| Objective:The purpose of this study was to investigate the mechanism of regulating synaptic balance disorders by targeting the P38/ELK-1 signaling pathway during the critical developmental period of autistic young mice,thereby improving the symptoms of autism.Methods:1.Establishment of a valproic acid-induced autism mouse model:(1)After conception,Wistar female mice were divided into two groups,10 mice in each group,and the same dose of solvent was injected intraperitoneally at 12.5 days of gestation:(1)Control group(control group,injected with normal saline),(2)VPA group(injected VPA 600 mg/kg,50mg/m L);(2)intervention:10 young mice were selected from the Control group(injected with normal saline),and 20 young mice from the pregnant mice of the VPA group were selected and divided into two groups:(1)SB203580group(injection of MAPK signaling pathway inhibitor SB203580,5mg/kg),(2)VPA group(injected with normal saline),the three groups of young mice were injected intraperitoneally with the same dose of the corresponding solvent for one week in the critical period of development for intervention treatment;(3)Growth and behavioral assessment:Eye test,negativity test,swimming test,body weight,tail deformity and postnatal mortality were used to understand the growth and development of young mice in Control group and VPA group;Three-box social behavior test,elevated plus maze,open field test,To understand the behavioral changes of the three groups of young mice after the intervention treatment through the self-combing experiment,the bead burying experiment and the water maze experiment;2.Understand the changes of pathways:(1)Western blot detection of P-P38 and P-ELK-1 in the P38/ELK-1 signaling pathway in the prefrontal cortex and hippocampus of the control group and VPA group on the first day after birth(PN1),PN7,PN14,PN21,PN28,PN35 brain tissue Protein expression,find the critical period of development;(2)Western blot and immunohistochemistry were used to detect the protein expression of each member of PFC and HPC signaling pathway in three groups of young mice after intervention treatment;3.Understanding synaptic and neuronal-related changes before and after intervention:(1)HE staining was used to observe the changes of nerve cells in the three groups of young mice;(2)Western blot,immunohistochemistry and q RT-PCR were used to detect the relative m RNA content and protein expression of PSD-95,SYN and Gephyrin in the PFC and HPC of the three groups of young mice;(3)The ratio of v GLUT1/v GAT in HPC CA1 and CA3 regions and primary neurons was observed by immunofluorescence.Results:1.The VPA-induced autism-like young mouse model was successfully established:(1)The VPA group had abnormal growth and development,as shown in the following: the time taken to turn around in the negative test was significantly increased(P<0.001),and the swimming ability was weakened(P<0.001),the time of eye opening was later(P<0.05～P<0.001),the growth weight was significantly decreased and continued to PN35(P<0.01～P<0.001).35% of the young mice in the VPA group had tail deformities(P<0.001),43.7% mortality rate(P<0.001);(2)The core symptoms of ASD in the VPA group were severe:social skills were impaired in the three-box social experiment,accompanied by social novelty deficits and the SB203580 group could partially improve their social deficits(P<0.05～P<0.001);In the elevated plus maze test and open field test,the VPA group had obvious anxiety,but the SB203580 group was better(P<0.05～P<0.001);In the self-carding experiment and the bead-embedding experiment,the VPA group had obvious stereotyped behaviors(P<0.001)and the SB203580 group decreased compared with the VPA group(P<0.01～P<0.001);In the learning and memory ability test,the latency time of the VPA group was relatively longer(P<0.001),and the number of crossing platforms was significantly less(P<0.001),while that of the SB203580 group was relatively improved(P<0.001);2.Pathway changes:(1)Western blot analysis showed that the levels of phosphorylated proteins in the P38/ELK-1 signaling pathway in PFC and HPC increased at the third week after birth(PN21)in young mice in the VPA group,which continued to adulthood;(2)Western blot and immunohistochemical detection results showed that the protein expression levels of each member of the P38/ELK-1 signaling pathway in the VPA group increased(P<0.01～P<0.001);3.Effects of interventional therapy on synapses and nerve cells:(1)Neurodegenerative changes were seen in the HE staining of PFC and HPC in the VPA group;(2)Western blot,immunohistochemistry and q RT-PCR detection results showed that the expression levels of PSD-95 and SYN protein and the relative m RNA content of PFC and HPC in VPA group increased(P<0.05～P<0.001),Gephyrin protein expression level and m RNA relative content decreased(P<0.05～P<0.001),compared with the VPA group,the SB203580 group showed the opposite(P<0.05～P<0.001);(3)The results of immunofluorescence detection of HPC CA1,CA3 area and primary nerve cells in VPA group showed that the ratio of v GLUT1/v GAT was increased(P=0.007,P<0.001,P<0.05).Compared with the VPA group,the ratio of v GLUT1/v GAT in the SB203580 group was decreased(P=0.006,P<0.001,P<0.05).Conclusions:1.VPA-induced autistic young mice have delayed neurodevelopmental milestones,growth retardation,high mortality,and severe behavioral symptoms;2.The third week after birth(PN21,equivalent to human early childhood)may be the development of the ASD nervous system critical period;3.The excitatory synapses increase and the inhibitory synapses decrease in young autistic mice,so that the balance of excitatory/inhibitory synapses is unbalanced;4.P38/ELK-1 is involved in the regulation of synaptic plasticity;5.During the critical period of development SB203580 treatment can regulate the abnormal activation of P38/ELK-1 signaling pathway and the imbalance of excitatory/inhibitory synapses,and further improve the behavioral symptoms of autism-like young mice. |
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