| Background:Long-term chronic stress can do harm to women’s reproductive health,but its mechanism remains to be further studied.Chaiyu-Dixian Formula(CYDXF)has the function of regulating HPO axis,monoamine neurotransmitter and BDNF protein level in human body.Therefore,this experiment studied the effect of CYDXF on abnormal follicular development induced by chronic stress,and explored the possible mechanism.Objective:1.The chaiyu-dixian Formula(CYDXF)was analyzed by UHPLC-MS to explore the main active components of its functions.2.Chronic unpredictable mild stress(CUMS)model was established to explore the mechanism of abnormal follicle development and its relationship with environmental stimulation by observing the effects of chronic stress model on body weight,behavioral performance and monoamine neurotransmitter in the brain of rats.By observing the serum levels of FSH,LH,P,E2 and PI3K-AKT-mTOR protein induced by BDNF in the ovaries of rats,we therefore studied the possible mechanism of CYDXF to improve the abnormal development of follicles caused by chronic stress.Methods:1.The decoction of CYDXF was condensed into freeze-dried powder for later use.Lyophilized powder was dissolved and the active components of CYDXF were analyzed by ultra high performance liquid chromatography mass spectrometry.2.Adult female rats were randomly divided into 5 groups:control group,CUMS model group,estradiol(E2)group,CYDXF low and high dose group.Except for the control group,CUMS was administered to all the other groups for 28 days to establish a chronic stress model.3.Drug intervention:estradiol suspension at 0.0001g/Kg concentration was given to the estradiol control group,2.73g/Kg concentration was given to the CYDXF low dose group,and 5.46g/Kg concentration was given to the CYDXF high dose group once a day,for continuous gavage for 28 days.Rats in the control control group and the model group were given 10mL/kg of normal saline.During the process of stress,the change of body weight,behavioral performance and the change of estrus cycle were observed and recorded.4.After the final behavioral test,paraffin sections of the ovaries were prepared,and morphological changes and follicular development of the ovaries were observed by hematoxylinin-eosin(HE)staining.Serum FSH,LH,P and E2 levels and DA and 5-HT levels in the hippocampus were measured by ELISA.The PI3K-AKT-mTOR pathway and brain-derived neurotrophic factor(BDNF)proteins were detected by western blotting(WB).Results:1.Tweleve active components in CYDXF were identified:lobetyolin,martynoside,isomartynoside,salvianolic acid B,cryptotanshinone,chrysophanic acid,icariin,epimedin A,saikosaponin D,glycyrrhizic acid,curcumenol,and poricoic acid A.2.After 28 days of chronic stress model,compared with the control group,the body weight of rats in the model group increased slowly,the immobility time in FST increased significantly.Total distance in OFT,the percentage of open arm entries in EPM,sucrose preference were decreased significantly,and the hippocampus DA and 5-HT levels were significantly decreased(P<0.01),suggesting that the stress model was successful established.Furthermore,the oestrus cycle was disordered,the proportion of antral follicles was decreased and the proportion of atresia follicles was increased(P<0.01).Serum FSH and LH levels were increased(P<0.01),P and E2 levels were decreased(P<0.01),and the expressions of BDNF,PI3K,p-AKT/AKT,p-mTOR/mTOR proteins in the ovaries were significantly decreased(P<0.05,P<0.01).3.After treatment with different doses of CYDXF group and estradiol group,all the indicators were improved to different degrees.In CYDXF group,the body weight of rats was increased,the immobility time in the FST had decreased significantly,the total distance in the OFT,the percentage of open arm entries in the EPM and the sucrose preference in the SPT were significantly increased.In addition to that,the contents of DA and 5-HT in the hippocampus were increased,with statistically significant differences(P<0.05,P<0.01),comparing to the model group.4.The estrous cycle was relatively regular,the proportion of antral follicles was increased,the proportion of atretic follicles was decreased,the serum levels of FSH and LH were decreased while the levels of E2 and P were increased.The differences were statistically significant(P<0.05.P<0.01).The expressions of BDNF,PI3K,pAKT/AKT and p-mTOR/mTOR were up-regulated in ovary,and the differences were statistically significant(p<0.05,p<0.01).On top of that,the estradiol group had no significant effect on the body weight of the rats(P>0.05),while the other indexes were statistically different(P<0.05,P<0.01).Conclusion:The 28-day chronic stress model could inhibit the activation of BDNF and PI3K/AKT signaling pathway and its downstream signaling molecule mTOR in the ovary,leading to the decrease of hippocampal monoamine neurotransmitter,the disorder of hypothalamic-pituitary-ovary(HPO)axis,the disorder of follicle production,development and maturation,and the acceleration of follicular atresia,thereby affecting ovarian function.CYDXF could raise the expression of BDNF,p-AKT protein,enhance the activity of PI3K/AKT signal pathway to alleviate the inhibitory effect of chronic stress,which may promote mTOR activation,thus could increase the rat hippocampal monoamine neurotransmitters,regulate ovarian autocrine and paracrine function of rats,and inhibit the apoptosis of follicle cells,promote the mature follicle growth and development,so as to restore ovarian function.The regulating function of CYDXF may be related to the detected active ingredients. |
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