Brain-derived Neurotrophic Factor Induce The Plasticity Of Colon Smooth Muscle Cells In Mice

BackgroundBrain-derived neurotrophic factor (BDNF) is one member of the neurotrophin family which is best known for its role in neuronal survival, differentiation, migration, and synaptic plasticity in central and peripheral neurons. BDNF mediates its biological functions by activating Tropomyosin-related kinase B (TrkB) receptor, which is a high-affinity receptor of BDNF. Recently, the positive role of BDNF in gastrointestinal (GI) motility has drawn increasing attentions. In animals, endogenous BDNF could enhance the colon peristaltic reflex by mucosal stimulation in mice. In humans, exogenous BDNF could stimulate human gut motility, accelerate colonic transit, and increase stool frequency. Smooth muscle cells (SMCs) are the direct effectors of colon contraction. However, the mechanisms underlying BDNF’s effect on the plasticity of colon SMC have not been fully characterized.ObjectiveThe ultrastructural and the functional (smooth muscle a-actin expression (α-SMA)) alterations of the smooth muscle were measured in BDNF+/- mice, and was compared with that in BDNF+/+ mice. After administrating of BDNF and TrkB receptor antagonists (K252a), the morphological, the functional (α-SMA expression), TrkB signal pathway proteins, and [Ca2+]i alterations of SMCs were observed.Method1.Heterozygous BDNF+/- mice (C57B1/6 background) and wild type BDNF+/+ mice were used in this study. Transmission electron microscopy (TEM) was used to measure the ultrastructural alterations of the smooth muscle cells in BDNF+/- mice, and was compared with that in BDNF+/+ mice. The a-SMA expression of colonic SMCs in the BDNF+/- mice was measured by Western blotting, and was compared with that in BDNF+/+ mice.2. Primary SMCs, originally derived from a C57b1/6 mouse distal colon, was used in the following studies. The expression of tropomyosin-related kinase B (TrkB) receptor was identified in the primary colonic SMCs of the mice by immune-fluorescence staining (IF). SMCs were intervened by BDNF and BDNF+ K252a,The SMCs were divided into three groups:the control group, the BDNF group and the BDNF+K252a group. The morphological alterations of SMCs were measured by IF.The nucleus size, the density and the skelemin’s fluorescence intension of SMCs were analyzed. Levels of a-SMA and TrkB-PLC signal pathway proteins were examined by Western blotting. After administrating of BDNF and K252a, the intracellular Ca+ concentration ([Ca+]i) alterations were measured by Ca2+ imaging.3. Statistical analysis: All data were expressed as mean ± SD using the SPSS 17.0 program. The level of a-SMA in mice colon of two groups and the [Ca2+]i alterations were measured by t tests. The size of cell neucleus, the density of SMCs, the level of a-SMA, and the levels of TrkB-PLC-Ca2+ signal pathway proteins were measured by one-way ANOVA, followed by Bonferroni tests. A value of p< 0.05 was considered statistically significant.Results1.In BDNF+/- mice, the interstitial gaps between adjacent SMCs were focally widened and filled with increased collagen and elastic fibres; the cellular borders of SMCs were characterized by multiple bulbous protrusions; the cell bodies frequently showed scrambled, fuzzy and less.2. Compared to the BDNF+/+ mice, the a-SMA expression in the BDNF+/- mice were significantly decreased in the distal colon.3. The expression of TrkB receptors were identifed in the primary distal colon SMCs.4. The size of SMCs’ nucleus and cell density were bigger, the fluorescence intension of skeleton protein was stronger than the control group and BDNF+K252a group.5. The level of a-SMA was significantly increased in BDNF group than in negative control cells and BDNF+K252a administrated group.6. The levels of TrkB and PLC were significantly increased in BDNF group than in the control group and BDNF+K252a administrated group. The Ca2+ influx was induced a significant increase under the effect of BDNF. And subsequently the Ca2+ influx induced fluorescence signal was significantly decreased after administrated of K252a.Conclusionsl.The ultrastructural and the functional alterations of the smooth muscle were measured in BDNF+/- mice colon which were controlled with that in BDNF+/+ mice.2.After administrated of BDNF and K252a, the morphological and the functional alterations of SMCs were observed.3.BDNF could directly induce the plasticity of the SMCs of the mice colon, through TrkB-PLC-Ca2+ signal pathway.