Clinical Efficacy Of Tiaochangzhishen Acupuncture On Autism And Bacteria/Brain Effect In Model Mice

Objective Observe the clinical efficacy of Professor Wu Xu's"tiao chang zhi shen",a cupuncture on autism spectrum disorde(ASD)and its influence on intestinal flora.Analy ze the correlation between the effect of acupuncture and the changes of intestinal flora,and explore the possible clinical mechanism of the treatment of "tiao chang zhi shen"acupuncture.At the same time,based on the bacteria-brain axis,we will preliminarily observe the effects of electro-acupuncture at Zusanli on the behavior,intestinal flora,p eripheral and central inflammation levels of ASD model mice,in order to provide scie ntific evidence for "tiao chang zhi shen" acupuncture to treat ASD through the bacteri a-brain axis.Methods ?Clinical study:A randomized control design was adopted.60 cases were ran domly divided into acupuncture group("tiao chang zhi shen" acupuncture combined wi th education and rehabilitation training)and rehabilitation group(education and rehabilitati on training),with 30 cases in each group.The Child Autism Rating Scale(CARS),Autis tic Child Behavior Scale(ABC)and "PEP-3 Psychological Educational Assessment for A utistic Children" were used to evaluate the efficacy before treatment and at the end of 3 months after treatment.?Intestinal microbial research:16S rDNA technology was used to analyze the difference in intestinal flora between ASD children and normal children,and the difference in in testinal flora in ASD children before and after acupuncture intervention or rehabilitation intervention.A simple linear regression was used to analyze the correlation between t he intestinal flora and the total score of PEP-3 before and after acupuncture,and scree n the intestinal flora related to the curative effect of acupuncture.?Experimental study:A mouse model of ASD was induced by intraperitoneal injection of valproic acid(VPA)into pregnant mice and randomly divided into blank group,mod el group,electroacupuncture Baihui group,electroacupuncture Zusanli group.4 weeks aft er treatment,the mice were sacrificed.Three-box social behavior test,open field test,water maze test,etc.are used as behavioral effect indicators,and the change of intesti nal flora structure,diversity index and similarity coefficient in the stool samples of AS D mice are used to observe the intestinal flora indicators,peripheral inflammatory fact ors TNF-?,IL-17a and hippocampal microglia are the observation indicators of central i nflammation.Screen the structure and level of intestinal flora that are closely related t o the difference in central inflammation,and to explore the effects of electroacupunctur e at Zusanli on the behavior,intestinal flora,peripheral and central inflammation levels of ASD model mice.Results Clinical research:?PEP-3 scale score statistics before and after treatment fo und that both groups can improve ASD children's cognition,language understanding and expression,large and small muscle,visual action mimicking ability,emotional expression,social interaction,non-verbal behavior and language behavior(all P<0.05).However,com pared with the rehabilitation group,the acupuncture group improved better in cognition,1 anguage expression,understanding,imitation,emotional expression and language behavior characteristics(P<0.05).?The statistical analysis of the difference between the total score s of the ABC scale before and after treatment found that the two groups can improve th e perception,communication,movement,language,and self-care abilities of ASD(P<0.05).However,the acupuncture group was better than the rehabilitation group in terms of sen sation,language,self-care ability and total score(all P<0.05).?The statistical analysis of t he difference between the total scores of CARS scale found that the two groups can imp rove the abilities of children with ASD to varying degrees(P<0.05).The acupuncture group was better than the rehabilitation group in terms of emotional response,auditory respons e,language communication,overall impression and total score after the intervention of th e bowel regulating and healing acupuncture method(P<0.05).Intestinal flora research:?There are significant differences in the composition and str ucture of the intestinal flora between normal children and ASD children:In the normal c hild group,the abundance of Bacteroides(phylum,class,order,family,genus)and Bacteroi des fragilis increased,while in the ASD child group,Firmicutes,Clostridium(class,order),Lacetospiraceae,Lactococcus,Faetobacter species,actinomycetes(phylum,class),Bifidobacteri um(order),Family,genus)are more abundant.?There are differences in the intestinal flora before and after acupuncture intervention:Compared with before acupuncture interventio n,after acupuncture intervention,the abundance of Bacteroides(phylum,class,order,famil y,genus),Proteobacteria(phylum,class),Alternomonas,and Marine bacteria(family,genus)increased;The abundance of actinomycetes(phylum,class)and bifidobacterial(order,family,genus)decreases.Among them,the abundance levels of Bacteroides in phylum,class,ord er,family,and genus changed significantly after acupuncture intervention,and tended to t he abundance level of normal children.In addition,after acupuncture intervention,the ab undance of Lactobacillus,Firmicutes,Clostridium,Lactococcus,and Rare Micrococcus de creased.?A total of five bacterial groups closely related to the acupuncture effect were screened out,namely Bacteroides,Bacteroides,Bifidobacteria,Clostridia,and Firmicutes.Among them,Bacteroides are positively correlated with acupuncture efficacy,while Bifido bacteria,Clostridium,Firmicutes are negatively correlated with acupuncture efficacy.Experimental research:?The three-box social experiment,the open field experiment,and the water maze experiment suggest that ASD model mice have obvious obstacles in social skills,obstacles in preference for new things,obstacles in exploration ability,stereotyped movement,and decreased spatial memory ability.Electroacupuncture at Zusanli or Baihui can all improve the above dysfunction.At the same time,electroacupuncture at Zusanli group can significantly improve the dysfunction of ASD model mice,and electroacupuncture at Baihui group can significantly improve the learning and memory ability of ASD model mice;?The levels of peripheral serum inflammatory factors TNF-? and IL-17a in ASD model mice were significantly increased,while electroacupuncture at Baihui or electroacupuncture at Zusanli could down-regulate the level of peripheral serum inflammation.At the same time,electroacupuncture at Zusanli group Peripheral anti-inflammatory effect of ASD is better than that of electroacupuncture in Baihui group;?The activity of microglia in the hippocampus of mice in the ASD model group is increased,and electro acupuncture in Baihui or Zusanli can inhibit the microglia in the hippocampus of ASD model mice,and the effect of electroacupuncture in Zusanli group in inhibiting central inflammation is better than that of electroacupuncture in Baihui group;?The experimental results of mouse intestinal flora show that:1)In the normal group of mice,the abundance of Bacteroides(phylum,class,order)and the Bacteroidales_S24_7group increased;while in the model group of mice,it was expressed as Firmicutes and Clostridia(Class,order),Ruminococcaceae,etc.abundance increased.2)The abundance levels of Bacteroides(phylum,class,order,family,genus),Verrucomicrobiae(phylum,class,order,family,genus),Akkermansia,etc.,increased after the intervention of the Zusanli group by electroacupuncture.The electro-acupuncture at Baihui point is mainly increase the Bacteroidales_S24_7group.Conclusion"Tiao chang zhi shen"acupuncture combined with conventional educati on rehabilitation training can effectively improve the clinical symptoms of ASD children,and it is better than pure education rehabilitation training in terms of cognition,languag e expression,understanding,language behavior characteristics,imitation,and emotional exp ression.There were no adverse reactions during the treatment.It is a feasible and extend able plan for the comprehensive treatment of autism with medical and education.The int estinal flora of ASD is different from that of normal children.The intestinal flora of AS D can be adjusted benignly by "tiao chang zhi shen" acupuncture.Electroacupuncture at Z usanli can improve the behavior,intestinal flora disorder,peripheral and central inflammat ion levels of ASD model mice.