小檗碱通过调控Smad3诱导自噬减轻脓毒症肠黏膜上皮细胞损伤的机制研究

Sepsis is a life-threatening organ dysfunction caused by the imbalance of the body's response to infection, and the gastrointestinal tract is the most vulnerable target organ of multiple organ dysfunction. The key to its treatment is how to protect intestinal mucosal epithelial cells and barrier function. Previous studies have found that berberine can reduce endotoxin induced intestinal mucosa epithelium cells injury and protect mucosal barrier function in rats. Its clinical efficacy is also obvious, but the specific mechanism needs further study. Our research group proposed the hypothesis: Berberine acts on the intestinal tract by regulating the expression of Smad3, inducing autophagy, thus reducing inflammatory reaction and sepsis-induced apoptosis of intestinal mucosal epithelial cells, and inhibiting organ damage after sepsis. Our hypothesis was verified by establishing sepsis rat model and in vitro cell experiments in this study. In addition, we used serum exosome gene chip technology and ileum tissue high throughput sequencing technology, and bioinformatics methods such as inter group difference analysis and cluster analysis to screen exosome miRNAs targeting to regulate Smad3, and verified it in vitro.In this study, the mechanism of Smad3 induced autophagy was discussed from different angles so as to lay the foundation for further study on the effect target of traditional Chinese medicine and searching for related serum miRNA as a diagnostic marker or therapeutic target for sepsis.

脓毒症是机体对感染反应失调而导致危及生命的器官功能障碍，而胃肠道是多器官功能障碍中最易受损的靶器官，其治疗关键是如何保护肠黏膜上皮细胞屏障功能。前期研究发现，小檗碱能减轻内毒素诱导的大鼠肠黏膜上皮损伤，保护黏膜屏障，且在临床上具有良好疗效，但具体机制有待于研究。我们课题组提出假说：小檗碱作用于肠道局部，通过调控Smad3表达，诱导自噬增加，降低炎症反应，减轻脓毒症肠黏膜上皮细胞凋亡，从而抑制脓毒症后的器官损伤。本研究通过构建脓毒症大鼠模型及体外细胞实验来验证我们的假说。另外本研究通过血清外泌体基因芯片及回肠组织高通量测序技术，采用组间差异及聚类分析等生物信息学手段筛选出靶向调控Smad3的外泌体miRNAs，并进行体外验证。本研究从不同角度对通过调控Smad3诱导自噬的机制进行了深入探讨，以期为后续研究相关中药作用靶点，以及寻找相关血清miRNA作为脓毒症诊断标记物或治疗作用靶点奠定基础。

胃肠道是脓毒症诱发多器官功能障碍时最易受损的靶器官；同时胃肠功能障碍在脓毒症的发病过程也发挥了重要作用，是脓毒症的启动器官，因此如何保护肠黏膜上皮细胞及屏障功能，减轻胃肠功能障碍，是改善脓毒症预后的关键问题。前期研究发现，小檗碱能减轻内毒素诱导的大鼠肠黏膜上皮损伤，保护黏膜屏障，但具体机制有待于研究。我们课题组提出研究假说：小檗碱作用于肠道局部，通过调控Smad3表达，诱导自噬增加，降低炎症反应，减轻脓毒症肠黏膜上皮细胞凋亡，促进细胞存活，从而抑制脓毒症后的器官损伤。我们从三部分实验验证了我们的假说：首先，通过动物实验以及体外细胞实验，探讨小檗碱对脓毒症大鼠及人肠黏膜上皮细胞自噬及黏膜屏障功能的影响。结果显示，小檗碱干预后脓毒症大鼠血清及人肠上皮细胞IL-1β、IL-17及TNF-α水平明显降低，肠道通透性指标改善，肠粘膜屏障功能改善，电镜下自噬泡明显增多，自噬相关基因及蛋白LC3、Beclin-1表达明显升高，p62表达明显下降，证明了小檗碱可通过诱导自噬减轻脓毒症肠黏膜上皮损伤。第二部分实验通过建立Smad3(-/-)大鼠脓毒症肠黏膜损伤模型及Smad3干扰人肠上皮类细胞模型，验证小檗碱可通过调控Smad3诱导自噬。结果显示：Smad3敲出或干扰成功后，IL-1β、IL-17及TNF-α水平较小檗碱组明显升高，自噬相关基因及蛋白LC3、Beclin-1表达明显下降，p62表达明显升高，说明小檗碱可通过调控Smad3诱导自噬。最后，通过借助外泌体基因芯片扫描等技术，针对靶向调控Smad3的外泌体miRNA进行研究，筛选出了目的miRNA，并进行了体外细胞实验验证，证实了外泌体miRNA调控Smad3从而诱导自噬减轻脓毒症肠黏膜上皮损伤的作用机制。本研究从不同角度对脓毒症患者肠黏膜上皮细胞的损伤机制及小檗碱作用靶点进行深入研究，证实了小檗碱通过调控Smad3表达诱导自噬增加，降低炎症反应，减轻脓毒症肠黏膜上皮细胞凋亡，促进细胞存活，从而抑制脓毒症后的器官损伤。本研究为临床工作中应用中医药解决脓毒症胃肠功能障碍问题提供客观、可靠、科学的基础实验和理论依据。