From the first part of the metabolomic and transcriptomic study, we found that the PFC PC content of DSS mice was decreased and the mRNA responsible for acetylcholine synthesis and secretion were increased. Moreover, the composition and function of gut microbiome in DSS group were significantly different from Control group. Since both acetylcholine and PC are choline metabolites, we hypothesized that the dysfunction of gut-brain axis in DSS mice may be caused by choline deficiency. In the second part of the experiment, the PC and acetylcholine levels of PFC were increased after the administration of CDP-choline to DSS mice, and the disruption of the gut microbiome was significantly reversed following CDP-choline treatment. From a behavioral standpoint, IBD mice exhibited increased behaviors consistent with mood dysregulation over control subjects. However, IBD mice treated with CDP-choline exhibited behaviors resembling the Control. Here we observed choline deficiency and choline metabolism disorder in gut-brain axis of IBD mice. CDP-choline treatment ameliorated PFC PC and acetylcholine content, which may underlie its antianxiety effects in IBD mice.
From a biochemical perspective, choline is an essential nutrient that plays a key role in acetylcholine synthesis in the central nervous system (CNS)(20). Acetylcholine has been linked to learning, memory, and emotion and is metabolized via the CDP-choline pathway to phosphatidylcholine (PC). PC is one of the most abundant phospholipids in ensuring cell membrane fluidity and integrity(21). As numerous studies indicated that supplementation with choline or choline metabolite-PC was effective in improving colonic inflammation(22–24), the efficacy of CDP-choline on the gut microbiome and behavioral improvements was expected. Previous studies have shown that patients with IBD have systemic abnormalities of lipid metabolism(25, 26). In particular, lipid metabolism has an important impact on brain function as the brain is a lipid-rich organ (27). Recent studies have revealed that animal models of depression or anxiety were associated with abnormal brain glycerophospholipid metabolism(28, 29). In this study, we also found abnormal lipid metabolism in DSS mice by metagenomics and PFC metabolomics. However, CDP-choline was able to effectively alleviate the abnormal lipid metabolism and increase PFC glycerophospholipid metabolite-PC content. These results suggest that lipid metabolism plays a key role in the symptoms and pathophysiology of IBD, especially PC metabolism.
Consistent with previous studies, a generalized dysbiosis was observed in DSS induced IBD mice model. In the first part of the experiment, we found that the content of Muribaculaceae in DSS mice were decreased, and the Muribaculaceae content showed a positive correlation with the PFC PC content. Muribaculaceae also designated as S24-7 is one of the dominant bacterial taxa in the human and mice gut microbial composition(30). A recent metagenomic analysis described the ability of Muribaculaceae to protect against oxidative stress and have genes for the production of succinic and propionic acids(31). The decrease of Muribaculaceae have been observed in clinical and basic experiments of IBD(32, 33), and the content of Muribaculaceae was negatively correlated with anxiety-like behaviours(34, 35). Yet, treatment with CDP-choline was able to reverse DSS induced decreases of Muribaculaceae. This was not the first observation that Muribaculaceae can affect CNS choline metabolite content, similar results were also observed in mice chronically exposed to dietary arsenic[35]. These mice showed a reduction in Muribaculaceae content and a statistically significant correlation between Muribaculaceae reduction and decreased CDP-choline content in the CNS(36). Moreover, Curcumin significantly increased the content of Muribaculaceae while increasing the PFC PC content in DSS-induced IBD mice(37). Above all, Muribaculaceae may be key in affecting choline metabolism in the gut-brain axis.
Previous studies have shown that dysfunction of the cholinergic system caused by IBD is systemic and affects multiple organs and tissues(38, 39). However, IBD-induced choline deficiency in the CNS has not been reported. Free choline in the brain originates from four known sources; ingestion from the plasma, release from PC in neuron membranes, hydrolyzed acetylcholine by AChE, or methylation of phosphatidylethanolamine through the phosphatidylethanolamine N-methyltransferase (PEMT) pathway(40). The free choline content in the brain is always in a state of dynamic equilibrium, and the body can consume choline metabolites in other organs to maintain the homeostasis of choline in the brain(20). Which would explain why we did not find any difference between the PFC choline content of DSS group and the Control group, and the ELISA results even showed that the free choline content in the serum of DSS mice was significantly higher than that of the Control group. In this study, we chose CDP-choline for treatment because CDP-choline also known as cytidine diphosphate-choline or Citicoline is a precursor of PC. CDP-choline occurs naturally in human and animal tissue cells, especially organ cells. Exogenous CDP-choline can directly cross the blood-brain barrier, or it can be rapidly hydrolyzed to cytidine and choline, which is effectively used in the synthesis of PC and acetylcholine(41). In various animal models of CNS disorders, CDP-choline enhanced membrane repair and neuronal functions(42, 43). Both PC and acetylcholine levels in the PFC of DSS mice were elevated after feeding CDP-choline, and free choline levels in the serum were significantly decreased, which was sufficient to demonstrate the presence of choline deficiency in PFC and systemic abnormalities of choline metabolism of the DSS-induced IBD mice model.
In the presence of choline deficiency, both PFC PC and acetylcholine content were significantly decreased in DSS mice. Previous studies have shown that neurodegenerative diseases also present simultaneous decreases in CNS PC and acetylcholine levels(44, 45). Moreover, the CNS cholinergic system is tightly correlated to age-related cognitive decline and dementia(46). This may explain why IBD patients are more likely to develop psychiatric degeneration than normal people(47, 48). To clarify the reason for the decrease in PFC choline metabolites in DSS mice, the content of choline transport proteins was inspected including high affinity choline transporter1 (CHT1/SLC5A7) and the intermediate-affinity choline transport proteins 1 (CTL1/SLC44A1). The results have shown that the content of CTL1 in DSS mice was not significantly different compared to control mice. However, the concentration of CHT1 was greatly increased. Choline uptake relies on carrier-mediated transport as cations cannot cross the cell membrane using passive transport. Choline transporter proteins are classified into three transporter protein families based on their affinity to choline(49): (i) The CHT1/SLC5A7, CHT1 is a Na+- and Cl- dependent cotransporter for acetylcholine synthesis(50); (ii) the low affinity organic cation transporters (OCTs). To date, three different OCTs have been identified, which are expressed mainly in the liver and kidney. They act through voltage-dependent and Na+-independent uptake mechanisms(51, 52); (iii) the intermediate-affinity choline transport proteins family (CTLs/SLC44). There are currently five members of the CTLs (CTL1-5) that are present in various types of human tissues(53–55). However, only CTL1 is significantly expressed in the nervous system(55). CTL1 is the main transporter protein of choline used for PC synthesis by the CDP-choline pathway(56). As such, CHT1 is the rate-limiting step for synthesis of acetylcholine(50). When acetylcholine synthesis demand is high, CHT proteins can be recruited from the reserve pool to mediate a compensatory increase in choline uptake(57). However, increased PFC CHT1 content in DSS mice did not increase PFC acetylcholine content. Due to this finding, we also examined key enzymes and proteins for acetylcholine metabolism. In the CNS, when choline is transported by CHT1 to the cytoplasm at the nerve endings, ChAT catalyzes the transfer of an acetyl group from acetyl coenzyme A to choline to form the Acetylcholine(58). Then acetylcholine is loaded into synaptic vesicles by VAChT and eventually secreted into synapses. Moreover, the content of AChE, which is responsible for acetylcholine degradation in the PFC of DSS mice, was significantly decreased compared with Control group. After receiving CDP-choline treatment, the content of AChE significantly increased. Above all, the change of CHT1 and AChE contents in the PFC of DSS mice were compensatory responses to maintain acetylcholine levels under choline deficiency. DSS mice may preferentially maintain PFC acetylcholine levels.
This study also examined key enzymes in the PC synthesis and degradation pathways. The main synthesis pathway of PC is through de-novo CDP-choline synthesis(59). In the CDP-choline pathway, free choline is first phosphorylated by choline kinase to generate phosphocholine, and then converted to CDP-choline by phosphate cytidylyltransferase. It is then finally synthesized with diacylglycerol on the endoplasmic reticulum by choline/ethanolamine phosphotransferase to produce PC (60). There were no differences in above key enzymes between the DSS and Control group. However, the PC degradation pathway was significantly enhanced as evidenced by a significant increase in PFC NET1 levels of DSS mice. The NTE1 is enriched in the brain and encodes PC-specific phospholipase B, which deacylates PC in the endoplasmic reticulum to produce GPC and two free fatty acid molecules(61). NTE1 was originally described as a neuropathic target esterase because its activity is inhibited by organophosphorus found in pesticides, leading to chronic neuropathy(62). Although PC can also be degraded through the cPLA2 and PLD pathways, we did not find that the PFC Cpla2 and PLD1 contents of DSS mice were increased compared with Control group. In some pathological conditions, the body increases the release of choline from PC, which is then used to make acetylcholine, this process has been termed autocannibalism(63). Autocannibalism may reveal the specific vulnerability of cholinergic neurons in some diseases by reducing the number of phospholipid molecules and neuronal membranes(63). After CDP-choline treatment, the content of NET1 in PFC and choline in serum of DSS + CDP group mice decreased significantly. Although CDP-choline does not prevent PC degradation through PLD1, CDP-choline treatment effectively reduced the phenomenon of autocannibalism, increased CEPT1 content and increased PC synthesis of PFC.
In conclusion, this study revealed that the biological basis of mood disorders caused by IBD are greatly affected by choline metabolism, and choline and its metabolites may play a key role in maintaining the stability of the gut-brain axis. Specifically, choline deficiency was seen in DSS induced acute colitis mice, and choline supplementation with CDP-choline was shown to relieve both choline deficiency and anxiety-like behaviors in DSS mice. As such, choline deficiency and choline metabolism disorders should be further investigated as a basis for mood disorders in IBD.