Chaihu Jia Longgu Muli Decoction In Improving Sleep In Menopausal Transition Mice Based On O-GlcNAc Modification Of ERα

ObjectiveThe menopausal transition period is a necessary life stage for women.With the abnormal fluctuation of hormone levels,the brain estrogen receptor（ER）network will also change,which is manifested in the decline of ER level and sensitivity,resulting in the emergence of a series of neuropsychiatric symptoms,especially insomnia.The incidence of incidence rate in the menopausal transition period is significantly higher than that in other life stages.Clinical research have shown that sleep surveys of menopausal transition women are mainly based on subjective scale tests,and are not consistent with the obj ective sleep time obtained from limited polysomnography monitoring.4-vinylcyclohexene diepoxide（VCD）is an ovarian toxic chemical,which can simulate the dynamic process of women from reproductive period,to menopausal transition,to menopause.It is widely used in the study of diseases related to menopausal transition,but the study of sleep-wake phase has not been reported.O-linkedβ-N-acetylglucosamine（O-GlcNAc）is a post-translational modification of monosaccharide in intracellular protein,which can modify ER and regulate the level and activity of ER.Chaihu Jia Longgu Muli Decoction,as a classic prescription commonly used in clinical practice of traditional Chinese medicine,and its mechanism is still unclear.Based on the above understanding,this study first used VCD-induced menopausal transition mice as animal models to clarify the changing characteristics of sleep-wake phase during menopausal transition.Then,on the basis of confirming that Chaihu Jia Longgu Muli Decoction can improve the sleep of menopausal transition mice,the mechanism of Chaihu Jia Longgu Muli Decoction to improve the sleep of menopausal transition mice was discussed based on the O-GlcNAc modification of ERa.MethodsPart I Changes of sleep-wake phase in menopausal transition miceThe 8-week-old ICR mice were randomly divided into six groups:VCD 20 d,VCD 35 d,VCD 52 d,VCD 63 d,Control 20 d,Control 35 d,Control 52 d,Control 63 d.Mice in the VCD and Control groups were intraperitoneally injected with VCD（160 mg/kg,0.05 mL/10 g）and sesame oil of the same volume at 8:00 a.m.every day,once a day,for consecutive 5 days a week and stopped inj ection for 2 days,continuously for 4 weeks,for a total of 20 days.At the 20th,35th,52nd and 63rd day after VCD or sesame oil injection,cortical EEG and EMG were monitored to analyze,episode number and average duration and length of wakefulness,non rapid eye movements（NREM）sleep and rapid eye movements（REM）sleep throughout the day,day and night.Part Ⅱ Expression of ERα,OGT,OGA and O-GlcNAc proteins in hypothalamus of menopausal transition miceThe experimental grouping and administration method are the same as part Ⅰ.The expressions of ERα,OGT,OGA and total O-GlcNAc protein in hypothalamus of mice in each group were determined by Western blot on the 20th,35th,52nd and 63rd day after injection of VCD or sesame oil.Part Ⅲ Effects of CJLM70E on sleep-wake phase in menopausal transition miceThe 8-week-old ICR mice were randomly divided into 4 groups:Control group,VCD group,CJLM70E group,and E₂ group.The Control group was intraperitoneally injected with sesame oil,and the other groups were intraperitoneally injected with VCD.On the 3 5th day after injection of VCD or sesame oil,CJLM70E group was given CJLM70E（48 mg/kg,0.2 mL/10 g）intragastric administration,and E₂ group was given estradiol（0.13 mg/kg,0.2 mL/10 g）intragastric administration.VCD group and Control group were given the same volume of 1%CMC-Na by gavage for four weeks.Cortical EEG and EMG were monitored at the 1st,2nd,3rd and 4th weeks after administration,and the episode number,average duration and length of 24 h Wake,NREM sleep and REM sleep were analyzed.Part Ⅳ Mechanism of CJLM70E in improving sleep in menopausal transition miceExperimental grouping and administration are the same as in part Ⅲ.At the first week,the expression of ERα,OGT,OGA and total O-GlcNAc protein in hypothalamus of mice in each group was determined by Western blot after administration.Immunocoprecipitation technique was used to verify the O-GlcNAc modification of ERα,Immunofluorescence technique was used to detect the expression of ERα and O-GlcNAc proteins in hypothalamus VLPO and PVN brain regions.ResultsPart Ⅰ Changes of sleep-wake phase in menopausal transition miceCompared with the corresponding Control group,the Wake time of VCD 20 d,VCD 35 d,and VCD 52 d mice was prolonged（P<0.05）,while the NREM and REM sleep times were shortened（P<0.05）,and the episode number of REM sleep was decreased（P<0.05）.The episode number of Wake and NREM sleep in VCD 63 day mice increased（P<0.05）,while the average duration of NREM sleep occurrence decreased（P<0.05）,Compared with VCD 20 day,VCD 35 day,and VCD 63 day mice,the Wake time in VCD 5 2 day mice was prolonged,while the REM sleep time and the episode number of REM sleep decreased（P<0.05）;Compared with VCD 20 days,VCD 35 days,and VCD 5 2 days,VCD 63 day mice showed an increase in the episode number of Wake and NREM sleep（P<0.05）,and a decrease in the average duration and Wake time of occurrence（P<0.05）.Part Ⅱ Expression of ERα,OGT,OGA and O-GlcNAc proteins in hypothalamus of menopausal transition miceCompared with the corresponding Control group,the expression of ERα protein in hypothalamus of VCD 35 d,VCD 52 d and VCD 63 d mice decreased（P<0.05）;The expression of OGT and O-GlcNAc proteins in the hypothalamus of VCD 35 d and VCD 52 d mice decreased（P<0.05）.Compared with VCD 20 and VCD 63 days,the OGT protein levels in the hypothalamus of VCD 35 d and VCD 52 d mice decreased（P<0.05）;Compared with VCD 20 d,VCD 35 d,and VCD 52 d,the expression of O-GlcNAc protein in the hypothalamus of VCD 63 d mice increased（P<0.05）.Part Ⅲ Effects of CJLM70E on sleep-wake phase in menopausal transition miceCompared with the corresponding Control group,the Wake time of VCD group mice on the 1st,2nd,and 3rd week was prolonged（P<0.05）,the NREM sleep time was shortened（P<0.05）.The REM sleep time and episode number of the first and second weeks were reduced（P<0.05）,and the average duration of Wake occurrence of the first week was shortened（P<0.05）;Compared with the VCD group,the Wake time of CJLM70E and E₂ mice at 1,2,and 3 weeks was shortened（P<0.05）,and NREM sleep time was prolonged（P<0.05）,the duration of REM sleep in the 1st and 2nd weeks was prolonged（P<0.05）,the episode number of REM sleep was decreased（P<0.05）,and the average duration of Wake in the 1st week was shortened（P<0.05）.Part Ⅳ Mechanism of CJLM70E in improving sleep in menopausal transition miceWestern blot results showed that compared with the Control group,the hypothalamic ERα of VCD group mice.The expression of OGT and O-GlcNAc proteins decreased（P<0.05）;compared with VCD group,the expression of ERα,OGT and O-GlcNAc in hypothalamus in C JLM70E and E₂ groups increased（P<0.05）.The results of co-immunoprecipitation showed that there was interaction between ERα and O-GlcNAc protein,and the interaction degree of VCD group was decreased,while CJLM70E and E₂ could increase the interaction degree.Immunofluorescence results showed that,compared with Control group,the expression of ERα and O-GlcNAc protein in VLPO and P VN brain region of VCD group was decreased（P<0.05）;compared with VCD group,ERα and O-GlcNAc protein expressions in VLPO and PVN brain regions of CJLM70E and E₂ groups were increased（P<0.05）.Conclusion1.The sleep quality of menopausal transition and menopausal mice decreased,and there was no obvious change of circadian rhythm in the sleep wake phase of menopausal mice.2.C JLM7 0E can improve the sleep quality of menopausal transition mice by regulating the O-GlcNAc modification of ERα.