基于Keap1-Nrf2-ARE通路维持黏膜屏障完整探讨舒胃汤防治胃溃疡的研究

Study on Shuwei decoction prevention and treatment of gastric ulcer by regulating Keap1-Nrf2-ARE signal pathway to maintain the integrity of gastric mucosa

目的通过观察胃溃疡患者治疗前后Kelch样环氧氯丙烷相关蛋白1-核转录相关因子2-抗氧化反应元件(Keap1-Nrf2-ARE)通路中相关指标及胃黏膜超微结构变化,探讨舒胃汤防治胃溃疡可能机制。方法采用自身前后历史对照设计,选择符合诊断标准、纳入标准及排除标准的胃溃疡患者30例,予舒胃汤连续干预6周,应用透射电镜观察治疗前后胃黏膜超微结构及上皮细胞凋亡情况,应用荧光定量PCR技术检测Keap1、Nrf2、ARE基因表达情况,应用免疫组化法检测ZO-1及Occludin蛋白表达情况。结果治疗前,电镜下可见胃黏膜上皮细胞微绒毛稀疏甚至脱落消失,细胞间复合连接松解消失,核固缩,出现核周隙,异染色质块状分布于核膜下;线粒体肿胀变大,嵴断裂;粗面内质网扩张,细胞处于坏死期,存在细胞大量自噬现象。治疗后,电镜下可见胃黏膜上皮细胞体积明显变小,核染色质边集、核固缩,核膜断核碎裂,细胞器明显扩张肿胀、碎裂,空泡化,凋亡小体形成,亦存在细胞自噬现象。治疗后,患者胃黏膜组织中Keap1、Nrf2、ARE表达水平,ZO-1及Occulidin蛋白阳性分布面积均明显高于治疗前(P均<0.05)。结论舒胃汤可能通过调控Keap1-Nrf2-ARE通路,减少氧化应激损伤,上调ZO-1及Occludin蛋白表达,促进黏膜屏障修复而发挥防治胃溃疡的作用。

Objective It is to explore the prevention and treatment mechanism of Shuwei decoction on gastric ulcer by observing the changes of Keap1- Nrf2- ARE signal pathway and the ultra structural in gastric mucosa before and after treatment. Methods It was a history control design trial. Thirty patients who conformed to diagnostic criteria,inclusion criteria and exclusion criteria were treated with Shuwei decoction for 6 weeks. The gastric mucosa ultra structure and apoptosis of epithelial cells were observed by electron microscope. The Keap1,Nrf2 ARE gene expression was detected by the fluorescence quantitative PCR. The expression of Occludin and ZO- 1 protein was detected by immunohistochemistry. Results Before treatment,gastric ulcer patients under electron microscope visible gastric mucosa epithelial microvillus sparse even shedding vanished,intercellular composite connections were loose or disappeared,nuclear pyknosis,nuclear week gaped,heterochromatin block distribution under nuclear membrane; mitochondrial swelling became larger,cristae fractured; rough endoplasmic reticulum expanded,cells were in the necrotic phase,there were a large number of cells autophagy. After treatment,gastric ulcer patients under electron microscope visible gastric mucosa epithelial cell volume became small,nuclear chromatin margination,nuclear pyknosis,nuclear membrane broken nuclear fragmentation,organelles marked dilation of the swelling,fragmentation,vacuolization,formation of apoptotic bodies,autophagy was also found. After treatment,the levels of Keap1,Nrf2,ARE expressions in gastric mucosa tissue,the positive distribution of ZO- 1 and Occulidin protein were significantly higher than that before treatment（ P〈0. 05）. Conclusion Shuwei decoction exerts a distinct effect on curing gastric ulcer by regulating Keap1-Nrf2- ARE pathway,reducing oxidative stress injury,up- regulating the expression of ZO- 1 and Occludin protein.