troglitazone  (MedChemExpress)

Journal: iScience

Article Title: Trigonelline activates NRF2 and awakens dormant ovarian follicles to promote pregnancy in aging mice

doi: 10.1016/j.isci.2025.113717

Figure Lengend Snippet: TRG-activated primordial follicles through the NRF2/EGF/AKT signaling pathway (A) Photos of mouse ovaries cultured in medium supplemented with TRG (0.5 μM) and perifosine (100 μM) (TRG+perifosine) at the same time. Scale bars: 200, 100, and 25 μm. (B) Photos of mouse ovary sectioning showing follicles in tissue treated with TRG and perifosine, and histological analysis revealed a decline in the activation of primordial follicles in cultured ovaries from the TRG+perifosine group. Mean ± SE; one-way ANOVA and Tukey’s multiple comparison test; ∗∗∗∗ p < 0.0001, ∗ p = 0.0399, and ∗ p = 0.0138. (C) Levels of P-AKT at the 473-serine site in cultured ovaries from the control, TRG, and TRG+perifosine groups. Mean ± SE; one-way ANOVA, and Tukey’s multiple comparison test; ∗ p = 0.022, ∗∗∗ p = 0.0004, and ∗∗∗∗ p < 0.0001. (D) Levels of P-AKT protein in non-cultured ovaries and ovaries cultured for 6 h from the control and TRG groups. Mean ± SE; one-way ANOVA, and Tukey’s multiple comparison test; ∗p = 0.0153 and ∗p = 0.0406. (E) Levels of FOXO3a protein in ovaries cultured for 7 days from the control and TRG groups. Mean ± SE; two-tailed t test. (F) Levels of P-FOXO3a protein in ovaries cultured for 7 days from the control and TRG groups. Mean ± SE; two-tailed t test. (G) Levels of PTEN protein in ovaries cultured for 7 days from the control and TRG groups. Mean ± SE; two-tailed t test. (H) Levels of MTOR protein in ovaries cultured for 7 days from the control and TRG groups. Mean ± SE; two-tailed t test. (I) Levels and fold change of pS6K/S6K proteins in ovaries cultures for 6 h from the control and TRG groups. (J) Level of EGF protein in cultured ovaries from the control and TRG groups. Mean ± SE; two-tailed t test; ∗ p = 0.0350. (K and L) Level of P-AKT after EGFR inhibition using erlotinib (5 μM and 10 μM) and (L) schematic mode of action of TRG in primordial follicles. TRG induces the nuclear translocation of NRF2 and upregulates NRF-responsive genes, including Egf , leading to increased levels of the EGF protein. EGF then autocrinally activates its receptor, EGFR, leading to phosphorylation and activation of AKT. The activated P-AKT phosphorylates FOXO3a, governing the cytoplasmic localization of FOXO3A and removing its inhibitory effect on cell proliferation, leading to the activation of primordial follicles.

Article Snippet: Ovaries were removed from cervical dislocated F1 (C57Bl/6JRj × CBA/JRj) mice at the indicated ages, randomly separated into control and treatment groups and cultured on inserts (pore size of 0.4 μm, 6.5 mm diameter, Corning, MA, United States) in 24-well plates (Corning, MA, United States) at 37°C in a humidified atmosphere of 5% CO 2 –95% air. α-MEM (Fisher Scientific, Cat# 22571-020 (Gibco)) supplemented with 10% foetal bovine serum (FBS) Fisher Scientific, Cat# 11550356); 5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml sodium selenite (1× ITS; Fisher Scientific, Cat# 12097549); 1× penicillin–streptomycin solution (Thermo Fisher Scientific Cat# 15140122); and 100 mIU/ml rFSH (Merck, Cat# GONAL-F75IE-IU) were used, as described., , Treatment included 0.1-20 μM TRG (Merck, Cat# T5509) (optimal concentration 0.5 μM in saline (TRG)), 1-100 μM perifosine (Selleck Chem Cat# KRX-0401) (optimal concentration is 100 μM) and 5-10 μM erlotinib (Nordic Biosite Cat# 33-A8234) (optimal concentration is 5 μM), as indicated.

Techniques: Cell Culture, Activation Assay, Comparison, Control, Two Tailed Test, Inhibition, Translocation Assay, Phospho-proteomics