生脉注射液对脓毒症肠黏膜屏障的保护及调控肠上皮紧密连接的分子机制研究

Intestinal barrier function (IBF) plays a central role in sepsis. The destruction of IBF is the main reason for the second hit to patients. Clinical evidences and our critical pre-tests suggested that Shengmai injection (SMJ) may play an protective role in sepsis through decreasing local proinflammatory mediators and reducing the damage to IBF. Thus, firstly, in vivo study using in situ single-pass intestinal perfusion for identifying the main site of action of SMJ on IBF is needed. Then, ex vivo study using Ussing chamber technique, and in vitro study using lamina propria cells (e.g. macrophages, monocyte, lymphocyte, etc.) and epithelial cells will be carried out. The intracellular molecular mechanisms studies for SMJ will focus on the route as follow: (immune cells) TLRs/NLRs → NF-κB/MAPK → proinflammatory mediators → (epithelial cells) MLCK → tight junction (e.g. ZO-1, occluding, claudin, etc.). The found targets will be further confirmed by an animal sepsis model. The aim of this study is to verify the hypothesis that “Yi Qi Wan Yin” Shengmai could prevent or protect the second hit in sepsis induced by intestinal mucosal barrier dysfunction and meanwhile to reveal the regulation mechanisms of Shengmai for the tight junction of epithelial cells. Our study not only provides a pharmacological basis for clinical rational drug use, but also have important academic value to clarify the scientific connotation for the ancient prescription, Shengmai.

肠上皮屏障功能在脓毒症发生发展中发挥了决定性作用，它的破坏是造成机体受到二次打击的主因。临床证据及关键预试提示生脉注射液(SMJ)在辅助治疗脓毒症中可能通过减少局部炎性因子并降低其对上皮屏障损害两条途径来发挥作用。项目以此为研究出发点，以在体单向肠灌流模型判断SMJ肠屏障保护的主要部位，体外与离体(尤斯室)实验则围绕肠粘膜固有层几种免疫细胞及上皮层组织与细胞进行。细胞内分子机制研究将以(免疫细胞)TLRs/NLRs→NFκB/MAPK→促炎因子→(上皮细胞)MLCK→TJ (ZO-1,occluding,claudin等)为轴心展开。找到的靶点再以脓毒症动物模型予以确认。以此求证“益气挽阴”的生脉在脓毒症中是通过预防或减轻因肠黏膜屏障功能损失所致 “二次打击”的假说，同时明晰其对肠上皮TJ调控的分子机制。该项目不仅可为临床合理用药提供参考，对阐明古方生脉救急扶危的科学内涵亦具重要的学术价值

肠上皮屏障功能在脓毒症发生发展中发挥了决定性作用，它的破坏是造成机体受到二次打击的主因。临床证据及关键预试提示生脉注射液(SMJ)在辅助治疗脓毒症中可能通过减少局部炎性因子并降低其对上皮屏障损害两条途径来发挥作用。本课题结合化学物质组学表征，基于UPLC指纹图谱技术结合两种药效相关的生物敏感活性指标的质量控制方法研究，建立了生脉注射液质量评价体系与药效指标相关、反映整体活性的质量控制方法；基于网络药理学和代谢组学技术，初步揭示生脉注射液治疗脓毒症的相关作用机制，表明生脉注射液对于脓毒症引发的机体炎症反应有一定的抑制作用，并具有明显的剂量依赖性，通过构建网络药理探讨了其有效成分发挥保护作用时可能机制，其中预测得到20个关键靶点涉及多个代谢通路，参与细胞周期、能量代谢、细胞内信号转导、转录调控及免疫系统发育等数十种生物学过程。通过研究脓毒症大鼠尿液代谢轮廓，发现生脉注射液对于疾病引发代谢紊乱的代谢通路具有回调作用，其中调节牛磺酸代谢、精氨酸和脯氨酸代谢等可能是其治疗脓毒症的关键机制。使用脓毒症模型验证网络药理学结果，发现生脉注射液主要是于空肠发挥作用，通过NF-κB→炎症介质→MLCK→occluding通路，降低炎症因子TNF-α、IL-2、INF-γ和 IL-10 发挥治疗作用，适宜的给药剂量为1.5 ml/kg。这将有助于保障生脉注射液的安全性和有效性，发现生脉注射液多成分多靶点多途径的特征优势，对其在临床的科学用药提供了新参考，对阐明古方生脉救急扶危的科学内涵亦具重要的学术价值。