Objective

2

To explore the possible mechanism of

Jianpi Yishen Huazhuo Formula

（健脾益肾化浊方， JYHF） in treating polycystic ovary syndrome （PCOS）.

Methods

2

Seventy SD female rats were randomly divided into blank group and model group with 15 rats each， metformin group， JYHF low-， medium- and high-dose groups with 10 rats each. Except for the blank group， all the other groups were fed with letrozole orally combined with high-fat diet for 21 days to establish the PCOS model. After successful modeling， the blank group and the model group were intragastrically administered with 10 ml/（kg·d） of normal saline， while the metformin group was administered with 100 mg/（kg·d） of metformin hydrochloride tablets by gavage， and the JYHF low-， medium- and high-dose groups were administered intragastrically with 1.275， 2.55， and 5.10 g/（kg·d） of JYHF suspension respectively， all for 30 days. The ovaries were weighed and the ova-rian index was calculated， and serum follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， testosterone （T）， anti-Müllerian hormone （AMH） and estradiol （E

2

） levels were measured through ELISA. HE staining was used to observe the morphological changes of ovarian tissue. Immunohistochemical staining was used to detect caspase-3， caspase-9 and phosphatidylinositol-3 kinase （PI3K）， protein kinase B （AKT）， mammalian target of rapamycin （mTOR） protein positive expression. Western blot was performed to detect relative expressions of rat ovarian tissue pro-apoptotic protein （BAX）， anti-apoptotic protein （Bcl-2）， Caspase-9， Cytochrome-C， PI3K， AKT， and mTOR proteins.

Results

2

Compared to those in the blank group， the ovarian tissue of the rats in the model group was dominated by cystic dilated follicles， and the granulosa cell layer was reduced， with obvious interstitial hyperplasia； the ovarian index， serum T， LH， AMH， as well as Caspase-3 and Caspase-9 average optical density， BAX/Bcl-2 value， the relative expression of Cytochrome-C and Caspase-9 proteins significantly increased， while serum E

2

and FSH levels， average optical density and protein expression of PI3K， AKT， mTOR in ovarian tissue significantly decreased （

P

＜0.05 or

P

＜0.01）. Compared to those in the model group， the polycystic changes of ovarian tissue in all JYHF groups and the metformin group were improved， with follicles and corpus luteum at all levels seen， and the granulosa cell layer thickened； the ovarian index， serum LH， AMH， average optical density and relative protein expression of Caspase-9 in the ovarian tissue were significantly reduced， while serum E

2

， FSH， ave-rage optical density of PI3K and AKT in the ovarian tissue， and relative protein expression of PI3K， AKT and mTOR significantly increased （

P

＜0.05 or

P

＜0.01）. The serum T level and Caspase-3 average optical density in the metformin group and JYHF medium- and high-dose groups were reduced than those in the model gorup， while mTOR average optical density increased significantly， and BAX/Bcl-2 value decreased in the JYHF low- and high-dose groups and metformin group than the model group； the expression of Cytochrome-C protein in ovarian tissue significantly decreased in the metformin group and JYHF high-dose group （

P

＜0.05 or

P

＜0.01）. Compared to the metformin group， the low-dose JYHF group had a significantly higher serum T level， and the medium-dose JYHF group had a significantly higher BAX/Bcl-2 value （

P

＜0.05）； the expression of Cytochrome-C protein in the high-dose JYHF group was significantly lower than that in the medium-dose group （

P

＜0.05）.

Conclusion

2

JYHF can inhibit the expression of apoptotic protein in ovarian granulosa cells of PCOS rats， thereby maintaining endocrine balance and improving ovarian reproductive function. The mechanism may be related to the activation of PI3K/AKT/mTOR signaling pathway.