Chaihu Jia Longgu Muli Decoction On Improving Anxiety- And Depression-like Behavior Of Sleep Deprived Mice During Perimenopause

ObjectiveAs a risk factor of new onset mental diseases（such as anxiety,depression,bipolar disorder,etc.）,insomnia shows a significant high incidence and susceptibility in perimenopause compared with other stages of women.Because of the limitation of perimenopausal animal model,whether perimenopausal insomnia aggravates the occurrence and development of mental diseases and whether the intervention of perimenopausal insomnia can delay the occurrence of menopausal related anxiety,depression and other emotional disorders is not very clear.In recent years,studies abroad have found that VCD induced mouse ovarian failure animal model can be used as perimenopausal animal model,but it is not clear whether it is suitable for ICR mice.Our previous study found that Chaihu Jia Longgu Muli Decoction can improve the sleep of ovariectomized animals and resist the anxiety-like behavior of ovariectomized combined sleep deprived mice.Therefore,on the basis of establishing the perimenopausal animal model of ICR mice induced by VCD,this study explored the effect and mechanism of Chaihu Jia Longgu Muli Decoction on improving anxiety-and depression-like behaviors of perimenopausal sleep deprived mice.The results of this study not only provide the best intervention period and measures for the prevention of menopausal and aging related mental diseases,but also provide the pharmacodynamic basis for the clinical application of Chaihu Jia Longgu Muli Decoction.Methods56 day old ICR mice were intraperitoneally injected with VCD（160 mg/kg,5d/w,4 w,i.e.20 days in total）or sesame oil of equal volume.The mice were vaginal smeared every day,and on the 20th,35th and 52nd after injection of VCD or sesame oil,the mice were tested with elevated plus maze and tail suspension,and the ovarian morphology,number of atresia follicles,uterine coefficient and ovarian coefficient,serum FSH,E₂ and CORT levels were measured and observed.The perimenopausal sleep deprivation model was established by VCD induced perimenopausal mice combined with treadmill sleep deprivation（3 s run,12 s stop,2.5 m/min,14 consecutive days）and the non perimenopausal sleep deprivation model was established by VCD induced perimenopausal mice combined with treadmill sleep deprivation 14 consecutive days before VCD injection.During sleep deprivation,the perimenopausal sleep deprived mice were given CJLM70E（48 mg/kg,0.2 mL/10g）,estradiol（0.13 mg/kg,0.2 mL/10g）and 1%CMC-Na of the same volume by gavage,while the non perimenopausal sleep deprived mice were given 1%CMC-Na of the same volume by gavage.In perimenopausal mice induced by VCD without sleep deprivation,only 1%CMC-Na of the same volume was given for 14 consecutive days during perimenopause.On the 52nd day after injection of VCD,all groups were tested by elevated plus maze and tail suspension,and HE staining was used to observe the ovarian morphology and the number of atretic follicles.The levels of FSH,E₂ and CORT in serum,E₂ and 5-HT in hippocampus were measured by ELISA and BDNF,TrkB,ERβand 5-HT_2AA in hippocampus were measured by immunofluorescence.ResultsFrom the 20th day after the injection of VCD,the estrous cycle of about 90%ICR mice showed the alternation of proestrus/estrus and diestrus,lasting for about 2 w until the 35th day after the injection of VCD.On the 52nd day after the injection of VCD,the vaginal smears of VCD mice showed a continuous diestrus,lasting more than 1 w,of which 80%of VCD mice lasted more than 2 w.The morphology of ovary was damaged,which showed that the primordial follicle and corpus luteum was rare,the oocyte was pyknosis,the granular cells were few and the arrangement was disorder,the number of ovarian atresia follicles was increased（p<0.01）on the 35th and 52nd day after the injection of VCD.The coefficient of uterus and ovary of mice decreased significantly（p<0.01）on the 35th day after VCD injection and the uterus coefficient（p<0.01）and ovary coefficient（p<0.05）decreased;The level of serum FSH was significantly increased on the 20th（p<0.05）,35th（p<0.01）and 52nd（p<0.01）day after the injection of VCD,and the level of E₂ was significantly decreased（p<0.01）.on the 20th and 52nd day after the injection of VCD,the CORT of mice increased significantly（p<0.01）.On the 20th（p<0.01）and 35th（p<0.05）day after the injection of VCD,the OE%and OT%of VCD mice decreased significantly;on the 20th,35th and 52nd day after the injection of VCD,the immobilized time of VCD mice in each group was significantly prolonged（p<0.01）.Compared with non perimenopausal sleep deprivation,the OE%and OT%of perimenopausal sleep deprived mice decreased significantly（p<0.01）,ovarian morphology was damaged,and the number of atresia follicles in ovaries increased（p=0.052）,ovarian coefficient decreased（p<0.05）,the level of serum E₂ decreased significantly（p<0.01）,the level of CORT increased（p=0.08）,areal density of TrkB decreased（p=0.07）,areal density of 5-HT_2AA increased significantly（p<0.01）.Compared with the perimenopausal mice without sleep deprivation,the OE%and OT%of perimenopausal sleep deprived mice decreased significantly（p<0.01）,the immobilized time increased significantly（p<0.05）.In the ovary,the oocytes were pyknosis and the zona pellucida was dilated,the number of ovarian atresia follicles increased（p<0.05）,the uterine coefficient and ovarian coefficient decreased（p<0.05）,the level of serum E₂ decreased（p<0.05）and the level of CORT increased（p<0.05）,and the level of E₂ and 5-HT in hippocampus decreased（p<0.05）.In addition,the expression of BDNF,TrkB and ERβin hippocampus of perimenopausal sleep deprived mice decreased significantly（p<0.05）,and the expression of 5-HT_2AA increased significantly（p<0.01）.CJLM70E（except for the increase of uterus coefficient）and estradiol（except for the increase of ERβexpression）can obviously antagonize the abnormality of the above indexes.ConclusionICR mice entered perimenopause on the 35th day and entered menopause on the52nd day after VCD injection.Sleep deprivation during perimenopause can increase the occurrence of anxiety-and depression-like behavior in menopausal mice and CJLM70E can effectively improve the increase of anxiety-and depression-like behavior in perimenopausal sleep deprived mice.The mechanism of action of CJLM70E may be through the ERβ/BDNF/TrkB/5-HT_2AA signal pathway in the hippocampus to affect the occurrence of negative emotions.