| Objective To observe the inhibitory effect of proanthocyanidins on autophagy of granulosa cells in rats with polycysticovary syndrome (PCOS), and to explore the regulatory effect of proanthocyanidins on p53 (p53)/adenosine monophosphate-activated protein kinase (AMPK) pathway.Methods Sixty adult female SD rats were assigned into N group, M group, P group, LD group, MD groupand HD group by the random digital table method, with 10 rats in each group. PCOS models were established except N group. Group Pwas given clomiphene citrate solution 5.21 mg/kg after successful modeling. LD group, MD group and HD group were given 25, 50, 100mg/kg proanthocyanidins respectively, while N group and M group were given the same amount of saline for 24 days. Serum sex hormone levels were compared before and after treatment. Histopathological changes of ovarian tissue were observed by hematoxylin-eosin (HE) staining. The autophagy rate of ovarian granulosa cells was measured by flow cytometry. Real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expressions of p53 and AMPK mRNA in ovarian tissues. The expressions of p53, microtubule-associated protein 1 light chain 3 (LC3 Ⅱ), AMPK protein and phosphorylation of AMPK (p-AMPK) were detected by Western blotting.Results After treatment, there was no significant difference in serum sex hormone level in group N in comparison with pre-treatment (P>0.05), while serum testosterone (T) and follicle-stimulating hormone (FSH) levels increased, and estradiol (E2) level decreased in group M, with statistically significant differences (P<0.05). Before treatment, serum T andFSH levels in groups M, P, LD, MD and HD were higher than those in N group, and E2 levels were lower than that in N group, with statistically significant differences (P<0.05). After treatment, the granulocyte autophagy rates in groups P, LD, MD and HD were (11.20±1.82)%, (13.45±2.10)%, (11.08±1.79)% and (9.86±1.05)% respectively. When comparing the levels of serum T and FSH and the granulocyte autophagy rates between any of the two groups, the orders from low to high were groups N, HD, MD/P, LD and M. Except thatthere was no statistically significiant difference between group MD and group P (P>0.05), there were statistically significant differences between any of the other two groups (P<0.05). When comparing the levels of serum E2 between any of the two groups after treatment,the orders from high to low were groups N, HD, MD/P, LD and M. Except that there was no statistically significant difference betweengroup MD and group P (P>0.05), there were statistically significant differences between any of the other two groups (P<0.05). In group N, follicles lined up neatly, with uniform size, clear structure and regular granular cells, reaching 8-9 layers. Cumulus oopholus couldbe seen in most of the follicles. The remaining 5 groups had different degrees of changes. Group HD was close to group N. There werestatistically significant differences in the expressions of p53 mRNA, p53, LC3Ⅱ protein and p-AMPK in ovarian tissues among all the groups(P<0.001). When comparing the expressions of p53 mRNA and p53 between any of the two groups, the orders from high to lowwere groups N, HD, MD/P, LD and M. Except that there was no statistically significant difference between group MD and group P (P> 0.05), there were statistically significant differences between any of the other two groups (P<0.05). When comparing the expressions of LC3 Ⅱ protein and p-AMPK, the orders from low to high were groups N, HD, MD/P, LD and M. Except that there was no significant difference between group MD and group P (P>0.05), there were statistically significant differences between any of the other two groups (P< 0.05). There were no statistically significant differences in the expressions of AMPK mRNA and protein among all the groups (P>0.05). Conclusions Proanthocyanidins can improve the level of sex hormones, alleviate pathological changes and inhibit autophagy of granulosa cells in PCOS rats. It is presumed that such influence can be achieved by regulating the p53/AMPK pathway, up-regulating the expressions of p53 gene and protein, inhibiting the phosphorylation of AMPK, and controlling the transformation of LC3 Ⅰ to LC3 Ⅱ. |
