Hy 103456

Four weeks after treatment, 60 rats (n = 20 per group) were randomly selected and equally divided into ERα group and ERβ group. Five rats were randomly selected from the SHAM group and OVX + E group of ERα group, the antagonists of ERα (AZD9496, HY-12870, MedChemExpress) were injected into POA, and the agonists of ERα (Propyl pyrazole triol, HY-100689, MedChemExpress) were injected into POA of the five rats randomly selected from the OVX group. Five rats were randomly selected from SHAM group and OVX + E group of ERβ group to inject antagonists of ERβ (PHTPP, HY-103456, MedChemExpress) into POA, and five rats from OVX group were randomly selected to inject agonists of ERβ (Liquiritigenin, HY-N0377, MedChemExpress) into POA. Other rats were given the same volume of cosolvent (10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline, MedChemExpress) in POA. After 72 h of drug injection, the POA was removed according to the above method, and the mRNA and protein expression of Vglut2 and Vgat in POA were detected by Western blot and qRT-PCR, respectively. The details on the primary and secondary antibodies of vglut2 and vgat are shown in Supplementary Table 1.1, and the details on the primers of vglut2 and vgat are shown in Supplementary Table 1.2.

The Ishikawa and RL95-2 cells were treated with estrogen (10⁻⁷ M, HY-B070, MedChem Express, NJ, USA), succinate (5 mM, Sigma-Aldrich; Merck KGaA, SL, MO, USA), ERα antagonist (MPP, 10⁻⁶ M, HY-103454, MedChem Express, Monmouth Junction, NJ, USA), ERβ antagonist (PHTPP, 10⁻⁶ M, HY-103456, MedChem Express, Monmouth Junction, NJ, USA), total ER antagonist Fulvestrant (ICI182780, 10⁻⁶ M, HY-13636, MedChem Express, Monmouth Junction, NJ, USA), and heme (0–100 μM, HY-19424, MedChem Express, Monmouth Junction, NJ, USA).
Both cell lines were seeded in 12-well plates at a density of 2.5 × 10⁵ cells/mL and cultured in DMEM/F12 medium (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS-charcoal-stripped (Gibco Cell Culture, Carlsbad, CA, USA) and 1% penicillin–streptomycin (HyClone, UT, USA). The relevant cells were cultured under 5% CO₂ in a 37 °C humidified incubator (Heal Force HF-90, Shanghai, China) for 48 h.