At present, the effect of lipid metabolism on the formation of CP has been discovered. The unsaturated triglycerides increased in the RA-treated mice could play an important role in the formation of CP . In the meantime, the gut microbiota was one of the causative factors affecting metabolic syndrome. It acted as a critical part in regulating dietary fat absorption and lipid metabolism by influencing bile acid metabolism, producing short-chain fatty acids, and regulating the intestinal endocrine system . Recent studies have proved that the maternal gut microbiome could promote healthy development by regulating metabolites entering the fetal brain , but the effect of maternal gut microbiome on the fetal cleft palate remains unclear.
In our research, since there was no significant difference in biodiversity between the two groups, we speculated that RA only affected the abundance of some species during cleft palate formation, but did not affect the appearance or disappearance of some species. Therefore, we shifted our focus on species abundance and function. Interestingly, we found that the expression of Lactobacillus was significantly increased in the RA group, including Lactobacillus intestinalis, Lactobacillus sp. ASF360, Lactobacillus paragasseri, and Lactobacillus amylovorus. As one of the most common probiotics, the antibacterial action of Lactobacillus reaps huge fruits. On one hand, Lactobacillus can colonize in the intestine and directly affect intestinal homeostasis. On the other hand, the antibacterial products of Lactobacillus reduce intestinal permeability by inhibiting pathogens . Lactobacillus decreased the level of serum cholesterol and reduced inflammation and oxidant damage by regulating gut-derived metabolites . It was previously found that gut microbiota also acted as a critical role in the reproductive system’s development . Lactobacillus made up 90–95% of the vaginal microbiota (VMB). Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri were the four most abundant species in VMB . Lactobacillus paragasseri represents a novel sister taxon of Lactobacillus gasseri. Its gene encoding the oxalate catabolism could catalyze the transfer of Coenzyme A from formyl-CoA to oxalic acid, such as the formyl-CoA transferase encoded by the frc gene, indicating that it has potential probiotic properties [30, 31]. That is maybe the reason that the metabolically related pathways esp. CAZymes were enriched after RA treatment in our study. Moreover, Lactobacillus intestinalis reduced menopausal symptoms by modulating the gut microbiota, involved in increased fat mass, decreased bone mineral density, increased pain sensitivity, depression-like behavior, and cognitive impairment . Crucially, these results inferred that we should pay attention to the role of Lactobacillus intestinalis as a probiotic drug in the treatment of menopausal symptoms.
The benefits of Lactobacillus to our health and the protection of disease have been fully verified, but an excessive increase in Lactobacillus can also be harmful, which could cause increased lactic acid. Lactobacillus-derived lactic acid as an essential metabolite triggering NADPH oxidase complex (NOX)-dependent reactive oxygen species (ROS) production and ISCs proliferation, which lead to premature aging . 16S rRNA gene sequencing demonstrated that Lactobacillus significantly increased in osteoporosis and type 2 diabetes (T2D) patients compared with controls [34, 35].In addition, the mother suffered from lactic acidosis, while her child presented with skull abnormalities such as larger frontal sinuses and thicker frontal bones .
In our study, we found the expression of Lactobacillus was significantly increased in the faeces of RA-induced pregnant mice, and the results of stereo microscope and hematoxylin-eosin (H&E) staining also discovered CP in RA-induced fetal mice. Current research has manifested that RA is one of the crucial trace elements in embryonic development, which plays an essential role in the regulation of morphology, cell proliferation and differentiation, and the production of extracellular matrix . The proliferation of palatal mesenchymal cells was inhibited by RA at E10.5, resulting in cleft palate and no apoptosis of palatal epithelial cells . In Alzheimer's disease, RA affects the intestinal flora by modulating immune cells, thereby affecting the function of neurons . These results indicated that the formation of fetal cleft palate was associated with the excess increase of Lactobacillus in the intestinal flora of RA-treated pregnant mice. And whether Lactobacillus is one of the causes of CP or the feedback of the pregnant mice to RA to prevent the formation of cleft palate in fetal mice needs further investigation.
Up to now, most studies only observed the changes of gut microbiota and metabolic phenotype between pregnant women and fetus [17, 40], but the concrete mechanism of cleft palate between them is not clear. Metagenomics is an effective way to clarify the relationship between gut microbiome and pathogenesis. In our work, to further explore the mechanisms involved in CP formation between pregnant mice and fetuses, the GO and KEGG enrichment analysis implied that metabolic-related pathways were significantly enriched in gut microbiome of pregnant mice, including metabolic pathways, TCA cycle, anaerobic, and so on. An earlier study on chicken and mouse embryos confirmed that energy metabolism was tightly regulated during development . Mouse early preimplantation embryos did not rely on glucose as their primary energy source, but participated in the TCA cycle and produce ATP using pyruvate and lactate . An important metabolic shift occurs during embryo implantation, resulting in increased glucose uptake and enhanced glycolysis activity. At this point, most of the glycolysis activity co-exists with an active TCA cycle and oxidative phosphorylation, causing the production of lactic acid. However, with the formation of organs, the intense glycolysis activity of embryos declined, and respiration became the main way of energy generation [43, 44]. In addition, maternal gut microbiota was associated with offspring metabolic phenotype. During pregnancy, the SCFAGPR41 and SCFA-GPR43 axes could pass on the mother's gut microbiota to offspring to make them resistant to obesity. GPR41 and GPR43 in the sympathetic nerve, intestinal tract, and pancreas of the embryo could sense SCFAs in the maternal gut microbiota, thereby affecting prenatal development of the metabolic and neural system .
CAZy analysis demonstrated that the percentage of GH and GT family enzymes in the control group and experimental group were higher than other enzymes. GT and GH play important roles in the formation of glycosylation . Many of the proteins produced by cells are attached to sugar molecules, and these additives were called glycosylation. The process of glycosylation facilitated the transport of proteins to the parts of the cell where the protein was needed . In some genetic disorders, individuals had abnormalities in glycosylation caused by genetic mutations that could lead to a variety of symptoms, including epilepsy, cleft palate, and heart defects . A report showed that in the process of mammalian organ formation, golgin subfamily B member 1 (Golgb1) as a large coiled-coil protein located at the cytoplasmic surface of the golgin apparatus, could involve in the early steps of regulating O-glycosylation . Golgb1 mutant embryos caused cleft palate in mice, which was due to reduce hyaluronan accumulation and impair protein glycosylation in the palatal mesenchyme . All of these results indicated that the formation of CP was related to metabolism, and maternal environment also affected the development of the fetal palate.
To sum up, our results suggested that changes in maternal gut microbiome could affect fetal palatal development, which might be related to changes in Lactobacillus and metabolic disorders. These results comparing the gut microbiome between normal and CP mice could lead to the discovery of new therapeutic approaches. Undeniably speaking, the limited samples may also be one of the reasons for the indifference of biodiversity which we will consider to enlarge the samples size in our subsequent experiments.