Lipoxin A4 inhibits the invasion and migration of endometrial stromal cells by down-regulating NF-κB signaling-mediated autophagy

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.