嗜酸乳杆菌对重型颅脑损伤后肠道ICC和平滑肌细胞间缝隙连接的影响及机制研究

Intestinal motility disturbance occurs at a high incidence in early severe head injury(SHI), which directly affected the treatment outcomes and clinical prognosis. Our previous studies showed that Lactobacillus acidophilus(La) can dramatically improve the condition of intestinal motility disturbance, but its underlying mechanism is still unrevealed. Researches showed that the gap junction channels formed by Cx43 ensure the harmonious contraction of the intestinal smooth muscle cells (SMC).In addition, other studies indicated Cx43 expression and phosphorylation level are closely related to the function of GJ. Our preliminary experiments showed that ICC and SMC were impaired and Cx43 expression level were decreased on SHI mice model. However, La reduced the damage degree of ICC and SMC after injury and up-regulated the expression of Cx43.So we speculate that the mechanism of La improving the intestinal motility after SHI would be related to its function in regulating the Cx43 expression between ICC and SMC, as well as the Cx43 phosphorylation level..To demonstrate our hypothesis, we would like to investigate the relationship between Cx43 phosphorylation level and intestinal motility before and after intervention. We Selected S386 as a phosphorylation site，constructed continuous phosphorylation and Cx43 mutant cell model by using site-directed mutagenesis. With this model we will fully elucidate the effect of Cx43 expression and its phosphorylation on cell communication under hypoxia. The current study will primarily reveal the mechanisms of La on the improvement of Intestinal motility disturbance after SHI and provide a theoretical basis for its application in trauma field.

重型颅脑损伤（SHI）早期肠动力不足发生率高，直接影响救治效果和临床预后。我们前期研究发现嗜酸乳杆菌（La）可明显改善SHI后肠动力不足，但机制尚不明确。研究表明Cx43形成的缝隙连接（GJ）通道保证了肠道平滑肌细胞（SMC）收缩的协调，Cx43表达量及磷酸化与GJ功能密切相关。预实验观察到SHI小鼠ICC与SMC受损，二者间Cx43表达减少；La减轻了ICC和SMC受损程度，上调Cx43表达。据此推测，La改善SHI后肠动力不足的机制与其调节ICC与SMC之间Cx43表达及磷酸化水平有关。为验证这一假说，本研究拟在前期基础上观察La干预前后Cx43表达及磷酸化水平与伤后肠动力的关系，挑选磷酸化位点S368以定点突变方法构建持续磷酸化和Cx43突变细胞模型，以充分阐明缺氧时Cx43表达及磷酸化对细胞通讯的影响，初步揭示La改善SHI后肠动力不足的机制，为益生菌在创伤领域的应用提供理论依据。

ICC、缝隙连接、平滑肌细胞及肠神经系统（ENS）是肠运动发生的核心要素，其中任一元素的损伤都将导致信号传递不畅，引起肠动力障碍。我们前期研究发现，嗜酸乳杆菌（La）可明显改善重型颅脑损伤（SHI）后肠动力不足，但机制还有待研究。本课题通过建立小鼠SHI后肠动力障碍（IMD）模型，采用生物信息采集和处理系统、激光共聚焦、Western Blot、透射电镜等技术与手段，观察La干预前后SHI小鼠肠道离体肌条收缩、ICC和平滑肌细胞间缝隙连接的形态学、Cx43表达量和蛋白磷酸化水平的变化，获得了以下研究成果：①创新改良的自由落体撞击法制备的SHI后IMD小鼠模型死亡率低，稳定性、重复性好。②SHI后肠肌条收缩幅度和频率降低、张力减弱，La可改善伤后肌条收缩障碍。③为了进一步分析La改善肠动力不足的机制，我们以肠动力起搏细胞ICC为研究对象，发现La能减轻SHI后肠肌丛ICC数量减少和网络受损的程度。④通过观察ICC和平滑肌细胞间网络的微观结构发现，La能减轻伤后ICC和平滑肌细胞的线粒体肿胀程度、缩小细胞间间隙、使缝隙连接增多。⑤为了深入探究La对缝隙连接的影响，我们对缝隙连接结构蛋白Cx43进行观察，发现伤后Cx43的蛋白表达量减少，给予La干预后其表达显著增加，提示La通过上调缝隙连接Cx43的蛋白表达，修复SHI后细胞间连接，使ICC和平滑肌细胞间信号传递障碍得以修复。⑥为进一步阐明La促进细胞间信号传递恢复的机理，我们对Cx43磷酸化水平进行了研究，发现La能减少SHI后Cx43蛋白磷酸化的阳性表达，表明La能够维护或修复缝隙连接的形态与功能。⑦同时在研究中发现，La对SHI后与ICC伴行的肠神经也有一定影响，为此进一步观察了La对肠神经递质阳性细胞的作用，发现La能恢复伤后受损AchE细胞的形态，降低NOS的活性，改善细胞数量比例的失衡。上述结果证实了我们提出的“La改善SHI后肠动力不足的机制与其调节ICC-SMC之间Cx43表达及磷酸化水平有关”的假说，为探讨La在严重创伤后患者中的应用提供实验证据。本课题发表中文论著3篇，正在撰写SCI论著1篇，培养硕士研究生4名。