Based PI3K / AKT Pathway Signal Added Guishen Pill Molecular Mechanisms Promoting Follicular Development

OBJECTIVE:With follicular development is closely related to the PI3K/AKT signaling pathway,so in this experimental study we put this signaling pathway as the breakthrough point, reproduce rat model of follicular development disorder caused by GTW on the basis of previous research.To further explore the molecular mechanism of Compound Herbal of Xinjiaguishenwan with the functions of Reinforcing Kidney and Activating Blood on stimulating follicle development from two aspects of cell proliferation and apoptosis. Tries to reveal the connotation of traditional Chinese medicine to improve ovarian function.METHODS:Reference to the pre-experimental study,the vaginal cytology smears of SD rats in child-bearing period were made every day. We selected54rats with normal estrous cycle as subjects according to the results of vaginal smears. Nine of them were randomly selected as normal control control group, the remaining45rats were were all given intra-gastric administration with GTW40mg/(kg-d) for10weeks to make the required models,and the blank control group rats were given intra-gastric administration with distilled water.Monitored the exchanges of estrous cycles of rats through the variation of vaginal cytology smears from the eighth week.When all the rats showed irregular estrous cycle,we believed the models were successfully made.Then we divided the45rats into5groups randomly-the model control group, the western medicine control group, three different dosages (high, medium and low) of herbal compound Xinjiaduishenwan groups, with the normal control group,there were6groups altogether in this experiment, every group with9rats. The normal control group and model control groups were filled daily with distilled water; The western medicine control group were treated with estrogen and progestin sequential through intra-gastric administration; three different dosages of herbal compound Xinjiaduishenwan group were given different dose of Chinese medicine in the same way. All groups were treated for2weeks ontinuously. Vaginal exfoliated cells smears were operated during the treatment to observe oestrous cycle of the rats.24hours after the last treatment,all the rats were killed after taken femoral artery blood. Then the rats’abdominal cavity were opened and both ovaries were taken out. We taken the weight of the ovary and calculated the ovary index, detected the concentration of serum "E2","P","FSH"and"LH" with RIA and ELISA. Stained the paraffin sections of ovarian with HE, counted primordial, scondary, antral, atresia follicles and corpus luteum under optical microscope,measured the thickness of the granulosa cells of secondary and antral follicles. Detected the expression of "p-AKT""p-mTOR"and "p-p70S6K" in ovarian with Western blotting; cyclinD2mRNA and BaxmRNA with RT-PCR;"PTEN","cyclinD2","p-AKT","Caspase-3" with immunohistochemical.RESULTS:GTW40mg/(kg-d) gavage for10weeks can result in follicular development disorder of the child-bearing period SD rats. They showed the disorders of estrous cycle, the decreasing of ovarian indexes, level of serum E2and P, thickness of the granulosa cells, number of growing follicles, number of corpus luteum, expression of p-AKT, p-mTOR, p-p70S6K, mRNA levels of cyclinD2in rat ovary;the increasing of number of atresia follicles, expression of Caspase-3, mRNA levels of Bax in ovary.The above indicators had significant difference compared with the normal control group (P<0.05or P<0.01).No changes of serum FSH, serum LH, PTEN, The above indicators had no significant difference (p>0.05).After treatment with Xinjiaguishenwan for two weeks,the model rats had obvious improvement in their general conditions. They showed normal or close to the normal estrous cycles, the increasing of ovarian indexes, level of serum E2, P, thickness of granular cell, number of growing follicles and corpus luteum, expression of p-AKT, p-mTOR p-p70S6K, mRNA levels of cyclinD2, and obvious decreasing of the number of atresia follicles, expression of Caspase-3, mRNA levels of Bax in ovary. The above indicators had significant difference compared with the model control group (P<0.05or P<0.01).The effect of high and medium dosages of herbal compound group were more obvious than low dosage.CONCLUSION:1. GTW40mg/(kg-d) gavage for10weeks can result in follicular development disorder of the child-bearing period SD rats.2. GTW probably inhibit the cascade activation reaction of AKT in the PI3K/AKT signaling pathway.On the one hand inhibit the activation of downstream signaling molecules-mTOR and p70S6K, block the mitosis signal transmission and down-regulate the expression of cyclinD2in granulosa cells.eventrually inhibit the proliferation of granulosa cells by blocking the cell cycle.On the other hand promote the apoptosis of granulosa cells by up-regulating the expression of Bax and Caspase-3.Therefore disorder the follicle’s normal development and damage the function of ovarian.3.Compound Herbal of Xinjiaguishenwan with the functions Reinforcing Kidney and Activating Blood can restore the activity of PI3K/AKT signaling pathway through up-regulating the expression of p-AKT.On the one hand activate mTOR and p70S6K,downlink the mitosis signal,up-regulate the expression of cyclinD2in granulosa cells.Eventrually promote the proliferation of granulosa cells by accelerating cell cycle process.On the other hand inhibite the apoptosis of granulosa cells by down-regulating the expression of Bax and Caspase-3.Therefore resume the development of follicle, renovate the damaged function of the ovaries.