Fig. 5

Resveratrol-βcd synergistically promotes autophagy in ovarian granulosa cells via the IL-6/sIL-6R pathway. (A) IL-6R levels on the surface of ovarian granulosa cells and primary macrophages; (B) After stimulating primary macrophages and ovarian granulosa cells with different concentrations of resveratrol-βcd for 24 h, culture medium was collected, and the levels of sIL-6R in the supernatant were detected using ELISA, n = 3; (C) Typical western blot result of the activation of JAK2 and protein levels of autophagy markers SQSTM1 and LC3 under different culture conditions, LC3-II/LC3-I and statistical analysis is shown in (D), n = 3; E. Typical images of primary granulosa cells transfected with mRFP-mGFP-LC3 lentivirus and cultured with different treatments for 24 h; (F) Quantification of the number of mRFP-mGFP-LC3 puncta from (E) and the ratio of autolysosomes (red, mRFP + GFP-) to autophagosomes (yellow, mRFP + GFP+) (G), n = 40 for each group. (H) Typical western blot results of SQSTM1 and LC3 under different culture conditions. LC3-II/LC3-I statistical analysis is shown in (I), n = 3. (J) After coculturing with macrophages and/or resveratrol or with anti-IL-6 antibody (K), the level of E2 in the supernatant of primary granulosa cells was measured after 48 h, n = 5. Statistical analysis was performed using one-way ANOVA, comparison was made 0 µM resveratrol-βcd vs. 1 µM, 5 µM or 10 µM resveratrol-βcd in B; comparison was made 12 h post IL-6 mono-treatment vs. each other groups in D; comparison was made resveratrol-βcd mono-treatment group vs. each other groups in F, G, J, K; comparison was made macrophage + BafA1 group vs. each other groups in I; ns, not significant