Autism spectrum disorder (ASD) is a group of severe neurodevelopmental disorders that present social impairments and repetitive behavior problems. Currently, the prevalence of the disease has reached 1/59; however, its etiology remains unclear, with few therapeutic breakthroughs observed (Saurman et al., 2020). While numerous medical comorbidities have been associated with ASD, gastrointestinal (GI) problems seriously affect the normal life and rehabilitation treatment of ASD individuals (Kang et al., 2017). A strong association is observed between GI symptoms and intestinal microbiota misbalance in ASD patients (Fu and Lee, 2021). The changes in bacterial diversity and community structure of the intestinal microbiome may affect the gut-brain neural network, gut immune system and neurotransmitters and then interfere with the function of the gut-brain axis, subsequently aggravating the behavioral disorders of ASD children (Wang and Wang, 2016; Ma et al., 2019; Sgritta et al., 2019). Insights into the mechanisms of microbiome-gut-brain communication may clarify the underlying pathophysiology of ASD and lead to the discovery of novel therapeutic targets.

Constipation, abdominal pain and diarrhea are common gastrointestinal problems in children with ASD that not only worsen the autistic behavioral symptoms but also cause stress and anxiety problems (Kang et al., 2017; Ferguson et al., 2019; Madra et al., 2020, 2021). These GI symptoms of ASD children may be associated with an imbalance of gut microbiota (GM) and poor intestinal motility (Gabriele et al., 2016; Pulikkan et al., 2019). Therefore, microbially mediated therapies, specifically probiotics and fecal microbiota transplantation, have shown promise in the treatment of GI symptoms in ASD children and potential benefits to the core behavioral symptoms of autism as well (Martínez-González and Andreo-Martínez, 2020; Tan et al., 2021).

Stigma maydis polysaccharide (SMPS) is a plant polysaccharide that participates in immune regulation and gastrointestinal motility. Intestinal flora represent an important bridge in the interaction between plant polysaccharides and the human body. Polysaccharides are digested into short-chain fatty acids by intestinal flora to aid absorption and utilization, produce active metabolites with pharmacological effects, and contribute to physiological function control (Sun et al., 2020; Chen et al., 2021; Zhou et al., 2021). Meanwhile, the abundance, species, and proportion of the host intestinal microflora can all be influenced by polysaccharides. Numerous studies based on the “gut-brain axis” theory have found that the occurrence of many neurological diseases is closely associated with intestinal microbiosis (Saurman et al., 2020; Socała et al., 2021). Interestingly, polysaccharides can modulate the synthesis of neurotransmitters (such as serotonin and dopamine) in the human gut and affect their involvement in the structure and function of the nervous system by boosting the growth of helpful intestinal bacteria while suppressing the growth of harmful bacteria (Settanni et al., 2021; Sun et al., 2021; Zhang et al., 2022). Recent human genetics revealed that bear long polysaccharides were associated with autism spectrum disorder and played an important role in synapse formation, neural plasticity (Kamimura and Maeda, 2021). One study reported that clostridium bolteae levels were overabundant in intestinal tract of ASD children suffering from gastric intestinal ailments. However, it could produce a conserved specific capsular polysaccharide which might be as a vaccine to reduce or prevent clostridium bolteae colonization of the intestinal tract in autistic patients, and hopefully defend the augmentation of regressive-autism related symptoms (Pequegnat et al., 2013). Additionally, Endreffy et al. found that total glycosaminoglycans (a family of linear, sulfated polysaccharides that are associated with central nervous system development, maintenance and disorders) were significantly higher in the urine of ASD children compared to healthy controls (Endreffy and Bjørklund, 2016). However, adequate evidence is not available to explain the correlation between polysaccharides and autism, especially if polysaccharides can influence the gut-brain axis by regulating the gut microbiota. In our previous study, we found that SMPS intervention could meliorate behavioral impairments and alleviate some inflammatory responses in the gut of VPA induced autism model rats. Based on the above, this study was aimed to clarify the effect and mechanism of SMPS on the gut microbiota of autistic rats by 16S rRNA sequencing and RNA-seq technologies.

In this study, after the SMPS intervention (from pregnancy to lactation) of VPA-injected pregnant rats, the diversity and structure of the GM in their pups were changed, especially the relative abundance of Prevotellaceae and Lachnospiraceae_NK4A136_group. The finding confirmed the supporting effect of plant polysaccharides on the intestinal microbiota. To authenticate whether it has initiated the gut-brain axis, we performed a transcriptomic analysis of the offspring’s hippocampal tissue. Interestingly, the overall findings showed that 496 genes were differentially expressed after induction by SMPS compared with the VPA group. Moreover, the GO analysis indicated that these DEGs were mainly involved in collagen fibril organization, precursor metabolite and energy generation, and purine ribonucleotide metabolic processes. The KEGG analysis showed that both upregulated and downregulated genes were mostly enriched in the ribosome pathway. The above findings provide scientific evidence to elucidate the relationship between ASD and the microbiome-gut-brain axis.

Numerous studies have shown that plant polysaccharides can improve the intestinal flora and regulate the body’s immune system (Gudi et al., 2019; Youn et al., 2020; Guo et al., 2021). It is worth noting that a large number of studies have shown a strong association between the etiology of ASD and its disturbance of immune function (Meltzer and Van de Water, 2017; Garcia-Gutierrez et al., 2020). Furthermore, the gut microbiome is an integral part of the gastrointestinal tract and involved in the absorption and digestion of polysaccharides in food, and it simultaneously influences human health and the occurrence of disease (Cockburn and Koropatkin, 2016). The GM has a strong connection with the central nervous system (Suganya and Koo, 2020). ASD, as a heterogeneous, behaviorally defined disorder, is a neurodevelopmental disease, and the observed behavioral and cognitive features are associated with pervasive atypicalities in the central nervous system (Shuid and Jayusman, 2020). Early life is a critical period for the formation and development of the nervous system, especially from maternal intervention (Foong and Hung, 2020). The nutrients and risk factors in early life are all associated with the mother, and they play an important role in the GM and neural development of the offspring (Buffington et al., 2016; Wang et al., 2017).

To date, many studies have reported alterations in the GM of ASD children and autistic animal models (Schneider and Przewłocki, 2005; Ma et al., 2019; Wong et al., 2021). However, due to the influence of the dietary structure and habits of ASD children, considerable heterogeneity is observed in the changes in the intestinal flora (Liu et al., 2019; Iglesias-Vázquez et al., 2020; Zou et al., 2020). Exposure to valproic acid (VPA) during pregnancy has been demonstrated to increase the risk of autism in children (Nicolini and Fahnestock, 2018). Moreover, rodents prenatally exposed to VPA display behavioral phenotypes characteristic of the human condition. At the same time, VPA-induced autism model rats are highly similar with ASD patients in terms of genetics, physiology and metabolism and have been widely used as an ideal model for studying the etiology of autism (Tartaglione et al., 2019; Mehra et al., 2022). In this study, the SMPS intervention was continuously administered to pregnant rats from pregnancy to lactation, and pup feces were collected at the end of lactation (PND21). In this way, the effect of SMPS on the VPA-induced intestinal flora of model rats can be more accurately explored. Through 16S rRNA gene sequencing analysis, we found that the richness and diversity of the GM showed significant differences among the SMPS, VPA and control rats, which is consistent with the findings of a previous study (Gu et al., 2021). A recent report found that the richness and diversity of gut microbiota did not differ between VPA and control groups, while the overall composition of gut microbiota was significantly different (Gu et al., 2022). In addition, the PCoA results revealed that the overall composition of the GM was different between the SMPS and VPA groups. At the phylum level, the taxa composition of the three groups mainly contained Bacteroidota, Firmicutes and Proteobacteria; however, the ratio of each group was different. The relative abundance of Firmicutes was decreased after SMPS intervention. A previous study based on gut microbiota of the ASD population found that the relative abundance of Firmicutes in the ASD group was increased, which is consistent with the findings for the VPA-induced model rats in the present study (Strati et al., 2017). Moreover, the ratio of Bacteroidetes to Firmicutes in the VPA group was lower than that of C group, which is agreement with the provious studies of ASD children (Tomova et al., 2015; Strati et al., 2017). This suggests that the gut microbiota characteristics of the VPA-induced model rats are similar to those of ASD children. It was worth noted that the B/F ratio was elevated after SMPS intervention compared to the VPA group. For this, it revealed that SMPS treatment ameliorated the intestinal microbiota of VPA induced rats.

To find key biomarkers in the gut microbiota of the SMPS and VPA rats, we conducted linear discriminant analysis, which is an analytical tool for discovering and interpreting high-dimensional biological identifiers (Segata et al., 2011). In the current study, the difference of taxa was significant between SMPS and VPA groups, especially in Prevotellaceae and Lachnospiraceae_NK4A136_group. One study has reported that Prevotellaceae was the specific causal microbe taxa for autism spectrum disorder (Ni et al., 2021). In comparative studies between ASD and normal children, the relative abundance of Prevotellaceae in the ASD children was significantly lower than that of control children, whereas our study indicated that SMPS intervention was able to increase the abundance of Prevotellaceae (Pulikkan et al., 2018; Qiao et al., 2018; Sun et al., 2019). Moreover, the change of Lachnospiraceae_NK4A136_group is significantly correlated with enhanced gut barrier function (Ma et al., 2020). Thus, SMPS may have an important role for regulating the dysbiosis of intestinal flora in the ASD. These findings were consistent with the effects of various plant polysaccharides on human and animal intestinal microbiota (Wang et al., 2020; Zhu et al., 2020; Cui et al., 2021; Sang et al., 2021). In addition, the functional enrichment analysis of 16S rRNA profile suggested that SMPS intervention affected biological metabolism (including starch and sucrose metabolism, biosynthesis of amino acids, galactose metabolism and other glycan degradation pathways) through regulating intestinal flora. Interestingly, the recent literature indicated that the gut microbiota had effect on brain functions through endocrine and metabolic pathways and the enteric network, especially in the onset and maintenance of neurodevelopment and neurodegenerative disorders (Marazziti et al., 2021). ASD is a neurodevelopmental disease. To further explore whether SMPS intervention regulates the gut-brain axis through the gut microbiota, we performed transcriptome sequencing of the hippocampal tissue.

The early stage of life is a critical period for the formation and development of the nervous system (Cisneros-Franco et al., 2020). In this study, SMPS intervention began in pregnancy and continued to the juvenile weaning period, which is the key period of brain and nervous system development in the young rats, and the nutritional source was mainly derived from the mothers. Through the transcriptomic sequencing results, we found 496 DEGs between the SMPS and VPA groups. Subsequently, GO analysis showed that these DEGs were mainly involved in collagen fibril organization, the precursor metabolite and energy generation, and purine ribonucleotide metabolic processes. Meanwhile, the KEGG analysis showed that these DEGs were enriched in oxidative phosphorylation, retrograde endocannabinoid signaling, thermogenesis, Parkinson disease, ribosome, protein digestion and absorption, renin-angiotensin system, calcium signaling pathway, phenylalanine metabolism, glycosphingolipid biosynthesis-ganglio series, and propanoate metabolism. Based on the enrichment analysis, we focused more on oxidative phosphorylation and ribosomes, which were both enriched in the GO and KEGG analyses. Some studies have reported that ASD patients possess abnormal oxidative phosphorylation, which is associated with autistic behaviors (including social interaction, abnormal behavior, and verbal communication; Legido et al., 2013; Napoli et al., 2014; Singh et al., 2014). Many ASD patients also exhibit mitochondrial disease, and the oxidative phosphorylation process occurs precisely in the mitochondria; therefore, abnormal oxidative phosphorylation may induce impaired mitochondrial function, followed by oxidative stress, resulting in excess reactive oxygen species with certain neurotoxicity, which may be a possible cause of the occurrence of ASD (Martinez-Finley et al., 2013; Rose et al., 2018; Bjørklund et al., 2020; Manivasagam et al., 2020). After the SMPS intervention, differentially expressed genes were enriched in the oxidative phosphate signaling pathway, including 22 upregulated genes (Uqcrb, Ndufa6, Ndufa3, etc.) and 14 downregulated genes (Mt-nd3, Cox7a2l2, Mt-cyb, etc.). Development of the nervous system is carried out by complex gene expression programs that are regulated at both the transcriptional and translational levels. The production of ribosomes is essential for protein synthesis. Mutations in several ribosomal components and trans-acting ribosomal biogenesis factors result in neurodevelopmental syndromes that present with autism, intellectual deficits and/or progressive neurodegeneration (Hetman and Slomnicki, 2019). One study of autistic etiology found that mice lacking the Eif4g1 microexon, which functions as a translational brake by causing ribosome stalling, displayed deficits in social behavior, learning, and memory as well as altered hippocampal synaptic plasticity (Gonatopoulos-Pournatzis et al., 2020). Additionally, a previous report indicated that the individual copy number of ribosomal genes is a factor associated autism risk and severity (Porokhovnik, 2019). In this study, the differentially expressed genes enriched in the ribosomal pathway included 43 upregulated genes (Rps4x, Rpl21, Mrps10, etc.) and 20 downregulated genes (Rpl39, Rps4y2, Rps13, etc.). In the KEGG enrichment analysis results, propanoate metabolism pathway also attracted our attention, which included 8 DEGs. The animal research revealed that propionic acid-treated rats display ASD-like repetitive, perseverative, and antisocial behaviors and seizure. Moreover, the neurochemical changes were consistent with findings in ASD patients, including mitochondrial dysfunction (Witters et al., 2016). Propanoate metabolism pathway has bioactive effects on neurotransmitter systems, calcium release, fatty acid metabolism (Macfabe, 2013). It is similarity to the GO and KEGG enrichment results of this study, which provided a scientific basis for the association of propionate metabolic pathway and ASD.

The above evidence strongly suggests that continuous SMPS intervention in the early life of autism model rats could change the diversity and composition of their GM and simultaneously affect the expression of a large number of genes. To date, this is the first study on the effects of plant polysaccharide intervention on the gut microflora and transcriptome of autism, which not only provides an important scientific basis for the role of the microbe-gut-brain axis in ASD research but also provides a new direction for the treatment of ASD.

The original contributions presented in the study are publicly available. This data can be found at: NCBI, PRJNA870709.

The animal study was reviewed and approved by Ethics Committee of Qiqihar Medical University (QMU-AECC-2021-62).

XY: conceptualization, project administration, and writing original manuscript. JiyL: methodology and statistical analysis. YZ: methodology. NZ: methodology and supervision. JicL: writing–review and editing. All authors contributed to the article and approved the submitted version.

This work was supported by the Natural Science Foundation of Heilongjiang Province (no. LH2020H131) and the National Natural Science Foundation of China (no. 82103869).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.