Study On The Effect Of Acupuncture On The Intestinal Flora Of Mice With Chronic Restraint Stress Anxiety

Anxiety disorder is the most common mental Disorder,known as Anxiety neurosis,divided into Generalized Anxiety Disorder and acute anxiety,characterized by anxious emotional experience.With the rapid development of social economy,more and more people are faced with the problem,psychological pressure and burden,so that the incidence of anxiety gradually on the rise.The intestinal system as a "second brain",participate in a wide variety of physiological activities in the human body,some existing experiments prove the existence of intestinal flora and anxiety,however,there are few studies on how anxiety can cause changes in intestinal flora,and acupuncture has unique advantages in anti-anxiety.Therefore,in this study,the anxiety model of mice was established by chronic restraint stress method,and acupuncture therapy was adopted to explore the changes of intestinal flora in anxiety model mice induced by acupuncture.ObjectiveBy studying the effect of acupuncture on the behavior of anxious model mice and the structure and diversity of intestinal flora,the anti-anxiety effect of acupuncture and its regulation effect on intestinal flora of anxious model mice were explored.MethodsTwenty-four male C57BL6 mice of SPF grade were randomly divided into four groups:control group,model group,tiao-shen groupandshu-gan group,6 mice in each group after one week of adaptive feeding.Anxiety model was established by chronic restraint stress method;Tiao-shen group was treated with acupuncture at Baihui and Yintang points once a day for 10 minutes for 9 consecutive days;Shu-gan group was treated with acupuncture at Hegu and Taichong points once a day for 10 minutes for 9 consecutive days;Behavior test in black and white box on the 8th day;Behavior test in elevated maze on the 9th day;Behavioral tests were performed to record the residence time in the white box,the proportion of times entering the open arm(OE)and the residence time in the open arm(OT);The changes of intestinal microflora structure and diversity in mice were analyzed by using 16S rDNA V3+V4 region high-throughput sequencing technology.Results(1)Residence time in the white box:compared with the control group,the residence time in the white box of mice in the model group is shortened(P<0.05).Compared with the model group,the residence time of tiao-shen group and shu-gan group in the white box was significantly longer(p<0.01).Compared with that of the tiao-shen group,the residence time of the two groups showed no statistical difference(p>0.05).(2)Open arm entry times(OE)and open arm residence time(OT):Compared with the control group,the number of entering into the open arm of the model group decreased(P>0.05),and the mice's residence time in the open arm increased(p<0.05);Compared with the model group,the number of entering into the open arm of the acupuncture tiao-shen group significantly increased(p<0.05),and the length of stay in the open arm was significantly extended(p<0.01);Compared with the model group,the number of entering into the open arm of the acupuncture shu-gan group increased(p>0.05),and the mice's residence time in the open arm increased(p<0.01).(3)Alpha diversity analysis:alpha diversity analysis showed that model group,observed-species index and shannon index of mice in the model group were higher than those in the control group(p<0.05),on the observed-species index,compared with that of the model group,that of the tiao-shen group increased to a certain extent(p>0.05),and that of the shu-gan group increased more than that of the model group(p<0.05),on the shannon index,compared with that of the model group,that of the tiao-shen group and that of the shu-gan group increased to a certain extent(p>0.05).(4)Beta diversity analysis:based on the Unweighted PCoAUnifrac distance analysis,model group and acupuncture group of each sample in the floor plan of the regional distribution and the control group significantly different,distance to each other,dividing line is clear,and the acupuncture group and model group are regional overlap,at close range between groups,the distanc.e between the tiao-shen group and the shu-gan group was closer than that between the model group and the control group.(5)Community composition analysis:Bacteroidetes and firmicutes main position of intestinal flora in mice,compared with the control mice,under the phylum level,the mouse intestinal flora in the model group showed a decrease in the ratio of bacteroidetes and a increase in the ratio of firmicutes.Under the genus level,the model group of unidentified lachnospiraceae,unidentified_ruminococcaceae,bacteroides,alistipes and roseburiaincrased,and the proportion of lactobacillus,candidatus saccharimonas and alloprevotella decreased.Compared with model group mice,under the phylum level,acupuncture group will make the bacteroidetes accounted for ascension,firmicutes proportion is reduced;Under the genus level,in the acupuncture siguan group the proportion of unidentified_Ruminococcaceae,unidentified lachnospiraceae and alloprevotellawas decreased,while the proportion of bacteroides,alistipes and roseburia was increased;inthe acupuncture tiao-shen group the proportion of unidentified lachnospiraceae,bacteroides and alloprevotella was decreased,while that of lactobacillus,alistipes,roseburia and candidatus saccharimonas was increased.LEfSe analysis(LDA threshold of 4)showed that,compared with the blank group,the model group had significant enrichment of muribaculaceae,rikenellaceae,ruminococcaceae with significant difference.Compared with the model group,the differences of the above three bacteria in tiao-shen group and shu-gan group disappeared.Compared with the control group,onlyrikenellaceae had significant enrichment in tiao-shen group and shu-gan group,with significant difference.Conclusion(1)acupuncture can effectively improve the anxiety-like behavior of anxiety model mice(2)acupuncture increased the number of probiotics in the gut and bacterial diversity of anxious mice,and can adjust the level of different flora.To sum up,acupuncture may alleviate anxiety-like behaviors by increasing the number of probiotics and the bacterial diversity in the intestinal tract of anxious mice and adjusting the level of different flora.