The Molecular Mechanisms Of Gut Motility In Mice

BackgroundIntestinal motility disorders are considered to play an important role in the pathogenesis of many functional gastrointestinal disorders. Intractable symptoms and treatment difficulties seriously impact on the quality of life of patients. However, the pathogenesis of abnormal intestinal motility has not as yet clear.Brain-derived neurotrophic factor (BDNF), as a member of the neurotrophin family, through binding with its high affinity tyrosine kinase receptor B-TrkB, activates phospholipase C-PLC/inositol triphosphate-IP3pathway and store-operated calcium entry to enhance signal transduction in neuron-muscular junction. Except for nervous system, abundant BDNF and TrkB were also fund expressing in the intestinal tracts including enteric nervous system (ENS) and intestinal epithelial of various species. And the concentrations of BDNF in the colon were higher than in the brain. Though the effects of BDNF on the visceral hyperalgesia have been examined to some degree, limited studies have focused on the effects of BDNF on gut motility.Several clinical and animal studies reported that recombinant human BDNF(r-HuBDNF) could accelerate colonic emptying and stimulate gut myoelectric activities characterized by enhanced spiking bursts. These studies implicated that BDNF might play a fundamental role in regulating gastrointestinal motility. However, the specific role of BDNF and the underline mechanisms on gut motility are still unclear.It is noteworthy that BDNF can act as a neuromodulator or neurotransmitter. In both central (CNS) and peripheral nervous system (PNS), BDNF has been detected co-existing with certain neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP). BDNF could enhance the release and the effect of SP/CGRP. These studies strongly suggests that BDNF as a neuromodulator or neurotransmitter might interact with SP/CGRP and play a modulatory role on the gut motility.AimsIn the present study, to explore the molecular mechanisms of mice gut motility, we examine the mice gut motility in vivo and in vitro. First, we investigate the effect of BDNF on gut motility of mice in vivo and in vitro. Second, the effect of TrkB-PLC/IP3pathway on the contraction of isolated longitudinal muscle (LM) strips induced by BDNF was examined. Third, the role of SP and CGRP on the contraction of LM strips of mice was clarified. Fourth, the effect of exogenous and endogenous BDNF on the contraction of LM strips induced by SP and CGRP was explored.MethodsMale C57B1/6BDNF+/+mice and heterozygous BDNF+/-mice were used in this study.1:gut motility in vivo1.1Frequency and water weight of stoolsThe stool frequency of BDNF+/+mice and BDNF+/-mice was measured as the number of stool beads for each mice after1h. After weighted after24h, the wet stools of each mice were dried and then weighted.1.2Total gastrointestinal transit and small intestinal transitA charcoal meal was orally administered to BDNF+/+mice and BDNF+/-mice. The time that each mice first defecate black feces was recorded and measured as the total gastrointestinal transit. After30min orally administered charcoal meal, the mice were sacrificed. The small intestine was removed and the distance traveled by charcoal was measured as well as the total length of the small intestine.2gut motility in vitro2.1Preparation of intestinal muscle stripsBDNF+/+mice and BDNF+/-mice fasted for12h before experiments were sacrificed. Segments of ileum, proximal and distal colon were immediately removed. Muscle strips (3mm wide,8mm long) that were cut parallel to the long axis of the longitudinal muscle layer were longitudinally mounted in an organ bath which were filled with5ml Krebs solution (37℃) and bubbled continuously with95%O2and5%CO2. Each muscle strip was placed under a resting preload of0.5g and allowed to equilibrate for30min with flushing every10min.2.2Experiment protocol2.2.1The intestinal LM strips was administrated with different doses BDNF, SP and CGRP. The mechanical responses were recorded to investigate the contractile effects of BDNF, SP and CGRP on the contraction of mice intestinal LM strips.2.2.2The strips were pretreated with the antibody of TrkB (TrkB-Ab), neomycin (an inhibitor of PLC) and heparin (an inhibitor of IP3receptors) before administration of BDNF. The mechanical responses were recorded to explore the influence of TrkB-PLC/IP3pathway on the contraction of LM strips induced by BDNF.2.2.3The strips were pretreated with BDNF before administration of SP or CGRP. The mechanical responses were recorded to explore the influence of exogenous BDNF on the contraction of LM strips induced by SP and CGRP.2.2.4Firstly, the mechanical responses of the muscle strips of BDNF+/-mice to SP and CGRP were recorded, and compared with those of BDNF+/+mice. Further, muscle strips of BDNF+/+mice were pretreated with TrkB-Ab before administration of SP or CGRP. The mechanical responses were recorded to investigate the influence of endogenous BDNF on the contraction of muscle strips induced by SP and CGRP.3Measurement of BDNF ReleaseAfter the LM strips of BDNF+/+mice, BDNF+/-mice and TrkB-Ab pretreatment BDNF+/+mice were stabilized, the Krebs solution were collected. Levels of BDNF in Krebs solution were measured by specific Enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocols. Each sample was analyzed in duplicate.4ImmunohistochemistryColon tissues from wild-type BDNF+/+mice were processed for TrkB immunohistochemistry using widely applied methods. 5Data analysisAll values were expressed as mean±SEM. Statistical evaluation of the data was performed by Student’s t-test for comparisons between two groups and one-way analysis of variance (ANOVA) for comparisons among multiple groups with SPSS16.0. P<0.05was considered statistically significant.Results1The frequency and water weight of stoolsCompared with BDNF+/+mice, the frequency and percentage of water weight of stools was significant decreased in BDNF+/-mice.2Total gastrointestinal transit and small intestinal transitThe time to the first black feces and small intestinal transit was significantly delayed in BDNF+/-mice, compared with that in BDNF+/+mice.3Effects of BDNF on the contraction of mice intestinal muscle stripsBDNF (10-7mol/L) significantly increased the contraction of isolated LM strips from the ileum, proximal colon and distal colon.4Effects of TrkB-PLC/IP3pathway in the excitatory effect of BDNF on LM stripsTrkB-Ab, neomycin and heparin dramatically attenuated the excitatory effect of BDNF (10-7mol/L) on the contraction of mice intestinal muscle strips.5Effects of SP and CGRP on the contraction of LM strips from BDNF+/+miceSP (1×10-8-1×10-6mol/L) dose-dependently increased the contraction of isolated LM strips from the ileum and distal colon. CGRP (1×10-8-1×10-7mol/L) increased the contraction of LM strips with a prior transient inhibition period. However, Higher concentration of CGRP (10-6mol/L) showed inhibitory effects.6Effects of exogenous BDNF on contraction of LM strips induced by SP and CGRPBDNF (1×10-8mol/L) remarkably enhanced the contraction of ileum and distal colon LM strip induced by SP (1×10-8-1×10-7mol/L) and CGRP (1×10-9-1×10-8 mol/L).7Effects of endogenous BDNF on contraction of LM strips induced by SP and CGRPThe excitatory effects of SP(1×10-8-1×10-6mol/L) and CGRP(1×10-8-1×10-7mol/L) on contraction of LM strips were significantly attenuated in BDNF+/-mice compared with those in BDNF+/+mice. TrkB-Ab (10-8mol/L) inhibited the spontaneous contractions of LM strips, and obviously attenuated the excitatory effect of SP and CGRP (1×10-7mol/L).8Spontaneous release of BDNFThe level of BDNF released from LM strips in BDNF+/-mice was approximately half that in BDNF+/+mice. However, there were no differences on the level of BDNF released from LM strips between TrKB-Ab pretreated and non-pretreated BDNF+/+mice.9Distribution of TrkB in mice intestineTrkB-immunoreactivity primary distributes in myenteric plexus of mice intestine. However, TrkB-immunoreactivity was not observed in muscle layer of intestine.Conclusions1BDNF could enhance the mice gut motility in vivo and in vitro through TrkB-PLC/IP3pathway.2SP dose-dependent excited the contraction of mice intestinal LM strips. CGRP showed stimulating effects with a prior transient inhibition period after administration on intestinal LM strips.3Exogenous and endogenous BDNF could modulate the excitatory effects of SP/CGRP on the contraction of mice intestinal LM strips.