| Objectives: This study aimed to investigate the impact of valproic acid(VPA)on the histone acetylation of acetaldehyde dehydrogenase 1A1(ALDH1A1)and the mechanism underlying VPA-induced autism-like behavior.Methods:(1)Establishment of VPA-induced autism-like model: after female adult rats were mated with males,they were randomly assigned to two groups: the VPA group and the CON group.At 12.5 days of gestation,the VPA group pregnant rats were intraperitoneally injected with VPA,and the CON group pregnant rats were injected with the same volume of 0.9%saline.Behavioral tests,including an open-field test and a three-chamber social test,were conducted on the offspring six weeks after birth.After the behavioral tests were completed(about 8 weeks),offspring's serum and brain tissues were collected to measure the electrophysiological and related biomakers levels.Electrophysiological experiments were performed to investigate long-term potentiation(LTP)in the prefrontal cortex(PFC).The expression levels of proteins associated with synaptic plasticity,including AMPA receptors(Glu A1 and Glu A 2),NMDA receptors(Glu N1,Glu N2 A and Glu N2B)and synapsin 1(SYN1),HDACs(including HDAC1?HDAC 2?HDAC 3 and HDAC 8),acetylated histone 3(Ac H3),retinoic acid receptor alpha(RAR?),and ALDH1A1 proteins in the PFC were measured by Western blotting(WB).The quantitative polymerase chain reaction(q PCR)was used to detect the HDACs(including HDAC1?HDAC 2?HDAC 3 and HDAC 8)and ALDH1As(including ALDH1A1?ALDH1A2 and ALDH1A3)m RNA levels in the PFC.ALDH enzyme activity in PFC tissue was detected using a Micro ALDH Assay Kit.Serum retinol levels were determined using high performance liquid chromatography(HPLC).The RA level in the PFC was measured using ultrahigh-performance liquid chromatography/tandem mass spectrometry.A chromatin immunoprecipitation(Ch IP)experiment explored the enrichment of Ac H3 on the ALDH1A1 gene promoter.(2)Design of MS-275(an HDACs inhibitor)intervention experiment: female rats were paired with males and randomly assigned to three groups: the CON+Saline group,VPA+Saline group and VPA+MS-275 group.During pregnancy,the VPA+Saline group and VPA+MS-275 group pregnant rats were intraperitoneally injected with VPA,and the CON+Saline group was injected with saline.Offspring from the VPA+MS-275 group received an intraperitoneal MS-275 injection after birth.Offspring from the CON+Saline group and VPA+Saline group were treated with saline for comparison.Behavioral experiments were conducted to test the autism-like behavior changes of offspring rats after MS-275 intervention.The LTP changes in the PFC were detected using electrophysiological experiments.The protein expression levels of Ac H3,ALDH1A1,RAR? and synaptic related proteins(Glu A1,Glu2 A and SYN1)in PFC were detected by WB.The expression level of ALDH1A1 m RNA in PFC was quantitatively detected by q PCR.A Micro ALDH Assay Kit was used to determine the change of ALDH enzyme activity in PFC.RA levels in PFC were detected by UPLC-MS/MS.CHIP assay was used to detect the enrichment of ACH3 in the ALDH1A1 gene promoter region.(3)Design of RA administration experiment: pregnant female rats were randomly assigned to three groups:the CON+Corn Oil group,VPA+Corn Oil group and VPA+RA group.During pregnancy,VPA+Corn Oil and VPA+RA groups were intraperitoneally injected with VPA,and the CON+Corn Oil group was treated with saline.Offspring from the VPA+RA group received an oral RA administration after birth.Offspring from the CON+Corn Oil group and VPA+Corn Oil group were treated with corn oil for comparison.Behavioral tests was conducted to test the test the autism-like behavior changes of offspring after treatment with RA.The changes of LTP level in PFC were detected using electrophysiological experiments.The protein expression levels of HDAC3,ACH3,ALDH1A1,RAR? and synaptic related proteins(Glu A1,Glu2 A and SYN1)in PFC were detected using WB.UPLC-MS/MS was used to detect the RA levels in PFC.Results:(1)In the open field test,there was no significant difference in the total distance traveled between the CON group and the VPA group(P>0.05);the VPA group offspring spent significantly less time in the central zone(P<0.001)and more time on self-grooming than CON group(P<0.001).In one three-chamber test(the stimulus: a strange rat vs.an object),the CON group offspring spent significantly more time in the strange rat zone than in the object zone(P<0.001),but the VPA group spent almost equal time between the stranger and the object zones(P>0.05).In another three-chamber test(the stimulus: a stranger rat vs.a familiar rat),CON group offspring showed more interest in the stranger rat than the familiar rat(P<0.01),while the VPA group spent equal time in the stranger rat and the familiar rat zones(P>0.05).Electrophysiological experiments showed that the LTP level of the VPA group offspring was significantly higher than that of CON group(P<0.001).The protein expression levels of synapse-related molecules in the VPA group offspring,such as Glu N2A(P<0.05),Glu A1(P<0.01)and SYN1(P<0.001),were significantly higher than those in the CON group;while the levels of other proteins such as Glu A2,Glu N1 and Glu N2 B were not significantly different between the two groups(P>0.05).Compared to the CON group offspring,the HDAC1(P<0.05),HDAC2(P<0.01),HDAC3(P<0.05)and HDAC8(P<0.05)m RNA levels in the nuclear of the VPA group were significantly increased,the level of HDAC3 protein was significant increased(P<0.05)and the ratio of Ac H3/H3 in the VPA group was significantly decreased(P<0.01).The levels of RA(P<0.05)and RAR?protein(P<0.01)in the VPA group offspring were significantly lower than those in the CON group,while there was no significant difference in serum retinol levels between the two groups(P>0.05).The levels of ALDH1A1 m RNA(P<0.01)and protein(P<0.01)and ALDH activity(P<0.01)in the VPA group were significantly lower than those in the CON group.The Ch IP experiment showed that the enrichment of Ac H3 on the ALDH1A1 gene of the VPA group was significantly lower than that of the CON group(P<0.05).(2)The MS-275 intervention experiment showed that after intraperitoneal injection of MS-275,the Ac H3 level in the PFC of the VPA group offspring was significantly increased(P<0.01).MS-275 treatment significantly increased the ALDH1A1 m RNA(P<0.05)and protein(P<0.05),ALDH activity(P<0.01),RA(P<0.05)and RAR? protein(P<0.05)levels in the VPA group offspring.Ch IP results showed that MS-275 treatment significantly elevated the combination of Ac H3 and ALDH1A1 gene promoter in the VPA group offspring(P<0.05).After MS-275 treatment,the Glu A1 protein level of the VPA group offspring was significantly decreased(P<0.01),while Glu N2 A and SYN1 protein did not change significantly(P>0.05).Electrophysiological experiments showed that MS-275 treatment significantly decreased the LTP level of the VPA group offspring(P<0.05).The open field test showed that the VPA group offspring' time spent in the central zone?? was significantly increased after MS-275 treatment(P<0.01),but the time on self-grooming did not change significantly(P>0.05).In one three-chamber test(the stimulus: a strange rat vs.an object),the offspring of the VPA group spent significantly longer time in the strange rat zone than the object zone after MS-275 treatment(P<0.001).Another three-chamber test(the stimulus: a stranger rat vs.a familiar rat)showed that after MS-275 treatment,the VPA group offspring spent the same time in the strange rat and the familiar rat zones(P>0.05).(3)RA supplementation experiments showed that RA supplementation significantly increased the levels of RA(P<0.05)and RAR? protein(P<0.01)in the PFC of the VPA group offspring.However,RA treatment did not significantly affect the levels of ALDH1A1 protein,HDAC3 protein and Ac H3 in the VPA group(all P>0.05).After RA treatment,the Glu A1 protein level of the VPA group offspring was significantly down-regulated(P<0.01),while Glu N2 A and SYN1 protein did not change significantly(P>0.05).Electrophysiological experiments showed that RA supplementation significantly reduced the LTP level of the VPA group offspring(P<0.001).The open field test showed that the VPA group offspring's time in the central zone increased significantly after RA treatment(P<0.001),but the self-grooming time did not change significantly(P>0.05).In one three-chamber test(the stimulus: a strange rat vs.an object),the offspring of the VPA group spent significantly longer time in the strange rat zone than the object zone after RA treatment(P<0.001).Another three-chamber test(the stimulus: a stranger rat vs.a familiar rat)showed that after RA treatment,the VPA group offspring spent significantly more time in the strange rat zone than the familiar rat zone(P<0.05).Conclusions: These findings suggest that VPA impairs histone acetylation of ALDH1A1 and downregulates the RA-RAR? pathway,thereby leading to autism-like synaptic and behavioural deficits,suggesting a possible molecular mechanisms of epigenetic modification underlying the VPA-induced ASD subtype. |
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