Enteric glial cells (EGCs) influence nitric oxide (NO)− and adenosine diphosphate (ADP)− mediated signaling in the enteric nervous system (ENS). Since Toll-like receptor 4 (TLR4) participates to EGC homoeostasis, this study aimed to evaluate the possible involvement of EGCs in the alterations of the inhibitory neurotransmission in TLR4−/− mice. Ileal segments from male TLR4−/− and wild-type (WT) C57BL/6J mice were incubated with the gliotoxin fluoroacetate (FA). Alterations in ENS morphology and neurochemical coding were investigated by immunohistochemistry whereas neuromuscular responses were determined by recording non-adrenergic non-cholinergic (NANC) relaxations in isometrically suspended isolated ileal preparations. TLR4−/− ileal segments showed increased iNOS immunoreactivity associated with enhanced NANC relaxation, mediated by iNOS-derived NO and sensitive to P2Y1 inhibition. Treatment with FA diminished iNOS immunoreactivity and partially abolished NO− and ADP− mediated relaxation in the TLR4−/− mouse ileum, with no changes of P2Y1 and connexin-43 immunofluorescence distribution in the ENS. After FA treatment, S100β and GFAP immunoreactivity in TLR4−/− myenteric plexus was reduced to levels comparable to those observed in WT. Our findings show the involvement of EGCs in the alterations of ENS architecture and in the increased purinergic and nitrergic-mediated relaxation, determining gut dysmotility in TLR4−/− mice.

Functional gastrointestinal disorders (FGIDs) are characterized by abdominal pain, bloating and bowel disturbances. FGID therapy is primarily symptomatic, including treatment with herbal remedies. Flower extract of Tilia tomentosa Moench (TtM) is occasionally used as an anti-spasmodic in popular medicine. Since its effect on intestinal response is unknown, we evaluated the influence of TtM extract on small intestine contractility. Ileal preparations from C57BL/6J mice were mounted in organ baths to assess changes in muscle tension, following addition of TtM extract (0.5–36 μg/mL) or a vehicle (ethanol). Changes in contractile response to receptor- and non-receptor-mediated stimuli were assessed in ileal preparations pretreated with 12 μg/mL TtM. Alterations in the enteric nervous system neuroglial network were analyzed by confocal immunofluorescence. Increasing addition of TtM induced a marked relaxation in ileal specimens compared to the vehicle. Pretreatment with TtM affected cholinergic and tachykininergic neuromuscular contractions as well as K+-induced smooth muscle depolarization. Following incubation with TtM, a significant reduction in non-adrenergic non-cholinergic-mediated relaxation sensitive to Nω-Nitro-L-arginine methyl ester hydrochloride (pan-nitric oxide synthase inhibitor) was found. In vitro incubation of intestinal specimens with TtM did not affect the myenteric plexus neuroglial network. Our findings show that TtM-induced intestinal relaxation is mediated by nitric oxide pathways, providing a pharmacological basis for the use of TtM in FGIDs.

Silver linden (Tilia tomentosa Moench, TtM) flowers possess several health-promoting properties, especially at the neurological level, such as intestinal relaxation activity associated with specific flavonols, particularly quercetin and kaempferol derivatives. However, such molecules are susceptible to degradation upon different triggers like heat, light and extreme pH values. To overcome the scarce stability of TtM flowers bioactive molecules and make them suitable for developing functional food and supplements, we applied microencapsulation. Spray-drying microencapsulation of TtM flowers extract was performed using three starch-derived wall materials: maltodextrin 12 DE (MD12) and 19 DE (MD19), and OSA-modified starch (OSA-S). The stability of total phenols, flavanols, and antioxidant capacity was monitored for 70 days under accelerated stress conditions (40 °C/70% RH) by HPLC and spectrophotometric methods, and the intestinal contractile activity was tested in a murine model. In comparison to MD12 and MD19, OSA-S stood out for the higher encapsulation efficiency of quercetin and kaempferol glycosides (+ 36–47% compared to MD12 and + 18–24% compared to MD19) and stability thereof (half-life on average + 30% compared to MD12 and + 51% compared to MD19). The intestinal contractile activity of OAS-S powders resulted comparable to the original extract, indicating that flavonols were biologically active and accessible. Our results underly the potential advantages of OSA-S encapsulated formulation as a functional ingredient for the development of nutraceutical products.

Background Alterations in Toll-like receptor 4 (TLR4) expression and changes in enteric neurotransmissions influence the onset and severity of inflammatory bowel disease (IBD). We aimed to assess the interaction between the enteric inhibitory pathways and TLR4 signaling in a mouse model of dinitrobenzene sulfonic acid (DNBS)-induced small intestine inflammation. Methods Male TLR4-/- mice (9±2 weeks old) were intrarectally treated with 2.5% DNBS. Pro-inflammatory cytokines (IL-6 and TNF-α) were measured in ileal samples. Changes in ileal muscle tension were recorded following exposure to: i) 10-Hz electric field stimulation in non-adrenergic-non-cholinergic (NANC) conditions (1 μM guanethidine + 1 μM atropine) with or without 10 µM 1400W (a inducible nitric oxide synthase (iNOS) inhibitor), or 0.1 µM Nω-nitro-L-arginine methyl ester (L-NAME; a pan-NOS inhibitor); ii) 30 μM dopamine or 30 μM SKF38393 (a dopamine 1 receptor (D1R) agonist) or bromocriptine (a D2R agonist). Immunoreactivity of neuronal (HuC/D) and glial (S100β) markers as well as nitrergic chemical coding (i.e. nNOS and iNOS) were determined in longitudinal muscle-myenteric plexus whole-mount preparations (LMMPs) by confocal microscopy. Results No changes in IL-6 and TNF-α ileal mRNA transcripts were found after DNBS administration. The development of ileitis did not affect the NANC-inhibitory response, that was partially reduced in presence of 1400W or L-NAME, suggesting that TLR4 signaling influences NO-mediated inhibitory responses. DNBS-treated mice showed a significant increase in dopamine-induced relaxation (+31%) and in D1R- and D2R-mediated inhibitory responses (by +57% and +75%, respectively). A significant reduction of the total number of HuC/D+ neurons (-25%), with a proportional increase in nNOS+ neurons (+11%) was found in LMMPs of DNBS mice, associated with a significant reduction of S100β immunofluorescence (-12%) and no changes in iNOS immunoreactivity, highlighting a potential involvement of TLR4 signaling in the development of neuroplasticity and reactive gliosis. Conclusion The present data suggest that the severity of small intestine ileitis and neuronal dysfunction depend on the interplay occurring between TLR4 signaling and enteric inhibitory pathways. This evidence further strengthens the importance of characterizing neuroimmune interactions to identify potential molecular mechanism(s) representing putative pharmacological targets for the development of efficacious IBD therapies.

Background Changes in serotonin (5-HT) levels, anomalies in serotonergic and cholinergic machinery and altered Toll-like receptor 4 (TLR4) expression have been shown in IBD in patients and related animal models. Thus, we aimed to assess the crosstalk of enteric serotonergic system and TLR4 signalling in a mouse model of dinitrobenzene sulfonic acid (DNBS)-induced colitis. Methods Male C57/Bl6 (WT) and TLR4−/− mice (9 ± 2 weeks old; N = 10 mice) were presensitised with 1% dinitrobenzene sulfonic acid (DNBS), and after 1 week was intrarectally instilled with 2.5% DNBS. Small intestine inflammation was measured by disease activity index and histological analysis. Changes in ileal muscle tension were isometrically recorded following: (1) cumulative addition of carbachol (CCh; 0.1–100 µM); (2) electric field stimulation (EFS, 0–40 Hz); (3) 60 mM KCl; (4) 30 μM 5-HT addition with or without 0.1 μM ondansetron (5-HT3R antagonist). Immunofluorescence distribution of the neuronal HuC/D and nNOS and glial GFAP markers were determined in longitudinal-muscle-myenteric plexus whole mounts (LMMPs) by confocal microscopy. Results In WT mice, DNBS treatment altered receptor and not-receptor mediated responses (+120% of Emax to CCh and +103% of contraction to KCl, respectively; p < 0.001, N = 5 mice/group) together with an altered cholinergic neurotransmission (−50% at 10 Hz; p < 0.01, N = 5 mice/group) and 2-fold increase to 30 μM 5-HT-mediated response (p < 0.001, N = 5 mice/group). After DNBS treatment TLR4−/− mice showed a significant increase in excitatory-mediated response (+98% of Emax to CCh; +80% of contraction to KCl; +120% at 10 Hz; p < 0.001, N = 5 mice/group) together with a significant reduction of 30 μM 5-HT-mediated response (−50%, p < 0.001, N = 5 mice/group). These changes were associated to a significant decrease of the total number of HuC/D+ neurons (−44% and −19% for WT DNBS and TLR4 DNBS mice, respectively) together with a 1.3-fold increase in S100b immunofluorescence in WT mice after DNBS treatment. Conclusion These findings not only suggest an important role of TLR4 in small intestine neuromuscular dysfunction during colitis but also provide novel information on the potential benefits of targeting TLR4 in various gut disorders that exhibit aberrant cholinergic and 5-HT signalling.