Study On The Mechanisms Of Insomnia In Rat Model Based On The Microbiota-gut-brain Axis Using Abdominal Massage

Objective: Taking the gut-brain interaction pathway " bacteria pathway " as an entry point,to explore the mechanism of effect of simple abdominal massage in the treatment of insomnia,and to clarify whether it is the core treatment technique for insomnia.Methods:Seventy-two(72)Wistar male rats which were randomly arranged in normal group(FK),model group(FM),simple abdominal group(FD),traditional abdominal therapy group(FC),drug group(FY)and drug + simple abdominal group(FT).12 rats from each FM and intervention group were injected intraperitoneally with para-chlorophenylalanine(PCPA)to prepare a model of insomnia,and pentobarbital sodium was used to evaluate the success of the co-sleep experiment.The success of the modeling was evaluated by pentobarbital sodium co-sleeping test.After successful modelling,no intervention was performed in the FK and FM groups;FD group applied abdominal massage to the rats;FC group after relaxing the soft tissues of neck and back,a series of abdominal massage manipulation was done on the abdomen;in the FY group,zolpidem tartrate tablets(0.92mg/kg)were administered by gavage;in the FT group,moistening was administered on top of the drug group once daily for 14 days.After the last intervention,tests were undertaken such as the following:(1)Sleep latency and sleep durability evaluation using pentobarbitone synergistic test for each group of rats;(2)Morris water maze tests were conducted for the purpose of verifying the memory capacity of different rats;(3)Collection samples from the rat faeces for analysis of the diversity and species specificity of each rat gut bacteria by 16 S r DNA sequencing technology;(4)The contents of the monoamine neurotransmitters 5-HT,DA and NE in the hypothalamus of rats were measured by ELISA.Results:(1)Sleep latencies were significantly extended in FM group compared to FK group,while sleep duration shortened significantly(P<0.05);sleep duration was prolonged by various treatment methods in rats(P<0.05),among which the sleep duration of rats in FY group was significantly longer than those in FY and FC groups(P<0.05),and a shorter sleep latency in FT group rather than FD group(P<0.05).(2)Compared with the FK group,the incubation period of rats in the FM group was significantly longer(P<0.05);significantly decreased period of incubation in FT group in comparison that in FM group(P<0.05);during the training period,there was no statistically significant difference in the incubation period of rats evasion between the intervention treatment groups(P>0.05).The difference in swimming speed between the groups was not statistically significant(P >0.05);the number of times rats crossed the first quadrant was significantly increased in the FT group compared with the FM group(P<0.05);the number of times rats crossed the second and third quadrants was not statistically significant between the groups(P >0.05);increased passage to the fourth quadrant was significantly greater in FC than FT group(P<0.05).(3)Comparison between the FM and FK groups indicated that the Alpha diversification Chao1,Shannon and Simpson indexes of rats had no statistical significance(P >0.05);the results of PCo A and NMDS based on Bray-Curtis analysis showed that there was a clear separation between the samples in the FK and FM groups;after the treatment with different methods of intervention,there was an overlap between the samples in each intervention group,and in the NMDS three-dimensional spatial distribution between the samples of rats in the FD,FY and FT groups,and between the samples of the FK and FC groups,the closer the distance,the higher the similarity and the smaller the difference.At the phylum level,faecal microorganisms from rats were dominated by the phylum Bacillus and Thick-walled Bacteria;the relative abundance of Bacillus,Prevotella,Lactobacillus,Trichosporon and Gastrodia was higher at the genus level.Contrast to FK group,the relative abundance of the Bacteroidetes,Firmicutes and Proteobacteria were significantly increased,and the abundance of the Actinobacteria,Prevotellaceae and Allobaculum were significantly decreased in the FM group(P<0.05);the abundance of Bacteroidetes,Parasutterella and Desulfovibrio was significantly lower in the FD group compared to the FM group,while the abundance of Firmicutes,Roseburia,Oscillibacter,Intestinimonas was significantly higher(P<0.05).According to LDA effect size(LEf Se)analysis,there were 28,4,4,9,16,8 significantly enriched species in the FK,FM,FY,FC,FD and FT groups at the phylum,order,family and genus levels respectively.(4)5-HT and DA level were reduced significantly,while NE level was elevated in hypothalamus of FM group compared to the FK group(P<0.05);hypothalamic 5-HT was significantly raised and NE markedly dropped compared to the FM group in each intervention group(P<0.05);DA levels showed a significant improvement in hypothalamus of FD,FY and FT groups compared to FM group(P<0.05),while the differences in the hypothalamic DA contents in the FD;the difference in DA content in the hypothalamus of rats in the FD,FY and FT groups was not statistically significant compared with that in the FM group(P>0.05).Conclusion:Abdominal massage is a central technique in the treatment of insomnia.The mechanism of effect may be related to the regulation of gut bacteria,affecting microbial metabolites in the gut,which in turn regulates the levels of neurotransmitters in the brain associated with gut bacteria,shortening the sleep latency and prolonging the duration of sleep.