Study On The Role Of Brain-derived Neurotrophic Factor In Colonic Mucosal Barrier Integrity

BackgroundBrain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is originally known to play an important role in the development, differentiation, regeneration of central and peripheral nervous system. Several studies have demonstrated that BDNF has been confirmed to be expressed in several developing and mature peripheral tissues, including the gastrointestinal tract, blood vessels and periodontal tissues. Several studies have demonstrated that various soluble factors secreted by peripheral nerve pericytes, including nerve growth factor (NGF), BDNF, and glial-derived neurotrophic factor (GDNF), may be essential for maintaining the integrity or permeability of blood-nerve barrier and modifying tight junctional molecules. BDNF synthesized by endothelial cells is highly effective in activating the receptor tyrosine kinase B (TrkB) to regulate blood vessel stabilization and functional integrity in the perinatal period. In adult vascular endothelial cells, BDNF has been shown to inhibit endothelial barrier dysfunction by increasing the expression of vascular endothelial (VE)-cadherin in response to the inflammatory mediators. VE-cadherin is one of the key intercellular junction proteins, which is known to play a pivotal regulatory role in the permeability of endothelial cells. In the gastrointestinal tract, certain neurotrophins such as NGF and neurotrophin (NT)-3 have been confirmed to be involved in the regulation of intestinal barrier integrity. As for BDNF, it is highly expressed in the intestinal mucosa. However, whether BDNF contributes to intestinal epithelial barrier formation or maintenance has not yet been explored.Objective1. To investigate the physiological role of BDNF in keeping intestinal epithelial barrier using BDNF knockdown mice model.2. To investigate the probable effects of BDNF on intestinal epithelial cells apoptosis and alterations of tight junction proteins (TJs) using BDNF knockdown mice model; To evaluate possible related molecules involved in BDNF induced IECs apoptosis in BDNF+/- mice;3. To inhibit the expression of BDNF in intestinal epithelial cells ht-29 and evaluate its effect on TJs expression;4. To assess how BDNF stimulation impacted on TJs expression in ht-29 intestinal epithelial cells.Methods1. Animal studiesColonic tissues from wild type BDNF+/+ mice and heterozygous BDNF+/- mice (C57B1/6 background) were prepared for this study. The colonic mucosa integrity was evaluated in Ussing chamber by measuring transepithelial resistance (TER). The colonic epithelial structure was detected by H&E staining and transmission electron microscopy (TEM). Apoptosis involvement was determined with TUNEL staining, active caspase-3 immunostaining and western blot for the protein expression of active casapase-3, Bax and Bcl-2. Expression and distribution of TJs (occludin and ZO-1) in the colonic mucosa were evaluated by immunohistochemistry. The expressions of TJs (claudin-1 and claudin-2) in the colonic mucosa were evaluated by western blotting.2.Cell studiesExperiments were performed with the human intestinal epithelial cell line ht-29. The siRNA was designed and synthesized according to BDNF. A siRNA against BDNF was used for knockdown of BDNF, with a scramble siRNA used as a control. After transfection, we performed real-time PCR and western blot to detect the gene and protein expression of BDNF and TJs (occludin, ZO-1, claudin-1 and claudin-2) in each experimental group.To analyze the effect of BDNF on TJs expression, ht-29 cells were incubated with 10-8 mol/L,10"9 mol/L or 10"10 mol/L of BDNF up to 2 days. Control cells were treated with PBS in culture medium. At 6h,12h,24h and 48h after treatment, cells were harvested and prepared for subsequent study. We further performed real-time PCR and western blot to assess how BDNF stimulation impacted on TJs gene and protein expression (occludin, ZO-1, claudin-1 and claudin-2) in each experimental group.2. Statistical analysisData are presented as the mean ± SEM using SPSS 20.0. Differences between two groups were analyzed using independent Student t test. One-way ANOVA followed by Dunnett’s test was performed to assess the potential influence of BDNF on the gene and protein expression of TJs. Differences were considered significant when P< 0.05.Results1. Animal studiesCompared with BDNF/+ mice, BDNF+/- mice displayed impaired colonic mucosal integrity and ultrastructural alterations in the colonic mucosa, which was characterized by diminished microvilli, mitochondrial swelling, epithelial cells apoptosis, widen intercellular space and bacteria invasion in the colonic mucosa. Altered intestinal barrier function was linked to excessive intestinal epithelial cells (IECs) apoptosis demonstrated by the significant up-regulation of TUNEL-positive apoptotic cells and enhanced caspases activities in BDNF+- mice. Increased expression of pro-apoptotic bax proteins and anti-apoptotic reduced bcl-2 expression were further detected in the colonic mucosa of BDNF+- mice. Besides, BDNF+/- mice also exhibited increased protein expression of claudin-2 and decreased protein expression of occludin, ZO-1 and claudin-1 in the colonic mucosa.2. Cell studiesA siRNA against BDNF in ht-29 cells could effectively suppress BDNF gene and protein expression, and subsequently reduce TJs gene and protein levels. When ht-29 cells were incubated with different doses of exogenous BDNF, a significant increase of TJs (occludin, ZO-1 and claudin-1) and decrease of claudin-2 protein were observed.Conclusion1. Compared with BDNF+/+ mice, alterations in expression of TJs and excessive apoptosis of IECs with corresponding intestinal barrier defects have been observed in the colon of BDNF+/- mice.2. After the knockdown of BDNF or the administration of BDNF, the altered expressions of TJs in the intestinal epithelial cells have been observed.3. BDNF may play a role in regulating intestinal epithelial barrier via affecting the expression of TJs and inhibiting IECs apoptosis.