| ObjectiveInsomnia is one of the mental diseases that seriously affect people's physical and mental health,often accompanied by depression.Chaihu Jialonggu Oyster Decoction(Chaihu plus Longgu oyster decoction)is a classic prescription for the treatment of insomnia,and its mechanism of action has not been fully elucidated.In this study,sleep-deprived rats were used to explore the role of brain-intestine-flora changes,providing a basic theoretical basis for the prevention and treatment of insomnia by traditional Chinese medicine.MethodsIn view of the fact that insomnia is often accompanied by the occurrence of depression,and multiple studies have shown that Chaihu plus Longgu oyster decoction can not only improve insomnia,but also adjust adverse emotions,70 SPF grade male rats(200±20 g)were randomly divided into the blank group,the model group,the low,medium and high-dose groups of Chaihu plus Longgu oyster Decoction plus or minus(traditional Chinese medicine),the diazepam group and escitalopram oxalate(antidepressant)group,with 10 rats in eAch group With the exception of the blank group,the other groups of rats were used to establish REM sleep deprivation model with modified multi-platform water environment,and pentobarbital sodium sleep synergy test was used to evaluate whether the model was successfully established After the rat model was established,the diazepam group was intragastrically administered with diazepam tablets at a dose of 0.45 mg/(kg·d),the antidepressant group was intragastrically administered with escitalopram oxalate tablets at a dose of 0.9 mg/(kg·d),and the low,medium,and high dose groups of traditional Chinese medicine were intragastrically administered with Chaihu plus Longgu oyster decoction at a dose of 5.8 g/(kg · d)in the low dose group,11.6 g/(kg·d)in the medium dose group,23.2 g/(kg·d)in the high dose group,and the blank model group was intragastrically administered with an equal volume of normal saline once daily.According to the results of the preliminary experiment,2 weeks after continuous gavage was selected as the observation time point,and the following tests were conducted:1.The sleep latency and sleep duration of eAch group of rats were evaluated by the sleep synergistic experiment with pentobarbital sodium;2.Behavioral tests(including field test and sugar water preference test)were used to evaluate the behavioral performance of rats in eAch group;3.The serum contents of 5-HT and NE were determined by ELISA4.The feces of rats in eAch group were collected,and at least one grain of formed feces was obtained from eAch mouse.After the collection,the samples were uniformly submitted for inspection.and the species and abundance of intestinal flora were detected by high-throughput sequencing technologyResults1 Results of collaborative sleep experiment with pentobarbital sodiumAfter 3 weeks of sleep deprivation,the righting reflex did not disappear in 13 rats,and the sleep latency time was prolonged and the sleep duration was shortened in the remaining sleep-deprived rats compared with the non-sleep-deprived rats(p<0.01,p<0.01).After 2 weeks of continuous gavage,the righting reflex did not disappear in 2 rats with diazepam.Sleep latency time:the model,blank groups prolonged(p<0.01);the traditional Chinese medicine medium-dose,model group shortened,shorter than traditional Chinese medicine low-dose group(p<0.01,p<0.05);the traditional Chinese medicine high-dose group,the diazepam group,the antidepressant,the model group shortened(p<0.05).Sleep duration:the model prolonged the blank group,and the high-dose,the model group(p<0.05,p<0.05)2 Behavioral test resultsThe horizontal and horizontal plus vertical scores of the model group were lower than those of the blank group(p<0.01,p<0.01);the horizontal and horizontal plus vertical scores of the medium and high dose groups of traditional Chinese medicine were higher than those of the model group(p<0.01,p<0.01);the difference between the traditional Chinese medicine low,medium and high treatment groups and between the traditional Chinese medicine treatment groups and the diazepam or antidepressant groups were not statistically significant(p>0.05)The preference rate of sugar water in the model group was lower than that in the blank group,and the preference rate of sugar water in eAch treatment group was higher than that in the model group,and the difference was not statistically significant(p>0.05).3 Serological results5-HT and NE:The model group,the low and high dose groups of traditional Chinese medicine were lower than the blank group(p<0.01).EAch treatment group was higher than the model group,among which the differences between the medium dose group of traditional Chinese medicine,the diazepam group,the antidepressant group and the model group were statistically significant(p<0.01).The Chinese medicine medium dose group was lower,and the high dose group higher,and the difference between the low and medium dose groups was statistically significant(p<0.01)4 High-throughput sequencing results4.1 Diversity of intestinal flora in eAch group of ratsAlpha diversity:observed otus index,chao1 index:those of the model group,the low-dose group of traditional Chinese medicine,the diazepam group,the antidepressant group were larger than the blank group.And those of the medium and high dose groups of traditional Chinese medicine were larger than the blank group(p<0.01,p<0.05).ACE index:Those of the model group and eAch treatment group were larger than the blank group(P<0.05).Shannon index:Those of the model group,the diazepam group and the antidepressant group were larger than the blank group,and the low and high dose groups of Chinese medicine were larger than the blank group(P<0.01,P<0.05).The simpson index:The model group and antidepressant group were larger than the blank group,the diazepam group was larger than the blank group(p<0.01,p<0.05).Beta diversity:Bray Curtis distance with significant difference:the blank group and other groups;the model group and the low,medium,high dose groups of traditional Chinese medicine,antidepressant group;the low dose group of traditional Chinese medicine and the medium dose group of traditional Chinese medicine,the diazepam group,the antidepressant group;the medium dose group of traditional Chinese medicine and the high dose group of traditional Chinese medicine,antidepressant group;the high dose group of traditional Chinese medicine and the diazepam group,the antidepressant group;the diazepam group and antidepressant group.Unweighted Unifrac distance with significant difference:the blank group and other groups;the model group and the high-dose group of traditional Chinese medicine;the low-dose group and the high-dose group of traditional Chinese medicine,the antidepressant group;the medium dose group and the antidepressant group;high-dose group and diazepam group,antidepressant group.Weighted Unifrac distance with significant difference:the blank group and the model group,the Chinese medicine low and high dose group,the diazepam group,the antidepressant group;the Chinese medicine medium dose group and the antidepressant group;the Chinese medicine high dose group and the diazepam group,the antidepressant group.4.2 Characteristic flora of rats in eAch groupPhylum level:the characteristic flora of rats in the model group were cyanobacteria,soft firmicutes and verrucous flora,which could help them sleep.Genus level:The characteristic flora of the blank group was oligobacterium,which included urethral oligobacterium and urinary oligobacterium.The former was a symbiotic bacterium of urogenital system,rarely pathogenic,and the latter also resided in the urethra,which was related to the long-term use of urethral catheter and urethral obstruction.The characteristic flora of the model group was Ekmania,Corynebacterium,Fiklum,Ruminococcus,among which Ekmania and Corynebacterium can ameliorate depression,while Fickramella and Ruminococcus are related to sleep restriction;Dorea and Faecalibacterium are the characteristic flora in the low-dose group of traditional Chinese medicine,which can reduce inflammation;Enterococcus and Twin coccus are the characteristic flora in the medium-dose group of traditional Chinese medicine,which can improve depression,and Enterococcus has a sleep-promoting effect;the High-dose group of traditional Chinese medicine can reduce inflammation.The characteristic flora of the dosage group were Brautella,Faecococcus,Collins,Corynebacterium anaerobicus and Holdermann,among which Corynebacterium anaerobicus could improve depression,and the rest were negatively correlated with high quality sleep.The characteristic flora of the antidepressant group were Bacteroides,Parabacterioides,Roxiella,Defluviitalea,Clostridium,which were associated with depression.Insomnia was positively correlated.4.3 Comparison of the relative abundance of the dominant flora in eAch group of ratsthe germs with significant difference:thick-walled germs:the traditional Chinese medicine medium-dose group was higher than the model group;the antidepressant group was lower than the blank group and the traditional Chinese medicine medium-dose group,and lower than the traditional Chinese medicine high-dose group(p<0.01,p<0.01,p<0.05).Proteus:The Chinese medicine medium dose group was higher than the blank group and model group,lower than the low dose group;The high dose group of traditional Chinese medicine was lower than the medium dose group,the diazepam group was higher than blank group and model group,higher than low and high dose groups of traditional Chinese medicine,and antidepressant group was higher than medium dose group of traditional Chinese medicine(p<0.01,p<0.05,p<0.01,p<0.01,p<0.05,p<0.01).Cyanobacteria:The model group was higher than the blank group,the traditional Chinese medicine high-dose group was lower than the model group,the diazepam group was lower than the model group,and the antidepressant group was higher than the traditional Chinese medicine high-dose group and higher than the diazepam group(p<0.05,p<0.01,p<0.05,p<0.01,p<0.05)bacterial genus with Significant Difference:Lactobacillus genus:model group,Chinese medicine low medium high dose group,diazepam group lower than blank group,antidepressant group lower than blank group(p<0.05,p<0.01).Brauteria:the high-dose group was higher than the blank group,the antidepressant group was lower than the high-dose group,and the diazepam group was lower(p<0.05,p<0.05,p<0.01).the group with low,medium and high dose of traditional Chinese medicine was higher than the model group and the antidepressant group(p<0.05).Alobarbiella:the antidepressant group was lower than the blank group and the medium dose group(p<0.01).Tremella:the high-dose group was lower than the model group and the diazepam group,and lower than the antidepressant group(p<0.05,p<0.01).Ruminococcus:diazepam group was higher than the blank group and the medium dose group,and the antidepressant group was higher than the blank group(p<0.05,p<0.05).Bacteroidetes the high-dose group was lower than the low-dose group,and lower than the diazepam group and the antidepressant group(p<0.05,p<0.01).Fecal coccus:the antidepressant group was lower than the model group and the medium dose group,and the higher dose group was lower than the model group(p<0.05,p<0.01).Dorea:the antidepressant group was lower than the blank group and the low,medium and high dose group(p<0.05).Proctella:the antidepressant group was lower than the diazepam group(p<0.05).Bacteroidetes:the diazepam treatment group was higher than the blank group,higher than the medium and high dose Chinese medicine group,and the antidepressant group was higher than the blank group and the high dose Chinese medicine group(p<0.01,p<0.05)p<0.05)ConclusionUsing improved multi-platform building method of water environment,for three weeks of intermittent sleep deprivation,can build a stable REM sleep deprivation model,medium and high dose of Chaihu Jialonggu Oyster Decoction can improve insomnia and its associated with bad mood,and between no statistical difference(p<0.05),but only in the dose group of 5-HT,NE level significantly increased compared with the model group,shows that the curative effect of Chaihu Jialonggu Oyster Decoction play main way not 5-HT,NE level adjustment.the medium dose group mainly increase the flora improve insomnia,adjust the mood,the high dose group of major damage to sleep,adjust the mood of flora,prompt Chaihu Jialonggu Oyster Decoction can be increased to improve the bacteria produce effect of the treatment of insomnia,depression,but after double dose effect did not ascend,may is associated with increased the destruction of the flora of sleep... |
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