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.

Keywords: Azithromycin; Cellular senescence; Endometriosis; IL-6; SASP; Senotherapy.

Fig. 1

AZM prevents endometriosis progression in murine models. (A) Our experimental protocol is shown. Four weeks after uterine tissue transplantation, recipient mice were treated with 50 mg/kg AZM twice weekly. (B-D) The numbers of endometriosis lesions were comparable in both groups, while volume per endometriosis lesion was reduced in the AZM group. (E) Representative Ki67 immunohistochemical images of endometriosis lesions. Scale bar: 100 μm. (F) Percentages of Ki67-positive cells in endometriosis lesions decreased in the AZM group in both epithelial and stromal cells. (G) Representative Masson trichrome staining analysis results of endometriosis lesions. Scale bar: 50 μm. (H) Fibrotic lesions within endometriosis cysts were significantly reduced in the AZM group. (I) Representative images of IL-6 immunohistochemistry in endometriosis lesions. (J) IL-6-positive areas in stromal cell lesions were significantly decreased in the AZM group (n = 6 in each group). AZM, azithromycin. Data are shown as means ± SEM. Data were analyzed by the Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant

Fig. 2

CSCs display characteristics of cellular senescence. (A) Representative images of nuclear swelling. Scale bar: 20 μm. (B) Representative images of cytoplasmic expansion. Scale bar: 200 μm. (C) The results of nuclear area quantitative analyses are shown in a box plot (n = 5 in each group). (D) The results of cytoplasmic area quantitative analyses are shown in a box plot (n = 5 in each group). (E) Representative SA-β-Gal staining images. Scale bar: 200 μm. (F) The proportion of SA-β-Gal staining positive cells in each group is shown (nESCs; n = 9, eESCs; n = 8, CSCs; n = 16). (G) Quantitation of SPiDER-β-Gal intensity is shown (n = 5 in each group). (H) Representative immunocytochemistry images of senescence markers; p16^INK4a, laminB1, and IL-6. Scale bar: 20 μm. Quantitative analysis is shown in (I) (n = 5 in each group). (J) Supernatant IL-6 concentration is shown in the box plot (n = 5 in each group). nESCs, normal endometrial stromal cells; eESCs, endometrial stromal cells with ovarian endometriosis (OE); CSCs, OE (chocolate cyst)-derived stromal cells; SA-β-Gal, senescence-associated β-galactosidase. (C, D and J) Horizontal lines centered within the boxes indicate median values. The bottom and top of the box indicate the 25th and 75th percentiles, respectively. The whisker ends indicate the respective minimum and maximum for all data. (F, G and I) Data are shown as means ± SEM. Data were analyzed by a one-way ANOVA (F and G) and the Student’s t-test (I). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; n.s., not significant

Fig. 3

Effects of AZM and ABT263 on cell viability and IL-6 secretion in CSCs. (A) AZM treatment reduces the viable cell fraction in CSCs (n = 5 in each group). (B) ABT263 treatment reduces the viable cell fraction in CSCs (n = 5 in each group). (C) Representative cytokine array (n = 4 in each group) images (upper panels), quantification of intensity of each spot (middle table). (D) The ratio of supernatant IL-6 concentrations before vs. after AZM treatment is shown in box plot form (n = 5 in each group). (E) The ratio of IL-6 concentrations before vs. after AZM treatment is shown in box plot form (n = 5 in each group). nESCs, normal endometrial stromal cells; eESCs, endometrial stromal cells with ovarian endometriosis (OE); CSCs, OE (chocolate cyst)-derived stromal cells; AZM, azithromycin; ABT263, navitoclax. (A and B) Data are shown as means ± SEM. (D and E) The horizontal line centered inside each box indicates the median. The bottom and top of the box indicate the 25th and 75th percentiles, respectively. The ends of the whiskers indicate the minimum and maximum of all the data. Data were analyzed by the Student’s t test. ∗P < 0.05; ∗∗P < 0.01; n.s., not significant