Biological Basis Of ERK1/2-CREB-BDNF Signaling Pathway On Liver-stagnation Pattern Of Perimenopausal Rats

ObjectiveFrom the perspective of prescriptions corresponding to syndrome,using Chaihu Shugan San to explore the biological basis of liver-stagnation pattern in perimenopausal rats through the ERK1/2-CREB-BDNF signaling pathway.Methods1.Forty-eight female 2-month-old SPF-grade SD rats were randomly divided into sham group,perimenopausal?PMS?group,liver-stagnation pattern in perimenopause group?model group?and Chaihu Shugan San?CSS?group according to body weight of rats after 1 month of adaptive feeding.The sham group removed the periovarian fat tissue,and the other groups underwent castration surgery.HE staining was used to observe the smears of the vaginal exfoliated cells of rats to determine whether the establishment of the perimenopausal animal model was successful.The model group and the CSS group were given chronic unpredictable mild stress?CUMS?for 21 days to establish the model of liver-stagnation pattern.2.Drug interventions:from day 1 of CUMS,the CSS group was simultaneously administrated with CSS(3.31 g·kg^-1·d^-1),meanwhile the sham group,the PMS group and the model group were perfused with an equal volume of saline once a day for 21 consecutive days.3.The changes of rat body weight,immobility time of forced swimming test and sucrose water preference test were observed.4.Serum estradiol,adreno-cortico-tropic-hormone,corticosterone,5-hydroxytryptamine,Dopamine and Norepinephrine contents were detected by enzyme-linked immunosorbent assay?ELISA?.5.Expressions of extracellular signal-regulated kinase1/2,cyclic-AMP response element binding protein,brain-derived neurotrophic factor and tropomyosin receptor kinase B mRNA in hippocampus and hypothalamus were measured by real-time quantitative PCR?q-PCR?.6.Protein levels of extracellular signal-regulated kinase1/2,cyclic-AMP response element binding protein,brain-derived neurotrophic factor and tropomyosin receptor kinase B in hippocampus were measured by western blot?WB?.7.The brain imaging changes of rats were collected by 7.0 T small animal functional magnetic resonance imaging,and the changes of neuronal activity in the brain regions of rats in each group were analyzed by Regional homogeneity?ReHo?.Results1.The smears of vaginal exfoliated cells in rats after castration prompt that the estrous cycle is disordered,indicating that the perimenopausal model has been successfully established.Compared with the sham group,the weight of rats in the PMS group increased?P<0.05?,and the growth rate of body weight increased significantly?P<0.01?.Compared with the PMS group,the weight of the rats in the model group decreased?P<0.05?,and the growth rate of body weight decreased significantly?P<0.01?.The body weight of rats in the CSS group was lower than that in the model group?P<0.05?.2.There was no significant difference in the forced swimming immobility time and sucrose water preference rate of rats in each group before modeling?P>0.05?.After intervention,compared with the sham group,the immobility time of forced swimming test was prolonged?P<0.05?,and the sucrose water preference rate was significantly reduced in the model group?P<0.01?.The sucrose water preference rate in the model group was significantly reduced when it compared with the PMS group?P<0.01?.The CSS group had a shorter immobility time of forced swimming than the model group?P<0.01?,while the sucrose water preference rate was significantly increased?P<0.01?.3.Compared with the sham group,the serum E₂ content in rats was significantly reduced in each group?P<0.01?.The serum ACTH and CORT contents in the model group were significantly higher than that in the sham group and the PMS group,while the CSS group was significantly lower than the model group.The contents of serum 5-HT,DA,and NE in the model group were significantly lower than that in the sham group and the PMS group,and the CSS group was significantly higher than the model group?P<0.01?.4.Expressions of hippocampal ERK1/2,CREB,BDNF and TrkB mRNA.Compared with the sham group,the expressions of hippocampal ERK1/2,BDNF and TrkB mRNA in the model group were reduced?P<0.05?,but there was no significant difference in the expression of CREB mRNA.Compared with the PMS group,the expressions of hippocampal ERK1/2,CREB,and BDNF in the model group were all reduced?P<0.05,P<0.01?,and there was no significant difference in the expression of TrkB mRNA.Compared with the model group,the relative expression levels of hippocampal ERK1/2,CREB,BDNF,and TrkB mRNA in the CSS group were significantly increased?P<0.01?.The expressions of ERK1/2,CREB,BDNF and TrkB mRNA in hypothalamus.The expressions of hypothalamic ERK1/2,CREB,BDNF,and TrkB mRNA in the model group was lower than that in the PMS group and sham group?P<0.05,P<0.01?.Compared with the model group,the expressions of hypothalamic ERK1/2,CREB,BDNF and TrkB mRNA in the CSS group were significantly increased?P<0.01?.5.The protein levels of ERK1/2,CREB,BDNF,and TrkB in hippocampus.The protein level of ERK1/2 showed changes in each group,but there was no statistical significance.Compared with the sham group,the protein levels of BDNF and TrkB in the model group decreased?P<0.05?,but the protein levels of CREB did not change significantly.The protein levels of CREB in the model group was lower than that of the PMS group?P<0.05?.The protein levels of CREB,BDNF and TrkB in the CSS group was higher than that of the model group?P<0.05,P<0.01?.6.Compared with the sham group,the resting state ReHo analysis results showed that ReHo of the PMS group decreased in the left hippocampus,left striatum,motor cortex,visual cortex and other brain regions of rats.The ReHo of the left hippocampus,amygdala,and motor cortex in the model group increased when it was compared with the PMS group.Compared with the sham group,the ReHo in the brain regions such as the posterior lobe of the cerebellum,the left pear cortex,and the left striatum decreased in the model group.After treatment with CSS,the ReHo in the brain regions such as posterior cerebellum,hippocampus,striatum,olfactory cortex,and sensory cortex increased in the CSS group.Conclusions1.There exists disorder of 5-HT,DA and NE in perimenopausal liver-stagnation pattern.2.Perimenopausal liver-stagnation pattern can cause abnormal ERK1/2-CREB-BDNF signaling pathways.Chaihu Shugan San can improve behavioral changes by regulating the expression of ERK1/2-CREB-BDNF signaling pathways and ameliorate the depression state of rats,in order to exert antidepressant effects.3.Pathological changes of perimenopausal liver-stagnation pattern may involve abnormal resting state function in the posterior cerebellum,striatum,hippocampus and other regions.Chaihu Shugan San may play an antidepressant role by regulating the spontaneous activity of the cortex-hippocampus-striatal-posterior cerebellar neural network.