The intestine is the body organ with the largest membrane surface area that serves to absorb nutrients and sense, and recognize and defend against pathogens, antigens, toxins, and other detrimental secretions. The intestine accomplishes these diverse functions through coordinated effects of the enteroendocrine system, the enteric nervous system (ENS), the gut immune system, and the non-immune defense system of the intestine (Ferraris et al., 1989; De Giorgio, 2006). Intestinal nerve cells and glial cells in the ENS secrete neuropeptides and immune factors to pass signals between the ENS and the central nervous system (CNS). These signals influence mucosal secretions and gastrointestinal peristalsis and affect rhythms and hormone secretion in the CNS (Rao and Gershon, 2018; Boesmans et al., 2019). The ENS plays an important role in intestinal diseases, such as colitis, irritable bowel syndrome, and inflammatory bowel disease (Lasrado et al., 2017; Resnikoff et al., 2019; Jarret et al., 2020). The ENS, which includes submucosal nerve plexus, myenteric nerve plexus, and glial cells, usually is functional and structurally mature during the early life of mammals (Montedonico et al., 2006; Parathan et al., 2020). The capacity to regulate neuropeptides is called neurochemical plasticity. Enteric neuroplasticity is an adaption to intestinal contents and the intestinal microenvironment and is pronounced during early development in mammals (Moeser et al., 2017).

The phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) signaling pathway regulates the activity ofglycogen synthase kinase-3β (GSK-3β), Tau protein, and N-methyl-D-aspartame receptor (NMDA), which promotes the development of nerve cells and regeneration of synapses (Iqbal et al., 2016; Majewska and Szeliga, 2017; Falcon et al., 2019). The PI3K/Akt signaling pathway also plays an important regulatory role in intestinal neurological diseases by reducing the oxidative stress and apoptosis of nerve cells, improving the secretion of neurotropic factors, regulating survival and differentiation of nerve cells, and promoting the proliferation of intestinal nerve cells and glial cells (Ichihara et al., 2004; Du et al., 2009; Becker et al., 2013).

Pyrroloquinoline quinone (PQQ), a water-soluble quinone compound, was discovered as a redox cofactor of methanol dehydrogenase in pseudomonas TP1 and other Gram-negative bacteria (Duine et al., 1979; Duine, 1991). We demonstrated previously that dietary supplementation with PQQ could regulate intestinal morphology, mucosal barrier function, colonic microbiota, and antioxidant status to reduce diarrhea and improve the growth performance of weaned pigs (Yin et al., 2019; Huang et al., 2020, 2021; Ming et al., 2021). PQQ is a potent neuroprotective nutrient in the CNS and peripheral nerves, which can mitigate sciatic nerve injury, reduce neurotoxin-induced neurotoxicity, and promote neuronal cell regeneration (Liu et al., 2005; Hara et al., 2007; Shanan et al., 2019). PQQ treatment can ameliorate signs of memory impairment in aging mice and schizophrenic rats via reducing the expression of phosphorylation Akt and maintaining the GSK-3β level in the hippocampus (Zhou X. Q. et al., 2018; Zhou et al., 2020).

We hypothesized that PQQ treatment could influence the ENS and regulate enteric neuroplasticity to improve intestinal health. We tested this hypothesis in the enteritis rat model via developed challenging rats with lipopolysaccharide (LPS). We detected neuropeptides in serum and intestine and Akt signaling pathway expression in the intestine to determine if PQQ could regulate enteric neuroplasticity via the Akt signaling pathway.

Pyrroloquinoline quinone is a natural antioxidant that has been used in the treatment of osteoporosis, muscle atrophy, radiation poisoning, and arthritis and promotes the growth of the organisms by regulating oxidation, repairing DNA damage, reducing apoptosis, and maintaining mitochondrial function (Kasahara and Kato, 2003; Huang et al., 2017; Wu et al., 2017; Xu et al., 2018; Geng et al., 2019; Jiang et al., 2019). We previously demonstrated that dietary PQQ could increase ADG and gain to feed ratio and reduce diarrhea incidence in weaned pigs (Yin et al., 2019; Huang et al., 2021; Ming et al., 2021). In this study, daily treatment with 5.0 mg/kg and 10.0 mg/kg PQQ increased ADG of rats compared with rats not supplemented with PQQ.

We developed the enteritis model by challenging rats with LPS and verified the existence of enteritis by evaluating jejunal morphology and cytokines in serum and intestine. LPS is a component of Gram-negative bacteria that can damage the intestinal barrier cause immune dysfunction, initiate apoptosis of intestine epithelial cells, and develop enteritis (Yoshioka et al., 2009; Li et al., 2018; Dong et al., 2020). In this study, rats challenged with LPS developed inflamed intestines as evidenced by damaged jejunal morphology, increased concentrations of IL-1β, IL-6, and TNF-α in serum and IL-1β and TNF-α in intestine, and decreased IL-10 in serum. These observations confirmed that rats challenged with LPS developed intestinal inflammation.

Intestinal morphology and immune function improved with PQQ supplementation of LPS-challenged rats. Intestinal damage reduces absorption and metabolism of nutrients and compromises the integrity of the intestinal barrier (Baumgart and Carding, 2007; Turner, 2009; Wang et al., 2015; Zhou et al., 2018). Intestinal morphology can be improved in weaned rats when they were supplemented with PQQ (Zhang et al., 2019). Our previous study showed that dietary supplementation with PQQ can increase intestinal VH and VCR in weaned pigs (Yin et al., 2019). In this study, PQQ treatment increased jejunal VH and VCR compared with rats challenged with LPS. PQQ has anti-inflammatory effects and can reduce arthritis by inhibiting the production of pro-inflammatory cytokines such as TNF-α and IL-6 (Harris et al., 2013; Liu et al., 2016). In our previous study, PQQ supplementation reduced gut inflammation which improved intestinal health and growth of weaned pigs (Yin et al., 2019; Huang et al., 2020). In this study, PQQ supplementation decreased concentrations of pro-inflammatory cytokines in serum and intestine and increased concentrations of the anti-inflammatory cytokine and IL-10 in serum, which confirmed that PQQ regulates immune responses.

Pyrroloquinoline quinone can regulate enteric nervous damage induced by LPS challenge. The ENS regulates intestinal secretions, peristalsis, and immunity through endocrine substances and neuropeptides secreted by myenteric neurons, submucosal neurons, and glial cells (Rao and Gershon, 2018; Boesmans et al., 2019). Normally, the neuropeptides are divided into inhibitory neurotransmitters and excitatory neurotransmitters to control intestinal relaxation and contraction, respectively (Rao and Gershon, 2018). When the intestine suffers pressure, mucosal damage, and inflammation, the ENS can release neuropeptides to regulate intestinal peristaltic and immune factors (Jakob et al., 2020). PGP 9.5 is a neuroendocrine marker and is reduced with intestinal inflammation and damage (Resnikoff et al., 2019; Heymans et al., 2020). In this study, the immunoreactive percentage of PGP9.5 in the jejunum was decreased by LPS treatment and increased by PQQ treatment, suggesting that PQQ reduced the damage of jejunal neurons caused by LPS.

Enteric neurochemical plasticity was regulated with PQQ treatment by regulating immunoreactive neurons and concentrations of neuropeptides. Neurochemical plasticity is the variable or modifiable changes of the nervous system elicited by adaptation to the environment. During the early development of rats, the ENS matures gradually and has the strongest neurochemical plasticity (Rza̧p et al., 2020). As the first isolated neuropeptide and known as the prototypic tachykinin (TK), SP stimulates systemic pain and vasodilation, regulates immunity, promotes contraction as an excitatory neurotransmitter, and inhibits the secretion of digestive juices in the intestine (Euler and Gaddum, 1931; Holzer, 1998; Engel et al., 2012; Nässel et al., 2019). The concentration of SP is enhanced in the blood of rats suffering from colitis (Holzer, 1998). As a systemic vasodilator, CGRP simulates pain and regulates intestinal immunity similar to SP (Holzer, 1998; Engel et al., 2012; Lai et al., 2020). Sensory neurons in the gut of mice release CGRP to defend against Salmonella infection, and neurogenic inflammation challenged with LPS can promote CGRP release via activation of Toll-like receptor 4 (TLR4) in sensory neurons (Meseguer et al., 2014; Lai et al., 2020). As an inhibitory neurotransmitter and neuroprotective agent in the intestine, VIP is also an immunomodulator (Gonzalez-Rey and Delgado, 2005; Arciszewski et al., 2008). Rat myenteric neurons challenged with LPS increase the expression of VIP (Arciszewski et al., 2008). Except for promoting differentiation, development, and survival of neurons, BDNF regulates the sensitivity of the colon and rectum to constipation (Delafoy et al., 2006). It is synthesized by sensory neurons to mediate inflammatory pain and regulate the sensitivity of visceral afferents in rats suffering from colitis (Matayoshi et al., 2005; Qiao et al., 2008). With systemic stress, NPY is released, regulates endocrine, behavior, stress, anxiety, appetite, and circadian rhythms, and functions in defecation and food intake (Adrian et al., 1983; Zhou et al., 2008). The level of NPY is increased in the hypothalamus and serum of mice suffering from colitis (Hassan et al., 2014; Reichmann et al., 2015). NGF is a nutrient protein for nerve cells and promotes the repair of damaged nerve fibers (Bilderback et al., 1999). At the site of inflammation, NGF concentration is increased, and cytokines promote the synthesis of NGF by neurons and other cells such as epithelial and endothelial cells (März et al., 1999; Minnone et al., 2017). In this study, concentrations of these neuropeptides were increased in serum and small intestine of rats in the LPS group. The increased levels were reduced with PQQ treatment. The optional PQQ dose approved to be 5.0 mg/kg b.w. The concentrations of IL-1β and TNF-α display a similar pattern to neuropeptides, suggesting that neuropeptides regulated immune factors concentrations. In the colon, SP and VIP concentrations showed the same trends as observed in the jejunum. This might be due to the main site of inflammation challenged with LPS. Additionally, P and VIP play the main role of immunomodulators in the nervous system. Treatments with PQQ increased concentrations of BDNF, NPY, and NGF in the colon which might be related to their neurotropic effects and the damage caused by the LPS challenge. These neuropeptides function in stress and neurotropic. When the intestine is damaged, the neuropeptides secreted by the ENS aim to promote neuronal survival and maintain normal functions (Holzer, 1998; Arciszewski et al., 2008; Qiao et al., 2008). We deduced that PQQ played a different role in different pathological states, and we would investigate further. In conclusion, the results reported in this study suggest that PQQ influenced the release of neuropeptides which regulated the small intestine. We conclude that PQQ regulated enteric neurochemical plasticity of SP-, CGRP-, VIP-, BDNF-, NPY-, and NGF-immunoreactive neurons of weaned rats.

Expression of p-Akt decreased in jejunum and colon with PQQ supplementation. Akt can be phosphorylated by PI3K. Activated Akt can reduce and improve the activity of glycogen synthase kinase-3β (GSK-3β) by phosphorylation of Ser9 and Tyr216 sites, respectively (Majewska and Szeliga, 2017). GSK-3β is associated with cell survival and apoptosis, which can promote peripheral nerve regeneration, improve regrowth of synapses after peripheral nerve damage, and play a role in neurodegenerative diseases (Kitagishi et al., 2014; Huang et al., 2017). Activated GSK-3β can induce Tau perphosphate. Tau protein, a microtube-related protein, mainly acts on the far end of the axon, participates in axon transport, and interacts with the microtube protein, Tubulin, to stabilize the microtube and regulate NMDA receptor signaling pathways (Goedert et al., 1996; Iqbal et al., 2016; Falcon et al., 2019). By regulating GSK-3β and Tau protein, the PI3K/Akt signaling pathway affects a variety of CNS diseases, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (Kitagishi et al., 2014; Rai et al., 2019). PQQ inhibits apoptosis in the rat hippocampus by regulating the Akt/GSK-3β pathway (Zhou et al., 2020). In addition, PQQ can regulate memory via maintaining activation of GSK-3β while reducing the expression of p-AKT (Zhou X. Q. et al., 2018). In this study, PQQ reduced the magnitude of increased p-Akt in jejunum and colon of rats caused by LPS treatment which suggests that PQQ might regulate the secretory functions and structure of the ENS via the Akt signaling pathway.

In conclusion, based on ADG, jejunal morphology, immune responses, and enteric neuropeptide expression, we conclude that the intestinal health of weaned rats was damaged by the LPS challenge. Dietary PQQ supplementation reduced inflammatory injury and regulated neurochemical plasticity via the Akt signaling pathway in the intestine of rats suffering from enteritis.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Laboratory Animal Welfare and Animal Experimental Ethical Inspection Committee of China Agricultural University (AW01211202-1-2).

CS: conceptualization, data curation, formal analysis, investigation, methodology, and writing—original draft. SX, CH, ZW, WW, DM, XY, and HL: data curation and methodology. FW: conceptualization, writing – review, supervision, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

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.

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