Fig. 3

Resveratrol-βcd can maintain the function of granulosa cells during POI progression. (A) On day 14 post treatment, primary ovarian granulosa cells were isolated and stained for FSHR using the indirect immunofluorescence method, with green fluorescence representing FSHR and blue fluorescence representing cell nuclei. The imaging parameters were consistent for all fields of view, scale bar = 40 μm. The mean fluorescence intensity (MFI) values of green fluorescence in three random fields were statistically analyzed (B); C. Typical western blot result and statistical analysis of SQSTM1 (D) and FSHR (E) expression levels in primary granulosa cells, n = 3; (F) The indirect immunofluorescence method was used to measure the level of LC3 in primary granulosa cells from different groups, scale bar = 40 μm; (G) A total of 1 × 10⁵ primary granulosa cells were cultured in a 12-well plate with phenol red-free DMEM/F12 medium (containing 10% charcoal-stripped fetal bovine serum), and 10 µM testosterone and 1 IU FSH were added. After 48 h of culture, the level of E2 in the supernatant was detected using the ELISA method, n = 5; (H) The lysosomal inhibitor bafilomycin A1 (BafA1) (100 nM) and 5 µM resveratrol-βcd were incubated alone or together with primary granulosa cells for 12 h, and western blotting was used to detect the expression levels of SQSTM1 and LC3. Statistical analysis is shown (I), n = 3. (J) A total of 1 × 10⁵ primary granulosa cells was cultured in a 12-well plate and treated with the indicated concentrations of resveratrol-βcd. After 24 h of culture, 10 µM testosterone and 1 IU FSH were added. After 48 h of culture, the level of E2 in the supernatant was measured, n = 5. Statistical analysis was performed using one-way ANOVA (B,D,E,G,I and J comparison was made POI vs. control, POI vs. resveratrol-βcd and POI vs. POI + resveratrol-βcd; ns, not significant