Mitigating Pro-Inflammatory SASP and DAMP With Urolithin A: A Novel Senomorphic Strategy

Abstract

Senescent cells are known to contribute to aging and age-related diseases. One key way they influence aging is by secreting senescence-associated secretory phenotype (SASP) factors along with several damage-associated molecular pattern (DAMP) molecules. Consequently, inhibiting SASP and DAMP signaling (senomorphics) has emerged as a therapeutic strategy. Urolithin A (UA), a gut-derived metabolite produced from ellagitannins and ellagic acid found in berries, nuts, and pomegranates, has demonstrated potent anti-inflammatory properties and protective effects against aging and age-related conditions in experimental models. Here we demonstrate that UA lowers the expression and release of pro-inflammatory SASP and DAMP factors, at least in part, by downregulating cytosolic DNA release and subsequent decrease in cGAS-STING signaling.

Keywords: Urolithin A; cGAS‐STING signaling; cytosolic DNA; senescence; senomorphic.

FIGURE 1

Doxorubicin treatment induces senescence in human IMR‐90 fibroblasts. (A) Representative images of SA‐βGal‐stained S‐dox and NS with or without the addition of Urolithin A (UA). (B) The proportion of cells staining positive for SA‐βGal in NS and S‐dox cells with and without the UA treatment. Four fields were quantified per well (n = 3). (C, D) Relative p16 (C) and p21 (D) expression in NS, S‐dox, and RS cells with and without UA treatment, n = 4. (E) IF staining for γ‐H2AX and HMGB1 in NS, S‐dox, and RS cells with or without the addition of UA, 10 days post‐induction. γ‐H2AX—green, HMGB1—red, and DAPI—blue. Scale bar = 30 μm. (F, G) The percentage of cells with two or more γ‐H2AX foci per cell (F) and with nuclear HMGB1 (G). (H–J) Fold change of IL6 (H), IL8 (I), and IL1α (J) gene expression in S‐dox and RS cells relative to NS control. n = 4. All data are presented as mean ± SEM. One‐way ANOVA. **p < 0.002, ***p < 0.0002 ****p < 0.0001.

FIGURE 2

UA decreases paracrine senescence through downregulation of c–GAS–STING signaling. (A, B) ELISA on conditioned media collected from NS, S‐dox, and RS cells with and without UA treatment after 24 h of incubation. IL‐6 (A) and IL‐8 (B) cytokine concentrations were normalized to 100,000 cells, n = 3. (C, D) Relative gene expression of CCL2 (C) and CCL5 (D) in NS, S‐dox, and RS cells with or without UA treatment, n = 3. (E) Proportion of SA‐βGal‐positive cells 6 days after they have been treated with media collected from NS, S‐dox, or RS cultured with or without UA for 6 days, n = 3. (F) Proportion of cells containing extranuclear pico green (PG)—stained DNA foci in NS and S‐dox cells with or without UA treatment. n = 3. (G) Quantification of the pSTING staining intensity in the NS and S‐dox cells, with and without UA treatment, n = 3. (H–J) Fold change in IL6 (H), IL8 (I), IL1α (J) gene expression in NS and S‐dox cells treated with UA, 9‐NO, or a combination of both. n ≥ 3. All data are presented as mean ± SEM. One‐way ANOVA. *p < 0.033, **p < 0.002, ****p < 0.0001.