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Title: [Lipoxin A4 inhibits the invasion and migration of endometrial stromal cells by down-regulating NF-κB signaling-mediated autophagy].
Author: Li YH, Geng YY, Liu L, Chen CY, Gao Y.
Journal: Zhonghua Fu Chan Ke Za Zhi; 2018 Aug 25; 53(8):547-553. PubMed ID: 30138965.
Abstract:
Objective: To investigate whether the suppressive effects of lipoxin A4 (LXA4) on endometriosis are mediated by the regulation of autophagic activity, and to further explore the actual molecular mechanism. Methods: (1) Eutopic and ectopic endometria were obtained from 13 patients with endometriosis, and 10 eutopic endometria collected from non-endometriosis patients were used as control. The expression of the autophagy-related biochemical markers [microtubule-associated protein 1 light chain 3 (LC3) and p62] were detected by western blot. Levels of LXA4 in the biopsies were measured by ELISA. (2) Primary human endometrial stromal cells (ESC) were isolated and cultured in vitro from eutopic endometria of infertility patients with endometriosis. After treatment with exogenous LXA4 or autophagy inhibitor 3-methyladenine (3-MA) or autophagy inducer rapamycin, cell migration and invasion were evaluated by transwell assay, and autophagy was detected by western blot. (3) ESC were treated with LXA4, the gene expressions of nuclear factor kappa B (NF-κB) etc. were examined by quantitative real-time PCR, and the activation of NF-κB signaling was detected by western blot. (4) ESC were incubated with 10 μmol/L NF-κB inhibitor BAY11-7080, the autophagic activation was detected by western blot. Results: (1) Autophagy-related marker, LC3-Ⅱ and LC3-Ⅱ/LC3-Ⅰ ratio, showed a significant up-regulation in ectopic lesions of endometriosis compared with eutopic endometria of affected or healthy women (all P<0.05) . However, the LXA4 level significantly decreased in ectopic tissue (P<0.05) . There was a significant negative correlation between LXA4 concentration and relative expression of LC3-Ⅱ in ectopic lesions (r= -0.780, P=0.002) . (2) 10 and 100 nmol/L exogenous LXA4 could significantly down-regulate the LC3-Ⅱ protein expression and up-regulate the p62 protein expression (all P<0.05) . LXA4 markedly inhibited the invasion and migration of ESC (P<0.05) ;while the reactivation of autophagy by rapamycin almost reversed the anti-invasion and anti-migration effects of LXA4. (3) After LXA4 treatment, the expression level of NF-κB gene significantly decreased (P<0.05) . Furthermore, the results of western blot analysis showed that the nuclear translocation of NF-κB p65 was markedly down-regulated under LXA4 treatment (P<0.05) . (4) The NF-κB inhibitor BAY11-7080 markedly suppressed the autophagic activation of LXA4 (P<0.05) . Conclusion: LXA4 could inhibit the invasion and migration of ESC by down-regulating the NF-κB signaling-mediated autophagy. 目的： 探讨脂氧素A4（LXA4）下调自噬活性对子宫内膜异位症（内异症）患者子宫内膜间质细胞（ESC）侵袭和迁移的影响，并进一步探索其分子作用机制。 方法： （1）收集2015年10月至2016年6月在华中科技大学同济医学院附属协和医院行腹腔镜手术的内异症患者共13例的在位内膜及异位病灶组织标本，以及非内异症患者共10例的子宫内膜组织标本；采用蛋白印迹法检测组织中自噬活性标志蛋白[微管相关蛋白轻链3（LC3）、p62]的表达，ELISA法测定组织中LXA4的浓度，并对异位病灶组织中LXA4的浓度与LC3-Ⅱ蛋白的表达水平进行相关性分析。（2）收集拟行体外受精-胚胎移植治疗的合并不孕的内异症患者的在位内膜组织，经体外分离培养获得ESC；外源性LXA4、自噬抑制剂3-甲基腺嘌呤或自噬诱导剂雷帕霉素干预ESC，细胞迁移和侵袭实验分析细胞的迁移和侵袭能力，蛋白印迹法检测细胞中LC3、p62蛋白的表达。（3）LXA4干预ESC后，逆转录实时定量荧光PCR技术检测自噬活性调控相关基因的表达水平，蛋白印迹法分析核因子κB（NF-κB）信号通路相关分子的表达。（4）NF-κB信号通路阻断剂BAY11-7080干预ESC，蛋白印迹法检测LC3、p62蛋白的表达。 结果： （1）与内异症患者及非内异症患者的在位内膜组织比较，内异症异位病灶组织中LC3-Ⅱ蛋白的表达水平（0.29±0.04）及LC3-Ⅱ/LC3-Ⅰ比值（0.43±0.03）均显著增加（P均<0.05）；而异位病灶组织中LXA4浓度却显著低于内异症患者和非内异症患者的在位内膜组织[分别为（20.6±5.7）、（39.1±8.7）和（44.3±9.3）ng/L；P均<0.05]。异位病灶组织中LC3-Ⅱ蛋白的表达水平与LXA4浓度呈显著负相关（r=-0.780，P=0.002）。（2）10、100 nmol/L的LXA4干预后ESC中LC3-Ⅱ蛋白的表达水平显著下调、p62蛋白的表达水平显著上调（P均<0.05）。LXA4干预能显著抑制ESC的迁移和侵袭能力，ESC的迁移细胞数及侵袭细胞数均明显减少（P均<0.05），而自噬诱导剂雷帕霉素预处理ESC后，可逆转LXA4的抗细胞迁移和侵袭作用，迁移细胞数及侵袭细胞数有差异（P均<0.05）。（3）LXA4干预后显著下调了ESC中NF-κB基因的表达水平（P<0.05）。LXA4干预也显著抑制了ESC细胞核内NF-κB p65蛋白的表达水平（P<0.05）。（4）NF-κB信号通路阻断剂干预ESC后可模拟外源性LXA4的干预效应，导致ESC中LC3-Ⅱ蛋白的表达水平显著降低（P<0.05）。 结论： LXA4可通过下调NF-κB信号通路介导的自噬活性抑制ESC的侵袭和迁移，进而发挥其抗内异症发生、发展的作用。.

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