Effect of Guizhi Fulingwan on Ovulation Dysfunction in PCOS-IR Rats by Regulating PI3K/Akt/mTOR Pathway / 中国实验方剂学杂志

ABSTRACT

Objective:To investigate the effect of Guizhi Fulingwan on ovulation dysfunction in rats with polycystic ovary syndrome with insulin resistance （PCOS-IR） induced by letrazole combined with high fat emulsion. Method:A total of 72 female SD rats were randomly divided into control group， model group， metformin group and Guizhi Fulingwan low， medium and high dose groups， with 12 rats in each group. Except for control group， rats were given letrozole 0.001g·kg^-1 combined with high-fat emulsion 15 mL·kg^-1 for 21 consecutive days to establish model of PCOS-IR. Guizhi Fulingwan low， medium and high-dose groups were administrated with Guizhi Fulingwan 0.31， 0.62， 1.24 g·kg^-1 respectively， metformin group was administrated with metformin 0.27 g·kg^-1， control group and model group were administrated with 12 mL·kg^-1 of normal saline daily for 30 days. Hematoxylin-eosin（HE） staining was used to observe ovarian tissue pathology morphology， and enzyme-linked immunoassay method （ELISA） was used to detect serum follicle stimulating hormone （FSH）， luteinizing hormone （LH）， testosterone （T）， fasting insulin （FINS） level，and LH/FSH and insulin resistance index （HOMA-IR） were calculated． Western blot was used to detect the expression levels of autophagy key molecular Atg6 yeast homologue （Beclin-1）， autophagy related gene 5（Atg5）， microtubule associated protein light chain 3 （LC3） Ⅱ proteins in the phosphatidylinositol 3-kinase/protein kinase B/rapamycin target protein （PI3K/Akt/mTOR） signaling pathway and autophagy related indicators in rat ovarian tissue. Beclin-1 and LC3Ⅱ protein expressions were detected by immunohistochemistry （IHC）. Result:Compared with control group， the thickness of follicles and follicular granulosa cells in the ovary of the model group also decreased， and the number of corpus luteum significantly decreased， while the white membrane thickness of the ovary increased， and the number of atresia follicles and cystic dilatation follicles increased significantly. Serum T， LH， LH/FSH， FINS， FINS， HOMA-IR were significantly increased （<italic>P</italic><0.01）. Phosphorylated （p） -PI3K， p-Akt， and p-mTOR proteins in ovarian tissue were all decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）. The relative expression levels of autophagy-related protein LC3Ⅱ and Beclin-1 were significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with model group， the number of follicles in the low， medium and high dose Guizhi Fulingwan group and the metformin group decreased， the number of follicles in atresia and atresia increased， and the follicular granulosa cell layer thickness increased. Serum T， LH， LH/FSH， FINS and HOMA-IR of Guizhi Fulingwan group were significantly decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）， and serum FINS and HOMA-IR of metformin group were significantly decreased （<italic>P</italic><0.01）. The expressions of p-PI3K， p-Akt， and p-mTOR proteins were increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression levels of LC3Ⅱ， Atg5 and Beclin-1 in the medium and high dose groups were significantly decreased （<italic>P</italic><0.01）. Conclusion:Guizhi Fulingwan can activate the PI3K/Akt/mTOR signaling pathway of granular cells， inhibit excessive autophagy of granular cells， improve ovarian function and insulin resistance， and restore ovulation， and the effect is better with high dose.