Intervention Effects And Mechanisms Of Sulforaphane In Subjects With Autism Spectrum Disorder

Background:Autism Spectrum Disorder（ASD）constitutes a heterogeneous group of pervasive developmental disorders with hallmarks of persistent social skills deficits along with repetitive stereotype behaviors.The complexity of pathological causes along with the heterogeneity of symptom phenotypes limit the development of therapeutic drugs and interventions,which requires further work to develop drugs that target the disease pathogenesis and improve the core symptoms of ASD.Objective:（1）A systematic review was conducted to characterize the use frequency and use type of complementary and alternative therapies in subjects with ASD.（2）An animal study was conducted to clarify the effects and mechanisms of sulforaphane on social interaction deficits and repetitive stereotyped behaviors in ASD model rats.（3）A clinical randomized controlled trial was conducted to investigate the effects of sulforaphane on social interaction deficits and repetitive stereotyped behaviors in subjects with ASD.Method:（1）A comprehensive literature search was performed in English databases（Pub Med,Web of Science,EMbase,ERIC,and Psyc INFO）and Chinese databases（China National Knowledge Infrastructure,Wanfang Database,VIP Database,and China Biomedical Literature Database）to locate all possible articles.Two reviewers independently performed the literature search,study selection,quality assessment and data extraction.Discrepancies were resolved through discussion,with the arbitration of a third reviewer where necessary.（2）Animal experiments were conducted as follows:Maternal immune activation（MIA）was adopted to build ASD model rats.Time-mated Sprague-Dawley（SD）female rats were injected intraperitoneally with lipopolysaccharide（LPS,150μg/kg）or saline on gestational day 15（G15）to induced ASD model rats or normal control rats.ASD model rats were randomly assigned to the experimental group and the control group at postnatal day 40（P40）.ASD model rats in the experimental group（LPS+SFN）received sulforaphane gavage and ASD model rats in the control group（LPS+NS）received equivalent amount saline gavage.Normal control mice received equivalent amount saline gavage was used as a blank control（NS+NS）.The intervention period lasted from postnatal day 45 to 72 and the sulforaphane dosage set at40mg/kg.A set of behavioral tests,open field test,three-chamber social interaction test,marble burying test,and novel object recognition test,were conducted to evaluate the effects of sulforaphane on aberrant behaviors in ASD model rats.Peripheral blood and brain tissues（prefrontal cortex and hippocampus）were collected for biological measures to investigate the mechanisms of sulforaphane on aberrant behaviors in ASD model rats.（3）Clinical study was conducted as follows:A total of 135 subjects with ASD aged 3～15 years were randomly assigned to the experimental group（n=68）and the control group（n=67）for a 12-week intervention.Subjects with ASD in the experimental group received sulforaphane supplements based on their body weight,while subjects with ASD in the control group received matched placebo tablets.A set of scales,Social Responsiveness Scale（SRS）,Repetitive Behavior Scale-Revised（RBS-R）,Autism Behavior Checklist（ABC）,OSU Autism Rating Scale-DSM-IV（OARS-4）,and Clinical Global Impression Scale（CGIS）,were used to examine the effects of sulforaphane on abnormal behaviors of subjects with ASD before intervention（0 week）,during intervention(4^thweek and 8^thweek),and after intervention(12^thweek).Blood and urine samples were collected for routine serum and urine laboratory chemistries drawn before intervention（0 week）and after intervention(12^thweek).Result:（1）The literature search returned a total of 1568 records,the adoption of selection criteria yielded a final inclusion of 23 articles.These included studies mainly used cross-sectional surveys to investigate the use frequency of complementary and alternative therapies in subjects with ASD.These included studies reported that the use frequency of complementary and alternative therapies in subjects with ASD ranged from 28%to 92%.These included studies mainly used questionnaires to investigate the use type of complementary and alternative therapies in subjects with ASD,special diets,vitamins/minerals,fatty acids,and dietary supplements constituted the most commonly used therapies.（2）Animal experiment results were as follows:Three-chamber test reported that sulforaphane improved social interaction deficits in ASD model rats,ASD model rats in experimental group spent more time in the chamber with a strange rat（stranger 1）than in the chamber with an empty cage（empty）,the difference was statistically significant（t=2.610,P=0.020）.Marble burning test reported that sulforaphane decreased repetitive stereotyped behaviors in ASD model rats,ASD model rats in experimental group buried less marbles as compared to ASD model rats in control group,the difference was statistically significant（t=2.83,P=0.01）.ELISA assay reported that sulforaphane reduced the levels of pro-inflammatory cytokines in ASD model rats,the levels of pro-inflammatory cytokine IL-1βin peripheral blood and brain tissue of ASD model rats in experimental group were lower than that of ASD model rats in control group,the difference was statistically significant（serum:t=2.71,P=0.016;PFC:t=2.57,P=0.02;Hip:t=3.23,P=0.006）.Immunofluorescence staining reported that sulforaphane reduced the activation of glia,the mean density of microglial marker IBA-1 in hippocampus of ASD model rats in experimental group were lower than that of ASD model rats in control group,the difference was statistically significant（t=3.68,P=0.02）.Golgi staining reported that sulforaphane reduced the density of dendritic spines,the density of dendritic spines in hippocampus of ASD model rats in experimental group were lower than that of ASD model rats in control group,the difference was statistically significant（t=10.77,P<0.001）.Immunofluorescence staining reported that sulforaphane increased astrocyte induced phagocytosis of inhibitory synapse,the co-localization of astrocyte marker GFAP and inhibitory postsynaptic protein Gephyrin in prefrontal cortex and hippocampus of ASD model rats in experimental group were higher than that of ASD model rats in control group,the difference was statistically significant（PFC:t=4.68,P=0.009;Hip:t=3.72,P=0.02）.Western blot reported that sulforaphane decreased the expression of Keap1,the expression of Keap1 in prefrontal cortex and hippocampus of ASD model rats in experimental group was lower than that of ASD model rats in control group,the difference was statistically significant（PFC:t=2.52,P=0.03;Hip:t=2.79,P=0.02）.（3）Clinical study results were as follows:Sulforaphane improved OARS-4 scores of subjects with ASD,the total OARS-4 scores,the impaired social interaction scores,and the communication barriers scores of subjects with ASD in experimental group was lower than that of subjects with ASD in control group,the differencewasstatisticallysignificant(totalscores:F_TR=10.2,DF=1,91,P=0.002^BH;impaired social interaction scores:F_TR=13.21,DF=1,91,P<0.001^BH;communication barriers scores:F_TR=9.73,DF=1,91,P=0.002^BH).Sulforaphane improved CGI-I scores of subjects with ASD,the CGI-I scores of subjects with ASD in experimental group was lower than that of subjects with ASD in control group,the difference was statistically significant(F_TR=22.80,DF=1,89,P<0.001^BH).Sulforaphane did not impact the SRS scores,RBS-R scores,ABC scores and CGI-S scores of subjects with ASD,there were no statistically significant differences in the SRS scores,RBS-R scores,ABC scores and CGI-S scores of subjects with ASD between the experimental group and the control group(SRS:F_TR=0.02,DF=1,91,P=0.885;RBS-R:F_TR=1.91,DF=1,86,P=0.171;ABC:F_TR=0.14,DF=1,89,P=0.706;CGI-S:F_TR=0.27,DF=1,91,P=0.604).Conclusion:（1）The use frequency of complementary and alternative therapies in subjects with ASD ranged from 28%to 92%,special diets and vitamins constituted the most commonly used therapies.（2）Sulforaphane reduced social interaction deficits and repetitive stereotyped behaviors,alleviate inflammation response and glial activation,regulate NF-κB and Nrf2 signaling pathway in ASD model rats.（3）Sulforaphane improved social interaction deficits and repetitive stereotyped behaviors in subjects with ASD.