BDNF及其受体TrkB在慢性应激大鼠结肠动力紊乱中的调节作用及其机制

Modulatory role and mechanism of BDNF and its receptor TrkB in colonic motility disorder in chronic stress rat

目的 观察脑源性神经营养因子（BDNF）及其受体酪氨酸激酶B（TrkB）在慢性应激大鼠结肠动力紊乱中的作用及其机制.方法 将20只Wistar雄性大鼠称重后按完全随机方法分成两组,避水应激组（WAS组）和假避水应激组（SWAS组）,每组10只.记录两组在慢性避水应激模型造模期间粪便粒数;酶联免疫吸附试验（ELISA）、实时定量PCR、Western印迹和免疫组化法检测BDNF和TrkB在大鼠血清及结肠肌层的表达.分别用河豚毒素（TTX）、BDNF和TrkB抑制剂K252a、BDNF孵育各组近端结肠肌条,记录近端结肠环形肌条肌张力的变化.结果 WAS组大鼠造模期间粪便粒数明显多于SWAS组（P＜0.05）.WAS组大鼠血清BNDF水平高于SWAS组[（158.30±9.82）比（84.68 ±7.80） pg/ml]、结肠肌层TrkB蛋白表达水平高于SWAS组（0.44±0.03比0.30±0.02）（均P＜0.05）,而肌层BDNF mRNA表达两组差异无统计学意义（P＞0.05）.TrkB主要在肌层神经元的细胞核表达,且WAS组表达明显强.WAS组近端结肠环形肌条收缩幅度明显高于SWAS组[（0.35±0.02）比（0.22±0.03）g,P＜0.05];加入TTX阻断肠神经作用后,WAS组环形肌条收缩幅度仍大于SWAS组[（0.89±0.07）比（0.53 ±0.06）g,P＜0.05];用BDNF孵育肌条,记录不同时间点的标准化率（R值）,其中孵育6、12 min时两组肌条R值差异有统计学意义（均P＜0.05）,BDNF能诱导收缩波峰;用K252a孵育环形肌条30 min后加入BDNF,导致BDNF引起的峰值延后且降低.结论 BDNF通过作用于TrkB受体在慢性应激大鼠结肠动力紊乱中发挥一定的调节作用.

Objective To investigate the role and mechanism of BDNF and its receptor TrkB in the disorder of colonic motility in chronic stress rats.Methods A total of 20 male Wistar rats were randomly divided into two groups using to completely random method after weighed,with 10 rats in each group.Chronic water avoidance stress model was established.Then the fecal pellets of water avoidance stress （WAS） group and sham water avoidance stress （SWAS） group were recorded.Enzyme linked immunosorbent assay （ELISA）,real time PCR,Western blot and immunohistochemistry were used to detect the expression of BDNF and TrkB in serum and colon muscle.The amplitudes of contractions of circular smooth muscle strips of each group were recorded after the treatment of TTX,BDNF and K252a.Results The number of fecal pellets had obviously increased in WAS group comparing with SWAS group （P ＜ 0.05）.The level of serum BDNF in WAS group was higher than that of SWAS group （（158.30 ± 9.82） vs （84.68 ± 7.80） pg/ml）.And the expression of TrkB in the colon muscle in WAS group was higher than that in SWAS group （0.44 ± 0.03 vs 0.30 ± 0.02,P ＜ 0.05）.There was no significant difference between the two groups in expression of BDNF mRNA in colon muscle （P ＞ 0.05）.TrkB was mainly expressed in the cell nucleus of muscular layer neurons,and the expression of TrkB had obviously increased in WAS rats.The amplitudes of contractions of circular smooth muscle in WAS rats had significantly increased compared with SWAS rats （（0.35 ±0.02） vs（0.22 ±0.03） g,P ＜0.05）.After adding TTX to block the function of enteric nervous,the difference was remaining （（0.89 ± 0.07） vs （0.53 ± 0.06） g,P ＜ 0.05）.BDNF was added to the bath and the R value at different time was recorded.The difference had statistically significant at 6 min and 12 min （both P ＜ 0.05）.BDNF could induced the contraction peak of the circular smooth muscle.The contraction peak induced by BDNF was delayed and reduced when K252a was added to the bath 30 min before adding BDNF.Conclusion BDNF plays a modulatory role in the disorder of colonic motility in chronic stress rat by acting on its receptor TrkB.