| Objective:In the part of theoretical research,we summarized the knowledge of insomnia,learning and memory disorders,Suanzaoren Decoction in traditional Chinese medicine,aiming to provide theoretical basis for Suanzaoren Decoction to prevent and treat the learning and memory disorders caused by insomnia;In the part of experimental research,we constructed the model of chronic sleep deprivation,and intervened with Suanzaoren Decoction to observe the changes of learning and memory function,synaptic plasticity,NLRP3 inflammasome before and after the intervention,so as to clarify the mechanism of Suanzaoren Decoction in the mediation of synaptic plasticity and neuroinflammation to improve learning and memory.Method:1.Theoretical part:Based on the ancient books of Traditional Chinese Medicine and recent literature,this paper summarized the disease name,etiology and pathogenesis of insomnia and learning memory disorders,expounded the relationship between insomnia and learning memory disorders,and provided theoretical basis for the prevention and treatment of learning memory disorders caused by insomnia by Suanzaoren decoction by combining the analysis of its composition and clinical experimental research.2.Experiment part:60 male rats were randomly divided into 5 groups: normal control group(NG),model group(MOD),estazolam group(EST),Suanzaoren Decoction low dose group,SZRDL)and Suanzaoren Decoction high dose group(SZRDH),12 rats in each group.Except NG group,the other four groups established chronic sleep deprivation model by multi platform water environment method for 12 hours and 30 days.Animal modeling and administration were performed at the same time.Pure water was administered to the NG group and the MOD group at a dose of 10 m L·kg-1· d-1· D-1 dose of oral estazolam solution,SZRDL group was administered at a dose of4.59 g · kg-1 · d-1,10 m L · kg-1 · d-1,SZRDH group Suanzaoren decoction was administered to the stomach at a dose of 18.36 mg · kg-1· d-1 and 10 m L · kg-1 · d-1.The general conditions of mental state,diet,water intake and body weight of rats in each group during modeling and administration were observed.After gavage,the following experiments were carried out: Morris water maze and platform experiment were used to evaluate the learning and memory ability of the rats in each group.Autonomic activity box was used to observe the autonomic activity ability of rats in each group.The changes of synapses in hippocampal CA3 region of rats in each group were observed by nese staining and golgi staining.Immunofluorescence and immunohistochemistry were used to observe the expression levels of PSD95,SYP and NMDAR1(NR1),NMDAR2A(NR2A)and NMDAR2B(NR2B),the key proteins of synaptic plasticity.The expression levels of IL-1βand TNF-αin serum and hippocampus were detected by ELISA.m RNA expression levels of IL-1βand TNF-α in the hippocampus were detected by real-time PCR.Protein and m RNA expression levels of NLRP3,ASC,caspase-1,IL-1βand IL-18 in the NLRP3 inflammasome pathway were detected by real-time PCR,immunohistochemistry and western bolt.Results:1.Theoretical research results Insomnia belongs to the category of "insomnia" in traditional Chinese medicine.The causes of insomnia in traditional Chinese medicine are complicated and various,such as abnormal emotions,abnormal diet,and excessive unhealthy qi.These causes cause imbalance between Yin and Yang,dysfunction of viscera and viscera,deficiency of qi and blood leading to insomnia.Learning and memory disorders belong to the category of "forgetfulness" in traditional Chinese medicine.Insomnia is closely related to learning and memory disorders.On the other hand,insomnia can cause learning and memory damage.Therefore,nourishing blood and calming the mind is an important way to prevent insomnia and learning and memory damage.Suanzaoren decoction is a representative prescription for nourishing blood and calming the nerves.In recent years,studies have suggested that suanzaoren decoction can not only improve sleep,reduce anxiety and depression,but also improve learning and memory,providing a theoretical basis for preventing insomnia and learning memory damage with suanzaoren decoction.2.Results of experiment 12.1 General conditions of the rats in each group: during sleep deprivation and administration,the NG group rats were generally in good condition,with dense and glossy hair,red and shiny tail and claws,good spirits and sensitive reactions,and no obvious abnormalities in food intake,water intake or defecation.Compared with the NG group,the rats in the MOD group experienced a slight increase in food intake and irritability in the 3 days before sleep deprivation,but after that there was a significant decrease in food intake and water intake,the hair was dull and easy to shed,the color of the tail and claws was light and dull,the spirit was listless,the expression was dull,the reaction was slow,and sometimes fell into the water.Compared with MOD group,the rats in SZRDL group and SZRTH group had significantly increased food and water intake,less hair loss,higher spirits and increased alertness.However,the EST group showed the tendency of listlessness,lethargy and lethargy.2.2 Weight changes of rats in each group: during sleep deprivation and administration,the NG group of rats rapidly gained weight.The rats in the MOD group gained a little weight,but the increase was significantly lower than the normal rats.The EST group,SZRDL group and SZRTH group also gained weight,but the increase was significantly lower than that of the sleep-deprived group.Compared with NG group,MOD group rats lost weight,but the difference was not statistically significant(P>0.05).Compared with the MOD group,the weight of EST group,SZRDL group and SZRTH group increased,and the difference was not statistically significant(P> 0.05).2.3Morris water maze experiment results: navigation test results: compared with NG group,the incubation period and total swimming distance of rats in MOD group increased significantly(P<0.01).Compared with MOD,both the SZRDL group and the SZRDH group had different degrees of decrease in the incubation period and total swimming distance on the platform(P<0.01,P<0.05),while the EST group had no decrease in the incubation period and total swimming distance on the platform(P>0.05).Compared with the SZRDL group,the incubation period of the upper platform and the total swimming distance of the rats in the SZRDH group decreased,but the difference was not statistically significant(P>0.05).Results of spatial exploration test: compared with NG group,the number of times that the rats crossed the platform and target quadrant in MOD group significantly decreased(P<0.01),while the time of the first arrival to the original platform significantly increased(P<0.01).Compared with MOD group,SZRDL and SZRDH group rats crossing platform number and target quadrant time increased(P < 0.01,P <0.05),while the first arrived in the original stage time has decreased(P < 0.05),the EST group rats rats crossing platform number and target quadrant time increased,while the first arrived in the original stage time has decreased,but the difference was not statistically significant(P > 0.05).Compared with the SZRDL group,The Times of crossing the platform,the target quadrant time and the first time of arriving at the original platform of the rats in the SZRDH group all changed,but the difference was not statistically significant(P>0.05).2.4 Platform jumping experiment results: compared with NG group,error frequency of rats in MOD group increased significantly(P<0.01),while latency decreased significantly(P<0.01).Compared with the MOD group,the number of errors in the SZRDL group and the SZRDH group decreased to different degrees(P<0.01,P<0.05),while the incubation period increased to different degrees(P<0.01,P<0.05).The number of errors in the EST group decreased and the incubation period increased,but the difference was not statistically significant(P>0.05).Compared with the SZRDL group,the error frequency and incubation period of the rats in the SZRDH group were changed,but the difference was not statistically significant(P>.05).2.5 Results of the autonomous activity experiment: compared with NG group,the activity time and movement distance of the rats in MOD group increased significantly(P<0.01),while the resting time decreased(P<0.05).Compared with the MOD group,the activity time and movement distance of the EST group,SZRDL group and SZRDH group decreased(P<0.01,P<0.05),and the resting time increased(P<0.01,P<0.05).Compared with the SZRDL group,the movement distance,activity time and resting time of the rats in the SZRDH group all changed,but the difference was not statistically significant(P>0.05).3.Results of experiment 23.1 Nissl staining results: the neurons in the hippocampus CA3 area of the NG group were clear and complete,arranged closely,with complete cell structure,clear intracellular nucleoli,and abundant neeldri bodies in the cytoplasm.In MOD group,the arrangement of neurons was irregular,the space around cells was widened,and the intracellular nidellosis was obvious.However,the arrangement of neurons in the hippocampal CA3 area of the SZRDL group and SZRDH group was improved after the administration of Suanzaoren decoction in the sleep-deprived rats,and the reduction of nainite was improved.However,there was no significant improvement in neuronal arrangement disorder and nainistenite reduction in the hippocampus CA3 area of EST rats treated with estazolam.Through the statistics on the number of neurons in the hippocampal CA3 region of the rats in each group,we found that the number of neurons in the hippocampal CA3 region of the rats in MOD group decreased significantly(P<0.01).Compared with MOD group,the number of neurons in hippocampal CA3 increased in SZRDL group and SZRDH group(P<0.01,P<0.05),while the number of neurons in hippocampal CA3 in EST group did not change significantly,with no significant difference(P> 0.05).Compared with the SZRDL group,the number of neurons in the hippocampus CA3 region of the rats in the SZRDH group did not change significantly,and the difference was not statistically significant(P>0.05).3.2 Golgi staining results: dendritic spines of neurons in the hippocampus CA3 region of NG group were abundant in number and high in density.Compared with NG group,the density of dendritic spines in CA3 area of rats in MOD group decreased significantly(P<0.01).Compared with the MOD group,the density of dendritic spines increased in the hippocampal CA3 area of the SZRDL group and the SZRDH group(P<0.01,P<0.05),and the density of dendritic spines increased in the hippocampal CA3 area of the EST group,but the difference was not statistically significant(P>0.05).Compared with the SZRDL group,the density of dendritic spines in the hippocampal CA3 region of the SZRDH group did not change significantly,and the difference was not statistically significant(P>0.05).3.3 Immunohistochemical test results: compared with NG group,IOD values of NR1,NR2 A and NR2 B in hippocampal CA3 of rats in MOD group were significantly decreased(P<0.01).Compared with MOD group,IOD values of NR1,NR2 A and NR2 B in hippocampal CA3 of rats in SZRDL group and SZRDH group increased(P<0.01,P<0.05),while IOD values of NR1,NR2 A and NR2 B in hippocampal CA3 of rats in EST group did not change significantly(P> 0.05).Compared with the SZRDL group,the IOD values of NR1,NR2 A and NR2 B in the hippocampal CA3 region of the rats in the SZRDH group did not change significantly,and the difference was not statistically significant(P>0.05).3.4 Immunofluorescence experiment results: compared with NG group,green fluorescence intensity and amount of PSD95 and SYP in hippocampus CA3 of rats in MOD group were significantly decreased.Compared with MOD group,green fluorescence intensity and fluorescence quantity of PSD95 and SYP in hippocampal CA3 of rats in SZRDL group and SZRDH group increased,while green fluorescence intensity and fluorescence quantity of PSD95 and SYP in hippocampal CA3 of rats in EST group did not change notably.Compared with SZRDL group,green fluorescence intensity and fluorescence quantity of PSD95 and SYP in hippocampal CA3 of SZRDH group did not change significantly.4.Results of experiment 34.1 ELISA results: compared with NG group,the expression levels of IL-1β、TNF-α in serum and hippocampus of MOD group were significantly increased (P<0.01).Compared with the MOD group,the expression levels of IL-1β、TNF-α in the serum and hippocampus of the SZRDL group and the SZRDH group were decreased to different degrees(P<0.01,P<0.05),while the expression levels of IL-1β、TNF-α in the serum and hippocampus of the EST group were not significantly changed(P>,0.05).Compared with the SZRDL group,the expression levels of IL-1β、TNF-α in the hippocampus of the rats in the SZRDH group were also not significantly changed,and the difference was not statistically significant(P>0.05).4.2 Real-time PCR results: compared with NG group,m RNA expression levels of IL-1β 、 TNF-α in hippocampus of rats in MOD group were significantly increased(P<0.01).Compared with the MOD group,the m RNA expression levels of IL-1β、TNF-α m RNA in the hippocampus of the SZRDL group and the SZRDH group were decreased to different degrees(P<0.01,P<0.05),while the m RNA expression levels of IL-1β、TNF-α m RNA in the hippocampus of the EST group were not significantly changed(P>,0.05).Compared with the SZRDL group,the expression level of IL-1β m RNA and TNF-α m RNA in the hippocampus of the SZRDH group was not significantly changed,and the difference was not statistically significant(P>.05).5.Results of experiment 45.1 Real-time PCR results: compared with NG group,the m RNA expression levels of NLRP3,ASC,caspase-1 and IL-18 in the hippocampus of the MOD group were significantly increased(P<0.01).Compared with the MOD group,the m RNA expressions of NLRP3,ASC,caspase-1 and IL-18 in the hippocampus of the SZRDL group and the SZRDH group were decreased to varying degrees(P<0.01,P<0.05),and the m RNA expressions of NLRP3,ASC,caspase-1 and IL-18 in the hippocampus of the EST group were also decreased,but the difference was not statistically significant (P>0.05).Compared with the SZRDL group,the m RNA expression levels of NLRP3,ASC,caspase-1 and IL-18 in the hippocampus of the SZRDH group were not significantly changed,and the difference was not statistically significant(P> 0.05).5.2 Immunohistochemical results: compared with NG group,IOD values of NLRP3,ASC,caspase-1,il-1 and il-18 in hippocampus of MOD group were significantly increased(P<0.01).Compared with the MOD group,the IOD values of NLRP3,ASC,caspase-1,IL-1β and IL-18 in the hippocampus of the SZRDL group and the SZRDH group decreased to different degrees(P<0.01,P<0.05),while the IOD values of NLRP3,ASC,caspase-1,IL-1β and IL-18 in the hippocampus of the EST group did not change significantly(P>0.05).Compared with the SZRDL group,the IOD values of NLRP3,ASC,caspase-1,IL-1β and IL-18 in the hippocampus of the SZRDH group did not change significantly,and the difference was not statistically significant(P>,0.05).5.3 Western bolt results: compared with NG group,the expression levels of NLRP3,ASC,caspase-1,IL-1β and IL-18 in the hippocampus of MOD group were significantly increased(P<0.01).Compared with the MOD group,the expression levels of NLRP3,ASC,caspase-1,IL-1β and IL-18 in the hippocampus of the SZRDL group and the SZRDH group were decreased to varying degrees(P<0.01,P<0.05),while the expression levels of NLRP3,ASC,caspase-1,IL-1β and IL-18 in the hippocampus of the EST group were decreased(P>0.05).Compared with the SZRDL group,the expression levels of NLRP3,ASC,caspase-1,IL-1β and IL-18 in the hippocampus of the SZRDH group were not significantly changed,and the differences were not statistically significant(P>,0.05).Conclusion:1.Theoretical research conclusion: Yin blood deficiency and spiritual displacement are important common pathogenesis of insomnia and learning and memory disorders,and suanzaoren decoction,as the representative prescription of nourishing blood and calming the mind,is an effective prescription to prevent insomnia and learning and memory disorders.2.Chronic sleep deprivation can lead to learning and memory disorders and anxiety-like behaviors in rats,while suanzaoren decoction can improve learning and memory disorders and anxiety-like behaviors caused by sleep deprivation;3.Sleep deprivation can lead to synaptic damage in hippocampal neurons in rats,while suanzaoren decoction can improve synaptic damage by regulating the expression levels of key synaptic plasticity proteins PSD95,SYP and key synaptic signaling proteins NMDAR1(NR1),NMDAR2A(NR2A)and NMDAR2B(NR2B).4.Sleep deprivation can cause inflammatory reactions in the serum and hippocampus of rats,and suanzaoren decoction can improve neuronal inflammation in the brain of sleep deprivation model rats by inhibiting the expression levels of IL-1β and TNF-α.5.Suanzaoren decoction can inhibit NLRP3 inflammatitis pathway and reduce neuroinflammation to prevent and treat learning and memory disorders and anxiety-like behaviors caused by sleep deprivation. |
