17 β estradiol 3 benzoate

Thirty adult C57BL/6 female mice (6–8 weeks, weight 20 ± 2 g) were purchased from the Laboratory Animal Facility of Fudan University and used for this study. Animal protocols were approved by the Ethics Committee of the Obstetrics and Gynecology Hospital of Fudan University. All mice were randomly assigned to one of the three groups. Intraperitoneal EMs-like lesions were surgically induced by injecting fragments of the uterine tissue into the peritoneal cavity. Each postoperative mouse received 17-β-Estradiol-3-benzoate (30 μ g/kg, Sigma) every 3 days for 14 days. Three days after surgery, each mouse in the experimental group received succinate (100 mg/kg, Sigma) intraperitoneally every 3 days for 14 days. PBS was used instead of succinate in the sham group. In the control group, no surgery was performed and PBS was used instead of succinate. Fourteen days after the operation, endometrial-like lesions were established, the mice were sacrificed, and the peritoneal lavage fluids and ectopic lesions were harvested. SUCNR1 and M1/M2 macrophage markers were measured and analyzed via FCM. MMP9 and ICAM-1 levels in the lesions were detected through IHC.

To assess the effect of estradiol on endogenous ghrelin levels, a cohort of females was divided into intact (n = 12) and OVX female groups (n = 24). Half of the OVX animals received estradiol replacement. Replacement began 4 days after surgeries. 17β-Estradiol-3-benzoate was purchased from Sigma Aldrich, dissolved in sesame oil, and administered subcutaneously at a dose of 2μg/100μl/animal every 4th day over a period of three months to mimic the natural fluctuations of the estrous cycle. Intact females and OVX controls received vehicle only. The estradiol replacement protocol was based on the work of Asarian and colleagues (25 (link)), where it was previously shown to successfully restore the body weight of OVX rats to the level of intact animals.

All animal handling and experimental procedures were approved by the Animal Experimental Ethics Committee of Harbin Medical University. A mouse model of endometriosis was established as previously described [30 (link)]. Seven-to-8-week-old C57BL/6 female mice were obtained and 17-β-estradiol-3-benzoate (30 µg/kg, Sigma) was administered to each mouse every day for 3 days. We removed uterine horns from the donor mice and added them to saline. Endometrium was cut into 1 mm² fragments. The endometrial fragments from each uterine horn were suspended in 0.3 ml saline and injected into the peritoneal cavities of recipient mice with an 18-gauge needle. At 8 days (5 days after the operation), endometrial-like lesions were established, and they were randomly divided into two groups (each group contained 12 mice). In the experimental group, each mouse received erastin (20 mg/kg/day) by intraperitoneal injection over a 7-day period. In the control group, DMSO was used instead of erastin. At 15 days, the mice were sacrificed and endometriotic lesions were collected (Fig. 7A). The length and width of ectopic lesions were measured and the volumes of lesions were calculated by the prolate ellipsoid geometric model: 1/2 (length × width²).