SIRT3通过抑制氧化应激介导雌激素保护食管粘膜屏障的机制

Estrogen and oxidative stress are associated with reflux esophagitis (RE) and its complications. It is reported that estradiol (E2) protected esophageal mucosa via antioxidant, however, the mechanism remains unclear. Binding of E2 to the receptor (ER) upregulates SIRT3. SIRT3 directly activates mitochondrial superoxide dismutase (MnSOD) or indirectly up-regulates MnSOD by activating FOXO3a through its deacetylase activity. MnSOD is a key enzyme regulating mitochondrial reactive oxygen species (mtROS). Our previous study confirmed that E2 inhibited mtROS and mucosal injury-induced by bile acid, and up-regulated SIRT3 in esophageal epithelial cells. We hypothesized that E2 activated MnSOD through the direct or/and indirect action in combination with ER. E2 inhibited mtROS and protected esophageal mucosa via antioxidant. Therefore, in the animal models and cell experiments, our study propose to verify sirt3-mediated E2 inhibition of mtROS induced by bile acid and protection of esophageal epithelial cell barrier through activation of MnSOD. Our study provides a potential targets for intervention in RE and its complications.

雌激素和氧化应激与反流性食管炎（RE）及并发症发病相关。雌二醇（E2）通过抗氧化作用保护食管粘膜，但机制不明。E2与受体（ER）结合可上调SIRT3，而SIRT3通过去乙酰化酶作用直接活化超氧化物歧化酶（MnSOD）或活化FOXO3a间接上调MnSOD，MnSOD是抑制线粒体活性氧自由基（mtROS）的关键酶。我们前期实验发现E2可抑制胆汁酸诱导的食管上皮细胞mtROS和屏障损伤、并上调SIRT3。因此提出假设：E2结合ER，上调SIRT3、通过直接或/和间接作用活化MnSOD、抑制mtROS、通过抗氧化作用而保护食管粘膜。本项目拟通过动物模型和细胞实验：探讨SIRT3通过活化MnSOD介导E2抑制胆汁酸诱导的mtROS从而保护食管上皮细胞屏障的分子机制，为RE及并发症的治疗新靶点提供依据。

研究背景：雌二醇(E2)通过抗氧化作用保护食管粘膜，但机制不明。文献中，MnSOD是关键的超氧化物清除剂；SIRT3是抗氧化和能量代谢的关键调控因子，通过去乙酰作用调控MnSOD的活性。.研究内容：通过动物模型和细胞实验，探讨SIRT3是否通过去乙酰化作用调控MnSOD的活性，进而减少胆汁酸（BA）诱导的氧化应激（ROS），介导E2的食管粘膜保护作用。.研究结果：1. 大鼠大体标本，反流性食管炎（RE）组食管粘膜见明显充血、糜烂性和溃疡，而RE+E2组食管粘膜轻度充血、仅散在糜烂；HE染色，RE+E2组炎症比RE组明显减轻；E2组大鼠食管粘膜SIRT3表达高于假手术组；RE+E2组Ac-K122-MnSOD表达明显低于RE组；免疫组化，RE组ERβ分布胞浆中，而RE+E2分布胞核中。2. BA刺激正常食管上皮细胞（Het-1a），随着时间延长，TER值逐渐下降而FITC-dextran浓度逐渐增加，而E2可改善此现象。3. BA诱导Het-1a细胞内ROS浓度明显增多，线粒体氧化酶和NADPH氧化酶抑制剂预处理后，细胞内线粒体ROS被抑制更明显、TER升高、FITC-dextran浓度降低。4. E2明显上调SIRT3表达和活性、下调Ac-K122-MnSOD表达和上调SOD活性。5. E2干预条件下，SIRT3 siRNA处理后，上调Ac-K122-MnSOD表达并下调SOD活性，但不影响总MnSOD表达、以及减弱E2抑制BA诱导的ROS和保护屏障功能的作用。6.与对照组相比，ERβ激动剂处理组，SIRT3表达明显升高，而ERβ siRNA转染组及Sp1 siRNA+ERβ激动剂组，SIRT3表达明显下降。.研究结论：E2通过ERβ和Sp1促进SIRT3活化，而SIRT3通过直接活化MnSOD介导E2抑制BA诱导的ROS从而保护食管粘膜。本研究加深对RE机制的理解；SIRT3选择性激动剂可能作为RE及并发症新的治疗新靶点。