| ObjectiveTo observe the effects of Jiaotai Pill on astrocytes and adenosine and its A1 receptor in rats with insomnia caused by multi-factor stimulation+intraperitoneal injection of PCPA,to analyze the mechanism of action of Jiaotai Pill in treating insomnia in terms of general condition,sleep phase,astrocyte markers and expression of adenosine and its A1 receptor,to provide experimental evidence for the clinical application and further development of Jiaotai Pill,and to provide reference for the improvement and development of insomnia diagnosis and treatment methods.MethodsFifty-five healthy male SD rats were randomly divided into normal and modeling groups using body mass as the grouping factor,15 rats for the normal group and 40 rats for the modeling group.5 rats in the normal group and 10 rats in the modeling group were randomly selected to undergo electrode implantation for sleep monitoring 5 days before modeling,while the rest of the rats were not operated.The rats in the normal group were routinely housed,and all rats in the modeling group were subjected to multifactorial stimulation+intraperitoneal injection of PCPA to construct a rat insomnia model.The rats received the same random stimulation at the same time every day,and after 14 days,PCPA suspension（300 mg/kg）was injected intraperitoneally at 10 m L/kg every day,and the normal group was injected intraperitoneally with an equal volume of weak alkaline saline once a day for 2 days.On the day of moulding,the moulded group with implanted electrodes was randomly divided into the model group and the Jiaotai Pill group according to body mass,with 5 rats in each group,and the remaining rats in the moulded group were randomly divided into the model group,Jiaotai Pill group and Eszopiclone group,with 10 rats in each group.The Jiaotai Pill group was gavaged with 10m L/kg of Jiaotai Pill solution,the Eszopiclone group was gavaged with10m L/kg of eszopiclone solution,while the normal and model groups were gavaged with10m L/kg of pure water once a day for 7 days.1.The rats’skin and hair,food intake,water intake,activity level,mental status and circadian rhythm were continuously observed at the end of the modeling period and during the administration period;the body mass of each group was recorded every other day.2.2h after the last gavage,the EEG and EMG of the rats were recorded by Sleep Sign Animal Sleep Analysis System for 24h,and the amount of time spent in each sleep phase per hour and the total amount of time spent in different sleep phases during the night and daytime were analyzed.3.Collect blood from the rats’abdominal aorta,brain tissue,bilateral adrenal glands and spleen.4.Calculate the adrenal index and spleen index of the rat.5.ELISA for serum ACTH and CRH levels.6.Immunofluorescence observation of basal forebrain GFAP positive expression;ELISA detection of serum GFAP and S100B levels;real-time fluorescence quantitative PCR detection of basal forebrain GFAP and S100B m RNA expression levels;Western Blot detection of basal forebrain GFAP and S100B protein relative expression.7.ELISA for serum basal forebrain adenosine;real-time fluorescence quantitative PCR for basal forebrain adenosine A1 receptor m RNA expression;Western Blot for basal forebrain adenosine A1 receptor protein relative expression.Results1.Comparison of general condition and body mass of rats:The rats in the normal group had smooth and lustrous hair,normal food intake and water intake,less daytime activity,more curled up,more nighttime activity,good mental state,and obvious circadian rhythm.Compared with the normal group,the rats in the model group had sparse and lackluster hair,reduced food intake,increased water intake,increased daytime activity,irritability,loss of circadian rhythm,and a significant decrease in body mass（P<0.01）.Compared with the model group,the rats in the Jiaotai Pill and Eszopiclone groups had more hair and regained luster,increased food intake and water consumption,increased daytime sleep,increased nighttime activity,significantly improved mental status,restored circadian rhythm,and significantly increased body mass from day 3 of treatment（P<0.01）.2.Comparison of sleep time phases in rats:In the 24h period,compared with the normal group,the total NREM and REM sleep time in the model group decreased significantly（P<0.01）and the total W time increased significantly（P<0.01）;compared with the model group,the total NREM and REM sleep time in the Jiaotai Pill group increased significantly（P<0.01）and the total W time decreased significantly（P<0.01）.At night,compared with the normal group,the total amount of NREM and REM sleep time in the model group was significantly decreased（P<0.01）and the total amount of W time was significantly increased（P<0.01）;compared with the model group,the total amount of NREM sleep time in the Jiaotai Pill group was significantly increased（P<0.01）and the total amount of W time was significantly decreased（P<0.01）.During the daytime,compared with the normal group,the total NREM sleep time of the rats in the model group was significantly decreased（P<0.01）and the total W time was significantly increased（P<0.01）;compared with the model group,the total NREM sleep time of the rats in the Jiaotai Pill group was significantly increased（P<0.01）and the total W time was significantly decreased（P<0.01）.Regarding the comparison of each sleep time in 24h in rats:Compared with the normal group,the NREM sleep of rats in the model group was significantly decreased in the time periods of 20:00-21:00,22:00-23:00,2:00-3:00,6:00-11:00,12:00-13:00,14:00-15:00 and 16:00-19:00（P<0.05）,REM sleep was significantly decreased in the time periods of19:00-20:00,22:00-2:00,3:00-5:00,6:00-7:00,8:00-9:00,10:00-15:00（P<0.05）,and W time was significantly increased in the time periods of 20:00-0:00,2:00-3:00,4:00-5:00,6:00-11:00,12:00-17:00,and 18:00-19:00（P<0.05）;compared with the model group,NREM sleep of rats in the Jiaotai Pill group was significantly increased（P<0.05）in the time periods of 20:00-21:00,22:00-23:00,2:00-4:00,7:00-8:00,9:00-11:00,12:00-15:00,and 16:00-17:00（P<0.05）,REM sleep was significantly increased in the time periods of 2:00-5:00,10:00-11:00,and 13:00-15:00（P<0.05）,and W time was significantly decreased in the time periods of 20:00-21:00,22:00-0:00,1:00-4:00,7:00-11:00,and 12:00-19:00（P<0.05）.3.Adrenal index and spleen index of rats:Compared with the normal group,the adrenal index of rats in the model group was significantly higher（P<0.01）;compared with the model group,the adrenal index of rats in the Jiaotai Pill group was significantly lower（P<0.01）.The difference in spleen index of rats in each group was not statistically significant（P>0.05）.4.Comparison of serum ACTH and CRH levels in rats:Compared with the normal group,serum ACTH and CRH levels in the model group significantly increased（P<0.01）;compared with the model group,serum ACTH levels in the Jiaotai Pill and Eszopiclone groups significantly decreased（P<0.01）,but the differences in CRH levels between the groups were not statistically significant（P>0.05）.5.The positive expression of GFAP in the basal forebrain of rats:Compared with the normal group,in the model group,in the basal forebrain had significantly increased astrocyte protuberance and GFAP（P<0.01）,significantly increased serum GFAP and S100B levels（P<0.01）,significantly increased basal forebrain GFAP and S100B m RNA expression levels and relative protein expression（P<0.01）;Compared with the model group,in the Jiaotai Pill and Eszopiclone groups,in the basal forebrain had significantly decreased GFAP（P<0.01）,serum GFAP and S100B levels were significantly decreased（P<0.01）,and basal forebrain GFAP and S100B m RNA expression levels and relative protein expression were significantly reduced（P<0.05）.7.Comparison of adenosine content and adenosine A1 receptor expression in the basal forebrain of rats:Compared with the normal group,the adenosine content in the basal forebrain of rats in the model group was significantly increased（P<0.01）,and the m RNA expression level and relative protein expression of adenosine A1 receptor were significantly increased（P<0.01）;Compared with the model group,the adenosine content in the basal forebrain of rats in the Jiaotai Pill and Eszopiclone groups was significantly decreased（P<0.01）,the adenosine A1receptor m RNA expression level and relative protein expression in the basal forebrain were significantly decreased（P<0.05）.Conclusion1.Jiaotai Pill can improve the general state of insomnia rats and increase body mass of insomnia rats.2.Jiaotai Pill can treat insomnia mainly by increasing NREM sleep time.3.Jiaotai Pill can reduce the adrenal index and serum ACTH level in insomnia rats.4.Jiaotai Pill down-regulates the expression of serum and basal forebrain GFAP and S100B,expression of basal forebrain adenosine and its A1 receptor attenuates insomnia-induced neurotoxic effects and exerts a protective effect on the central nervous system,while increasing endostatic sleep pressure to improve insomnia. |
PDF Full Text Request