A novel senotherapeutic strategy with azithromycin for preventing endometriosis progression.

Abstract

Background: Endometriosis is an estrogen-dependent chronic inflammatory disease, however the mechanisms underlying inflammation remain unclear. Non-hormonal drugs that can prevent endometriosis progression and resolve endometriotic infertility are urgently required. We thus focused on cellular senescence as a novel feature of endometriosis. Senescent cells cause chronic inflammation via the senescence-associated secretory phenotype (SASP) factor. It has been reported the effects of senolysis for various diseases in recent years. The aim of this study was to validate the involvement of cellular senescence in endometriosis and as the effects of senolytic drug to develop a novel non-hormonal therapeutic strategy for endometriosis.

Methods: The senescence markers were assessed by morphological features and semiquantitative immunofluorescence staining (senescence-associated b-galactosidase [SA-b-Gal], the cyclin-dependent kinase inhibitor 2 A locus [p16^INK4a], and laminB1) to compare among cell types (normal endometrial stromal cells [nESCs], endometrial stromal cells with endometriosis [eESCs], and ovarian endometriosis [OE] cyst-derived stromal cells [CSCs]). Expression of SASP markers was examined in cell culture supernatants using a cytokine array. In addition, the effects of senolytic drugs (azithromycin [AZM] and navitoclax [ABT263]) on endometriosis were evaluated in vitro and in vivo. The in vivo study used the endometriosis mice model.

Results: CSCs exhibited stronger senescence markers. Semi-quantitative SA-β-Gal and p16^INK4a staining intensities were significantly increased, and that of LaminB1 was decreased in CSCs compared to those in nESCs and eESCs (SA-b-Gal, P < 0.001; p16^INK4a, P < 0.05; LaminB1, P < 0.05). Cytokine array analysis revealed elevated SASP-related cytokine levels, including interleukin-6 (IL-6), in CSC supernatants compared to those in nESCs. AZM and ABT263 reduced the viable fraction in CSCs (AZM: P < 0.001, ABT263: P < 0.01). Furthermore, AZM suppressed IL-6 expression in CSC culture supernatants (P < 0.05). In murine model, AZM administration reduced endometriotic lesion volume compared to that in vehicle (P < 0.05). Proliferative activity, IL-6 expression levels, and fibrosis within endometriotic lesions also decreased (Ki67, P < 0.01; IL-6, P < 0.001; fibrosis, P < 0.001).

Conclusions: Our findings show that cellular senescence is involved in the pathogenesis of endometriosis and that AZM may be useful for preventing endometriosis progression by suppressing the secretion of IL-6 as a SASP.