外源性硫化氢对重症感染性休克肠屏障及多脏器功能障碍的影响及分子机制

Under the condition of severe septic shock, intestinal barrier injury causes a large number of microorganisms and other antigenic substances from the intestinal lumen to enter the internal milieu, and promote the occurrence of multiple organ dysfunction. Hydrogen sulfide has a wide range of physiological functions. Recent studies have shown that it has anti-inflammatory and anti-apoptotic effects. We have previously demonstrated that hydrogen sulfide has protective effects on the intestinal barrier damage induced by inflammatory cytokines. Therefore, we hypothesized that exogenous hydrogen sulfide can reduce the incidence of multiple organ dysfunction in severe septic shock model and improve the survival rate". In this study, we first observe the characteristics of hydrogen sulfide metabolism in intestinal epithelial cells in severe septic shock. The effects of hydrogen sulfide on the intestinal epithelial tight junction proteins, apoptosis, oxidative stress and energy metabolism are investigated. Effects of exogenous hydrogen sulfide on multiple organ dysfunction and survival rate in model animals will be further investigated. By interfering with the expression of key signaling molecules in the MLCK-P-MLC pathway, the specific targets of hydrogen sulfide will be investigated. The relationship between hydrogen sulfide metabolism and intestinal barrier injury and its effect on multiple organ function in septic shock will be explored in the organ, cellular and molecular level in this project, aiming to provide novel insights into the treatment of severe septic shock.

重症感染性休克状态下，肠屏障损伤使得源自肠腔的大量微生物及其他抗原性物质得以进入人体内环境，促进多脏器功能障碍的发生。最新研究发现硫化氢具有抗炎、抗凋亡的作用。前期我们已证实，硫化氢对由炎症因子导致的肠屏障损伤具有保护作用。据此，我们推测，“外源性硫化氢可通过保护肠屏障减少重症感染性休克模型动物多脏器功能损伤的发生并提高生存率”。本课题首先观察重症感染性休克状态下肠上皮细胞硫化氢代谢的特点，同时观测外源性硫化氢对肠屏障的保护作用及其作用机制。重点关注其对肠上皮紧密连接蛋白以及细胞凋亡、氧化应激和能量代谢的影响。同时评估外源性硫化氢对模型动物多脏器功能障碍及生存率的影响。通过干预MLCK-P-MLC通路关键信号分子的表达，拟证明硫化氢作用的具体信号通路。本课题在器官、细胞及分子水平上探讨硫化氢代谢与肠屏障的关系及其对感染性休克状态下多脏器功能障碍的影响，为重症感染性休克的治疗提供新思路。

既往研究表明肠屏障损伤在结肠炎及重症感染性休克中起关键作用。本课题组已证实，H2S对由炎症因子导致的肠屏障损伤具有保护作用。据此推测H2S可保护肠屏障而减弱重症感染性休克动物模型的炎性风暴和多脏器功能损伤的发生。.首先，我们对溃疡性结肠炎（UC）患者结肠上皮的非炎症部位和炎症部位的H2S合成的关键酶CBS及H2S含量变化进行研究。与UC结肠上皮的非炎症部位相比，炎症部位CBS表达降低显著；在表观遗传学方面，在UC患者的炎症部位CBS启动子甲基化水平升高。UC患者炎症部位H2S的含量显著降低。可见CBS表达高低与H2S含量多少在UC患者结肠上皮中协同变化。 .其次，在体外实验方面，通过TNF-α/IFN-γ处理的Caco-2细胞单层模拟人体肠上皮损伤模型，抑制CBS的表达可显著降低H2S生成，佐证了CBS与H2S协同变化；TEER和FD-4通透性实验证实抑制CBS（基因沉默和AOAA活性抑制）显著加剧Caco-2单层屏障功能损伤；拯救实验发现，GYY4137，H2S缓释供体，显著抑制上述情况发生。在紧密连接蛋白表达方面，抑制CBS表达对紧密连接蛋白ZO-1、occludin和claudin-1的表达没有影响。在紧密连接蛋白定位方面，免疫荧光实验证实抑制CBS可显著加重TNF-α/IFN-γ诱导的Caco-2单层中ZO-1和occludin的定位改变。可见加重的紧密连接蛋白错误定位可能是抑制CBS对TNF-α/IFN-γ诱导的屏障损伤的破坏作用的主要原因。分子机制方面，抑制CBS可显著增加MLCK的表达和 MLC-2的磷酸化，加剧F-actin“鸡丝”样的典型膜相关定位向不规则转变。总之，MLCK-P-MLC信号通路激活以及F-actin的无序分布可能与CBS-H2S轴抑制诱导的肠屏障功能易损性增加相关。.CBS敲除小鼠实验证实，敲低CBS可显著加剧脂多糖LPS诱导的肠屏障损伤，重症感染性休克模型动物多脏器（肺、脑、肝）功能损伤的程度及炎性风暴水平；GYY4137显著抑制上述情况的发生。考虑到LPS诱导的炎症反应与COX-2之间的相关性，我们证实LPS诱导的实验性结肠炎小鼠结肠上皮中COX-2和HuR表达增加显著；GYY4137可显著抑制COX-2表达增加，而且增加HuR的二硫化修饰；H2S介导的HuR Cys13二硫化修饰可能是CBS-H2S轴对伴随实验性结肠炎的局