| Autism spectrum disorder(ASD)is a group of neurodevelopmental disorders characterized by difficulties in social interaction and communication,narrow interests,and stereotyped behavior.The treatment of ASD is facing great challenges.Drugs and behavioral intervention have obvious limitations in improving the core symptoms and accompanying symptoms,and Chinese medicine is increasingly widely used in clinical treatment of ASD.As a traditional non-pharmacological treatment method,Acupuncture has great application value in the clinical treatment of autism.Clinical studies have shown that Jin’s three-needle can be used to improve the core symptoms of ASD.The treatment of the core symptoms of social interaction disorder of ASD is the focus of our attention.Our clinical studies have demonstrated that "Jin’s three-needle technique" can improve somatosensory abnormalities and tactile defensiveness exhibited by children with autism in various contexts,promoting their social interaction and communication.In terms of basic research,our team has conducted long-term series studies using the intervention of "Jin’s three-needle technique" on a rat model of autism and found that endogenous oxytocin(OT)may be a key factor in acupuncture improving social interaction behaviors in individuals with autism.Oxytocin(OT)is an important molecule that mediates synaptic signal transmission in the brain’s sensory cortex and plays a crucial role in regulating social behavior in mammals.However,there is a need for further exploration into the relationship between the intervention of ’Jin San Zhen’ acupuncture and changes in oxytocin concentration in individuals with autism spectrum disorder(ASD),as well as the alterations in neuronal synapses within relevant brain regions following acupuncture treatment,and potential changes in gene protein expression.Research focused on these aspects will shed new light on the molecular biology underlying acupuncture therapy for ASD.Therefore,conducting mechanistic studies on’Jin San Zhen’ acupuncture,oxytocin,and its receptor(OXTR)to gain deeper insights into how acupuncture regulates oxytocin levels and elucidate the neuronal and genetic protein expression changes induced by oxytocin are crucial steps towards understanding the effectiveness of acupuncture treatment for ASD.This research not only holds significant implications for non-pharmacological interventions to improve core symptoms of autism clinically but also provides essential scientific evidence supporting the efficacy of acupuncture therapy for autism.ObjectiveThe purpose of this study is to elucidate the effects and mechanisms of "Jin’s three-needle" therapy on social interaction behaviors in individuals with autism spectrum disorder(ASD)through clinical and experimental research,providing theoretical basis and experimental evidence for its clinical application.In the clinical part,we will investigate the improvement of core symptoms,sleep disorders,and anxiety disorders in preschool-aged children with mild to moderate ASD using measures such as ABC(Autism Behavior Checklist),SRS(Social Responsiveness Scale),CARS(Childhood Autism Rating Scale),CSHQ(Children’s Sleep Habits Questionnaire),and SCAS(Spence Children’s Anxiety Scale).We will also measure scrum oxytocin levels.In the experimental part,we will explore behavioral differences of"Jin’s three-needle" in VPA-induced ASD animal models and OXTR-RNAi animal models from a behavioral perspective.At the molecular biology level,we will examine mRNA expression differences in the amygdala between control group,VPA model group,and "Jin’s three-needle therapy" group rats.Changes in serum OT levels before and after intervention will be measured.Additionally,we will assess the impact of "Jin’s three-needle " on ultrastructure of amygdala neurons as well as expression of OXTR gene and protein in the amygdala.Methods(1)Clinical study:60 preschool children with autism were randomly divided into two groups using a simple randomization method.The observation group consisted of 30 cases,while the control group consisted of another 30 cases.The control group received treatment combining applied behavior analysis therapy with structured education for 4 hours per day,5 days per week,continuously for 3 months.In addition to receiving the same treatment as the control group,the observation group also underwent "Jin San Zhen" acupuncture treatment based on traditional Chinese medicine differentiation and acupoint selection according to ASD(Autism Spectrum Disorder).This treatment was administered for 5 days per week,continuously for 3 months.Before and after the three-month treatment period,both groups were assessed using various scales including ABC(Autism Behavior Checklist)for assessing autism behaviors in children,SRS(Social Responsiveness Scale)for measuring social interaction abilities,CARS(Childhood Autism Rating Scale)for evaluating the severity of autism symptoms,CSHQ(Children’s Sleep Habits Questionnaire)to assess sleep disorders,and SCAS(Spence Children’s Anxiety Scale)to evaluate social anxiety levels in children with autism.Additionally,changes in serum oxytocin levels before and after treatment were measured in both groups.(2)Experimental study:①The rat model of autism was made by intraperitoneal injection of sodium valproate(VPA)during pregnancy,then carried out Jin’s three-needle,and the mRNA transcriptome analysis of amygdala in control group,VPA model group and Jin’s three-needle group was compared.②The rat model of OXTR gene with low expression was made by stereoscopic injection of OXTR-RNAi.③Through behavioral tests,ultrastructural observations,detection of oxytocin receptor(OXTR)gene and protein expression,a comparative analysis was conducted on social anxiety,social initiative,social cognition,neuronal synaptic ultrastructure differences in the control group,the two animal model groups,the acupuncture group and the sham acupuncture group.The study aimed to evaluate the impact of OT/OXTR on rat social interaction behavior and explore the role and changes in oxytocin in improving autistic social interaction behavior through Jin’s three-needle.Results(1)Clinical study:A total of 54 children completed the study.①Core symptoms:compared with pre-treatment,the total scores of ABC,factors and CARS were significantly decreased in both groups after treatment(P<0.05).Compared with the control group,the total score of ABC and social interaction factor were significantly lower in the observation group after treatment(P<0.05).②Social interaction ability:compared with pre-treatment,the total score of SRS and the scores of all factors were significantly decreased in both groups after treatment(P<0.05).Compared with the control group,the scores of social cognition,social communication,social motivation,autistic behavior and SRS were significantly lower in the observation group after treatment(P<0.05).③Sleep quality:Compared with pre-treatment,CSHQ scores and total scores in both groups were significantly decreased(P<0.05).The sleep impedance score,sleep duration,sleep anxiety score,daytime sleepiness score,sleep wake score and CSHQ score of experimental group were lower than those of control group(P<0.05).④Anxiety disorder:compared with pre-treatment,the scores of anxiety disorder factors and total scores in the observation group were significantly decreased(P<0.05),the physical injury fear score in control group was significantly decreased(P<0.05),social fear score was higher than before treatment(P<0.05),Compared with the control group,the physical injury fear score of the observation group after treatment was lower than that of the control group(P<0.05).⑤Serum oxytocin level,compared with pre-treatment,the serum oxytocin level was increased in both groups after treatment(P<0.05);Compared with the control group,the serum oxytocin level in the observation group was higher than that in the control group after treatment(P<0.05).(2)Experimental study:①Behavioral analysis:Compared with model group and sham acupuncture group,the control group and acupuncture group showed more positive prosocial behaviors in social anxiety,social initiative and social cognition(P<0.05).②mRNA transcriptome sequencing:Jin’s three-needle affected the oxytocin synapses,GABA synapses and other related neurosynaptic signaling pathways in amygdala neurons of VPA model mice,and regulated Arc synaptic plastic regulatory gene and Perl sleep rhythm gene.③Ultrastructure of amygdala neurons:Through the ultrastructure observation of amygdala neurons of rats in each group by electron microscopy,we found that the organelles of amygdala neurons of rats in the control group,VPA+acupoint and OXTR-RNAi+acupoint group had clear structure,complete synaptic structure,and many synapses and vesicles.On the contrary,the amygdala neurons and mitochondria in the VPA,VPA+nonacupoint and OXTR-RNAi+nonacupoint groups had edema and even vacuolization,lysosomes increased,organelles decreased,rough endoplasmic reticulum structure irregular arrangement,particle loss,synaptic structure blurred,and vesicles decreased.After acupuncture intervention,the number of organelles in amygdala neurons increased,the organelles’ structure was complete,the synaptic structure was clear,and the number of vesicles increased.④Serum oxytocin level:The serum oxytocin level of VPA model rats and OXTR-RNAi model rats is lower than that of control rats(P<0.05),and Jin’s three-needle can improve the serum oxytocin level of VPA model rats and OXTR-RNAi rats(P<0.05).⑤OXTR gene and protein expression:the expression levels of OXTR gene and protein in the amygdala of rats were significantly decreased after the interference of VPA and OXTR-RNAi(P<0.05).Jin’s three-needle up-regulated the OT/OXTR gene and protein expression levels in the amygdala of rats with OXTR-RNAi interference(P<0.05).The expression of OXTR gene and protein in amygdala of rats in sham acupuncture group did not show significant changes.ConclusionClinical studies have shown that Jin’s three-needle intervention can reduce the core symptoms of preschool children with mild to moderate ASD,improve their social ability,improve their sleep quality,d anxiety disorders,and raise peripheral blood oxytocin levels.The experimental study showed that Jin’s three-needle affected oxytocin synapses,GABA synapses and other related synapses in amygdala neurons,regulated Arc synaptic plastic regulatory gene and Perl sleep rhythm gene,which may improve the social interaction behavior of autism,improve sleep and anxiety disorders,and reshape the ultrastructure of synaptic cells.It is one of the important mechanisms to increase the level of serum oxytocin and increase the expression level of oxytocin and its receptor genes and proteins of autism. |
