The Mechanism Of N-acetylcysteine In Improving Autistic-like Behavior In Autism Model Induced By Valproic Acid

BackgroundThe etiology of autism is complex and there is currently no cure for autism.N-acetylcysteine(NAC)has been reported to improve social behavior,irritability,selfinjury,and anxiety-like behavior in autism,but the mechanism by which NAC ameliorates autism-like behavioral abnormalities remains elusive.The abnormal autophagy function and Notch signaling pathway play an important role in the pathogenesis of autism.Whether the improvement of N-acetylcysteine on autism-like behavioral abnormalities is related to the regulation of autophagy and Notch signaling pathway remains to be studied.ObjectiveTo investigate whether NAC can improve the autism-like behavioral abnormalities of valproic acid(VPA)-induced autism animals by regulating autophagy and Notch signaling abnormalities.MethodsThe autism model was established by intraperitoneal injection of VPA at 12.5 days of gestation,repeated and rigid behavior and social interaction behavior of offspring of pregnant Wistar rats were detected by open-field and three-chamber experiment,respectively,to identify whether the autism model was successfully established.The autism model group was randomly treated with NAC and normal saline,and divided into two groups: the intraperitoneal injection of NAC(VPA+NAC)group;VPA group was intraperitoneally injected with the same amount of normal saline.The open field experiment was used to identify the repetitive and rigid behavior changes in VPA+NAC group and VPA group.The expression differences of autophagy related molecules Beclin-1,LC3 B and P62,as well as Notch intracellular domain of Notch receptor(NICD),Notch-1receptor(Notch 1)and downstream target gene Hes1 in the prefrontal cortex(PFC),hippocampus(HC)and cerebellum(CB)were detected by Western Blot.The protein expression and localization of Hes-1 were detected by double immunofluorescence assay.Results1.The results of open field experiment showed that,compared with the control group,self-grooming time and number increased in the VPA group;In the three-chamber experiment,the time of entering the chamber of the stranger mouse 1 in the control group was higher than that of staying in the empty chamber.There was no statistical difference in the time of VPA group in the chamber of the stranger mouse 1 and empty chamber.In addition,the time spent in the chamber of stranger 2 in the VPA group was no different from the time spent in the chamber of stranger 1,while the time spent in the chamber of stranger 2 in the control group was higher than that in the chamber of stranger 1.2.Western Blot results showed that compared with the control group,the expressions of Notch 1,NICD and Hes1 in PFC,HC and CB brain regions of VPA rats were increased.3.Double immunofluorescence assay results showed that compared with the control group,the expression of Hes-1 in VPA-induced autistic animals was increased in both PFC and CB brain regions,and tended to increase in HC brain region.Hes-1 was mostly expressed in the cytoplasm or membrane.4.After NAC treatment,the results of open field experiment showed that compared with the VPA group,the number of crossing the central region increased and self-grooming time and number decreased in the VPA+NAC group.5.After NAC treatment,Western Blot results showed that the expressions levels of Notch 1,NICD and Hes1,related molecules of Notch signaling pathway in PFC,HC and CB brain regions in the VPA+NAC group were decreased compared with VPA group.The expression levels of autophagy marker LC3 B and Beclin-1 protein in the above-mentioned three brain regions were increased,while the expression of autophagy marker P62 was decreased,and the differences were statistically significant.Conclusions1.VPA-induced autism animals possessed hyperactivity of the Notch-1/Hes-1signaling pathway.2.NAC could improve the repetitive and rigid autism-like behavioral abnormalities.3.NAC can improve autophagy deficiency and down-regulate the Notch-1/ Hes-1signaling pathway.The above-mentioned conclusions suggest that NAC may improve autism-like behavioral abnormalities by regulating autophagy and Notch-1/Hes-1 signaling pathway.