significant difference(p<0.05 or p<0.01).② Laboratory indicators:compared with the normal group,the blood levels of 5-HT,DA and NE in rats of model group were significantly decreased,and the levels of E and β-EP were significantly increased(p<0.01 or p<0.05).Compared with the model group,the levels of 5-HT and NE in the blood of H group were significantly increased,and the levels of E and β-EP were significantly decreased(p<0.01 or p<0.05),the level of DA in the L group was significantly increased(p<0.01).Compared with the L group,the levels of 5-HT and NE in the blood of H group were significantly increased,and E was significantly decreased(p<0.01 or p<0.05).(2)The index of hypothalamic-pituitary-adrenal cortical axis:Compared with the normal group,the hypothalamic CRH,serum ACTH and CORT were significantly increased in the model group(p<0.01).Compared with the model group,the CRH and ACTH of the hypothalamus were significantly decreased(p<0.01),and the CRH and ACTH in L group were significantly decreased(p<0.01 or p<0.05),and the CORT showed a decreasing trend but has no significantly differences(p>0.05).Compared with the L group,the CRH of hypothalamus and serum CORT in the H group were significantly decreased(p<0.01 or p<0.05).(3)Hypothalamus-pituitary-thyroid axis index:Compared with the normal group,the level of TRH in the hypothalamus,and levels of T3,T4,TSH in the model group were significantly lower than those in the normal group(p<0.01 or p<0.05).Compared with the model group,the TRH in the hypothalamus and T3,T4 of the H group were significantly decreased,the level of TSH was significantly increased(p<0.01),in L group,even though the level of hypothalamic TRH,blood level of T3,T4 were decreased,the level of TSH was increased,however,only the level of T4 showed a significant difference(p<0.05).Compared with the L group,the hypothalamus TRH and the level of T3 and T4 in H group were decrease,TSH was increased(p<0.01 or p<0.05).(4)Hypothalamic-pituitary-gonadal axis indicators:①changes in estrous cycle:during the experiment,the emotional cycle of rats in normal group is stable,while in the model group is unstable,showing an extended or delayed period with "proest-estrus-met-diest".The apoptotic rate was 100%in the fourth week of the experiment.Compared with the model group,the rate of disturbance of emotional cycle were 20%and 80%in H and L Group,respectively,and the disorder rate of H group was significantly decreased(p<0.01).Compared with the L group,the disturbance rate of the estrous cycle in H group was significantly decreased(p<0.05).② Compared with the normal group,the changes of GnRH,FSH,LH,PRL,P,T and E2in the hypothalamus-pituitary-gonadal axis of model group were significantly higher(p<0.01 or p<0.05).Compared with the model group,the levels of GnRH,FSH,LH,PRL,P and T in the blood of H group were significantly decreased(p<0.01)and the trend of E2 was decreased.The FSH,LH,PRL,P and E2 were significant decreased(p<0.01),GnRH and T has a trend of decrease.Compared with the L group,the GnRH and FSH in the blood of H group were significantly decreased(p<0.01),and the other indexes were not statistically significant(p>0.05).4 ConclusionsThe appearance and laboratory indexes related to TCM syndromes in the rat model of liver depression induced by chronic restraint stress were changed,accompanied by hyperfunction and estrous cycle disorder of adrenal cortical axis,thyroid axis and gonadal axis,suggesting that the syndrome of liver depression in female has neurological-endocrine multiple system dysfunctions.Chaihu Shugan Powder,a classic formula which used to treat the syndrome of liver depression,has a good effect on the TCM syndrome of the female liver depression model rats and the related indexes of the neuroendocrine system.The effect of high dose group is superior than the low dose group.The results of this study provide some experimental evidence for the study of the modern pathophysiology of female liver depression syndrome and the theory of corresponding laws between prescriptions and syndromes(corresponding between prescription for dispersing stagnated liver qi and liver depression syndrome),also is conducive to the clinical application of Chaihu Shugan Powder. |
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