| BackgroundAutism,short for autism spectrum disorder(ASD),is a complex neurodevelopmental disorder.The core symptoms are social and language disorders and repetitive rigid actions.The incidence rate of ASD is rising rapidly worldwide,and the social burden is huge.The etiology of ASD is still unclear,which may involve the complex interaction of genetic and environmental factors.The vague understanding of pathophysiology of ASD hinders the development of effective treatment methods.The intestinal microbiota of ASD patients is unstable and immature at the early stage of life,which is easily disturbed by many factors and leads to ecological imbalance,this is one of the reasons for the dysfunction of neural function development in ASD patients.Due to the maturation and stability of intestinal microbiota and the development of nervous system are in the same critical time window as the early stage of life,regulating intestinal microbiota in the early stage of life is hopeful to become a promising treatment strategy for ASD.Melatonin is an endogenous hormone produced and released by pineal gland,which can participate in the regulation of circadian rhythm.It is also necessary for normal nerve development and embryo growth.Clinical studies have confirmed that ASD patients suffer from a decline in melatonin levels.In addition to pineal gland,most melatonin is secreted by gastrointestinal tract.Many studies have confirmed that melatonin can participate in repairing the ecological imbalance of intestinal microbiota in many diseases.Base on the above background and foundation of research,we assume that melatonin supplementation in early life can improve ASD social defects by regulating intestinal microbiota.In this study,we explored the effects and the gut-brain axis mechanism of melatonin supplementation during early life on social behavior and intestinal microbiota in a mouse model of autism induced by valproic acid(VPA).We found new mechanism of melatonin supplementation during early life to rescue the social defects of VPA by regulating intestinal microbiota,which provided a biological basis for the ASD therapy based on intestinal microbiota.Objective1.To explore whether melatonin supplementation during early life can rescue the social defects in VPA autistic mice.2.To explore whether melatonin supplementation during early life can regulate the intestinal microbiota in VPA autistic mice.3.To explore the effects and mechanisms of Akkermansia muciniphila supplementation during early life can rescue the social defects in VPA autistic mice.MethodsPart Ⅰ Early melatonin supplementation rescued social dysfunction and upregulate the abundance of Akkermansia spp.in the VPA-induced mouse model of autism 1.1 AnimalsPregnant C57BL/6J mice were randomly separated into four groups.The control group(CTL)received equal volume of saline,the remaining groups were injected with VPA at E 12.5.The VPA-injected dams were further grouped and treated as follows:VPA alone;VPA+MELS with melatonin on P0,and VPA+MELL with melatonin right after VPA injection.Pregnant females were allowed to raise their neonates till weaning on day 21.1.2 Social Behavior ExperimentsThree-Chamber social test and olfactory habituation test1.3 Gut microbiota analysisThe gut microbiota analysis was performed using 16s rRNA sequencing with collected mouse fecal samples.Total bacterial DNA was isolated from mouse feces.The region V3-V4 of the 16 S rRNA genes was amplified,and the resulting amplicons were purified and DNA libraries were constructed.Quality-qualifed libraries were sequenced with Illumina HiSeq 2500(Illumina,Inc,San Diego,CA,USA).The demultiplexed paired-end reads were joined to produce raw tags.After quality filtering and chimerism removal,the resulting effective tags were then assigned to OTUs and finally aligned,further diversity analysis,species classification annotation and difference analysis were carried out.Part Ⅱ A.muciniphila rescued the social deficits in the VPA-induced mouse model of autism2.1 AnimalsThe pregnant C57BL/6J mice were randomly divided into three groups.On the E12.5 day,they were given a single intraperitoneal injection of VPA,and orally administered A.muciniphila,as VPA+Akk group;in the VPA group,the same volume of glycerol was given orally after injection of VPA;the control group was injected with the same volume of saline,and the same volume of glycerol was given orally.In addition,the pregnant C57BL/6J mice were randomly divided into another three groups:after the offspring mice orally administered A.muciniphila from P7 to P21,AAV-PHP.eb was injected via the tail vein,then Clozapine N-oxide(CNO)was intraperitoneally injected at the age of 6 weeks,as the Akk+CNO group;in Akk+Sal group,the same volume of saline was injected intraperitoneally at the age of 6 weeks;in the Veh+Sal group,AAV-PHP.eb was injected after orally administration of the same volume of saline from P7 to P21,then saline was injected intraperitoneally at the age of 6 weeks.2.2 Social Behavior ExperimentsThree-Chamber social test,olfactory habituation test,open field test,and elevated plus maze test.2.3 Gut microbiota analysisThe gut microbiota analysis was performed using 16s rRNA sequencing with collected mouse fecal samples.Total bacterial DNA was isolated from mouse feces.The region V3-V4 of the 16s rRNA genes was amplified,and the resulting amplicons were purified and DNA libraries were constructed.Quality-qualifed libraries were sequenced with Illumina HiSeq 2500(Illumina,Inc,San Diego,CA,USA).The demultiplexed paired-end reads were joined to produce raw tags.After quality filtering and chimerism removal,the resulting effective tags were then assigned to OTUs and finally aligned,further diversity analysis,species classification annotation and difference analysis were carried out.2.4 ImmunohistochemistryThe mice were anesthetized 1.5 h after social interaction test with intraperitoneal injection of avertin.The brains were removed and postfixed in 4%paraformaldehyde.Expression of dopaminergic neuronal marker TH and the neuronal activity marker cfos staining and their co-localization was observed.2.5 Serum and cecal content metabolites analysisSerum and cecal contents were collected for metabolomic analysis by LC-MS.Results:Part Ⅰ Melatonin supplementation during early life rescued social deficits and upregulate the abundance of Akkermansia spp.in the VPA-induced mouse model of autism1.1 Melatonin supplementation during early life rescued social deficits in the VPAinduced mouse model of autism in adolescenceIn the three-chamber social test during six-week-old:the interaction time between the offspring mice of CTL group and the social object(S1)was significantly longer than that with the wired cage(E)(P<0.0001),and the interaction time with the new social object(S2)was significantly longer than that with the old social object(S1)(P<0.01).There was no significant difference in the interaction time between S1 and E in the offspring mice of VPA group,nor between S1 and S2(P>0.05).Compared with the offspring mice of VPA group,the interaction time of offspring mice with S1 was significantly longer than that with E in the VPA+MELS group and VPA+MELL group(VPA+MELS:P<0.0001;VPA+MELL:P<0.0001),and the interaction time with S2 was significantly longer than that with S1(VPA+MELS:P=0.0088;VPA+MELL:P=0.0001);Moreover,the social preference index and social novelty index of offspring mice in the VPA+MELL group were significantly better than those in the VPA group(social preference index:P<0.01,social novelty index:P<0.05).In the olfactory habituation test at the age of 6 weeks:All mice spent more time exploring the novel chemical odor during trial 1 and habituated during trials 2 and 3.When presented with urine odors taken from the same-sex mice(social odor),all mice showed normal habituation and dishabituation responses.However,the VPA exposed offspring showed reduced exploratory activity towards the social odors in comparison with that of mice from the other three groups(CTL vs.VPA:P<0.001;VPA vs.MELL:P<0.05).Melatonin supplements showed a clear rescue effect on the time spent exploring the social odors in the VPA-exposed offspring(CTL vs.VPA:P<0.01;VPA vs.MELS:P<0.05).1.2 Melatonin supplementation during early life rescued social deficits in the VPAinduced mouse model of autism in adulthood.In the three-chamber social test at the age of 8～10 weeks:the interaction time with S1 was significantly longer than that with E(P=0.0001),and the interaction time with S2 was significantly longer than that with S1(P<0.05)in the offspring mice of CTL group.There was no statistical difference between the interaction time with S1 and E,nor the interaction time with S1 and S2(P>0.05)in the VPA group offspring mice;Compared with the CTL group,the social preference index in the VPA group decreased significantly(P<0.05).The interaction time with S1 was significantly longer than that with E in VPA+MELS group and VPA+MELL group(VPA+MELS:P=0.0001,VPA+MELL:P<0.0001),and the social preference index in VPA+MELS group was significantly improved than that in VPA group(P<0.01).1.3 Melatonin supplementation in early life restores gut microbiota dysbiosis in mice with prenatal exposure to VPAThe a diversity of gut microbiota showed that VPA exposure significantly reduced Chao index than the CTL in 3-week-old offspring(P=0.014),The Chao index and Shannon index in the offspring mice of the 6-week-old VPA group were significantly higher than those of the CTL group(Chao:P<0.001,Shannon:P<0.01).The Chao index and Shannon index in the offspring mice of VPA+MELL group at the age of 3 weeks were significantly higher than those of VPA group(Chao:P<0.001,Shannon:P<0.01).There was no significant difference in the Shannon index among the CTL group,VPA group,VPA+MELS group,and VPA+MELL group in the 6-week old and the 3-week old offspring mice(P>0.05)The β-diversity analysis of gut microbiota presents that the VPA group separated significantly from the other three groups on the unweighted UniFrac plot of the gut microbiota of both 3-and 6-week-old offspring.Principal Coordinate 1 of unweighted UniFrac separates the VPA groups from the other three groups(CTL,VPA+MELS,VPA+MELL)in three-week-old mice and in six-week-old mice.The shared and specific OTUs analysis among the different groups showed the VPA group had the largest number of unique OTUs.1.4 Melatonin supplementation in early life upregulates the abundance of Akkermansia spp.in the gut microbiota of mice exposure to VPAThe abundance of Akkermansia spp.in the intestinal microbiota of 3-week old offspring mice in the VPA+MELS group and VPA+MELL group was significantly higher than that in the VPA group(VPA+MELS:P<0.05,VPA+MELL:P<0.01).The abundance of Akkermansia spp.in the intestinal microbiota of the 6-week-old offspring mice in VPA+MELL group was significantly higher than that in VPA group(P<0.01)Part Ⅱ A.muciniphila supplementation alleviated the social deficit in the VPAinduced mouse model of autism2.1 A.muciniphila supplementation during postnatal development alleviates the social deficit in the VPA-induced mouse model of autismIn the three-chamber social test:The interaction time with S1 was significantly longer than that with E(P<0.0001),and the interaction time with new social object(S2)was significantly longer than that with old social object(S1)in the offspring mice of CTL group(P<0.001).There was no significant difference in the interaction time between S1 and E,nor between S1 and S2 in the offspring mice of VPA group(P>0.05).Compared with the offspring mice of VPA group,the interaction time with S1 was significantly longer than that with E(P<0.0001),and the interaction time with S2 was significantly longer than that with S1(P<0.0001)in the VPA+Akk group;the social preference index of mice in VPA+Akk group was significantly better than that in VPA group(P<0.01).In the olfactory habituation test:There was no statistical difference in the three groups of mice for the taste of water and vanilla(P>0.05).The VPA group mice spent significantly less time than that of CTL group(P<0.01).The VPA+Akk mice spent significantly more time sniffing and licking the cotton swab coated with social odor than the VPA mice.2.2 A.muciniphila supplementation during early life did not alter the diversity and the overall structure of intestinal microbiota in the VPA-induced mouse model of autismThere was no statistical difference of Chao index and Shannon index in the αdiversity analysis between VPA+Akk group and VPA group(P>0.05);The β-Diversity analysis showed the VPA group showed no difference with VPA+Akk group on the unweighted UniFrac plot of the gut microbiota.2.3 A.muciniphila supplementation during early life augmented the activation of VTA dopaminergic neuronsThe total number of c-fos+in VTA dopaminergic neurons in VPA+Akk group was significantly higher than that in CTL group(P<0.05).The proportion of activated dopaminergic neurons(TH++c-fos+)in VTA of mice in VPA+Akk group was significantly higher than that in VPA group(P<0.05).2.4 Silencing the activity of dopaminergic neurons in the VTA partially blocked the role of A.musiniphila in alleviating social behaviorSingle chamber social interaction experiment:there was no statistically significant difference in the time spent exploring empty iron cages in the three groups of mice(P>0.05).The time spent by Akk+Sal group in exploring the chamber with social objects was not significantly different from the other two groups,but showed an upward trend;At the same time,the social preference index of Akk+Sal group mice was significantly higher than that of Veh+Sal group(P<0.05).The social preference index of Akk+CNO group mice was not significantly different from that of Veh+Sal group mice(P>0.05).In the open field experiment,there was no statistical difference in the total activity distance in each group(P>0.05),and there was no statistical difference in the residence time in the center of the open field(P>0.05).In the elevated plus maze experiment,there were insignificant differences among groups in terms of open arm entries,total arm entries,and the time spent in the open arm of the elevated plus maze(P>0.05).2.5 A.muciniphila supplementation during early life modulated the serum and cecal metabolome in the VPA-induced mouse model of autismIn the cecum contents and serum of mice,there were significant differences in levels of various metabolites between the VPA+Akk group and the VPA group.Compared with the VPA group,the caecum content and serum taurine levels in the VPA+Akk group mice were significantly increased(caecum content:P<0.01,serum:P<0.05).Conclusion1.Melatonin supplementation during early life can improve voluntary initiation of social interaction and the ability to discriminate social novelty in the offspring VPA autistic mice.2.Melatonin supplementation during early life can restore the ecological imbalance of intestinal microbiota in offspring VPA autistic mice,stabilize their overall diversity and community structure,and significantly upregulate the abundance of Akkermansia spp.3.A.muciniphila supplementation during early life can rescue social deficits in autistic mice.The effects of A.muciniphila on social behavior is mediated by VTA dopaminergic neurons.4.The caecum and serum metabolites may be involved in the mechanism of gutbrain axis underlying the regulatory effects of A.muciniphila on social behaviors. |
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