Fig. 7

Acetylsalicylic acid treatment markedly inhibits multiple beneficial functions of endometrial stem cells in vivo. A schematic representation of the experimental procedure as described in the Materials and Methods section is shown (A). The mice were treated intravenously with acetylsalicylic acid (50 mg/kg daily for 7 consecutive days), and the endometrial stem cells were isolated from the mouse uterine endometrium using our primary culture technique. The isolated endometrial stem cells were cultured in vitro either under continuous exposure to acetylsalicylic acid (2.5 mM) or in non-acetylsalicylic acid culture conditions to mimic the physiological environment of acetylsalicylic acid exposure in vivo. The subsequent inhibition of the self-renewal capacity of mouse endometrial stem cells was analyzed by MTT assays. The stem cell proliferation rates (%) were assessed by representing the viability of the acetylsalicylic acid-treated cells as a percentage of the viability of the vehicle-treated cells (B). The acetylsalicylic acid -induced inhibition of migratory potential in vivo was then analyzed by Transwell migration/invasion assays (C) and western blotting with antibodies against MMP-2 and MMP-9 (D). The acetylsalicylic acid-induced inhibition of adipogenic (E) and osteogenic (F) differentiation in vivo were analyzed by oil red O and alizarin red staining, respectively. The cytoplasmic calcium concentration and lipid droplet (LD) formation within differentiated cells were assessed by measuring the absorbance values of the solubilized cells at wavelengths of 500 nm and 570 nm, respectively. The acetylsalicylic acid-induced inhibition of the expression of various pluripotency/stemness markers (NANOG, OCT4, and SOX2) in vivo was evaluated using real-time PCR (G). Mice from each group were sacrificed by cervical dislocation. Uterine endometrial tissue samples from acetylsalicylic acid-treated mice were collected and fixed in 10% formalin for hematoxylin and eosin (H&E) staining. The histological evaluation revealed that the functional layer of the mouse endometrium was significantly decreased by acetylsalicylic acid treatment in vivo (H). β-actin was used as an internal control to normalize protein expression. All experiments were performed in triplicates. HPRT was used as a reference gene to normalize gene expression. Significant differences are presented. *p < 0.05, **p < 0.005, and ***p < 0.001 (One-way ANOVA)