| Objective:Polycystic ovarian syndrome (PCOS), the common female reproductive endocrine disease, associated with anovulation, hyperandrogenism and insulin resistance, is one of the main reasons for female anovulatory infertility. PCOS incidence is 5-10% among reproductive women, which leads to 70% anovulatory infertility. With age, PCOS increases the risk for type 2 diabetes and cardiovascular diseases. The etiology of PCOS is incompletely understood. Dysfunction of hypothalamus-pituitary-ovarian axis (HPOA) likely plays key role in it.Clinical researches indicate that Chinese medical therapies have good effects on endocrinopathy. Chinese medical therapies may restore PCOS patients' reproductive function and rectify their endocrinal and metabolic disorders without side or adverse effects. The tutor, Professor Li Lierong, with 40-year clinical experience in gynecopathy, has rich experiences on complicated diseases treated with Chinese medicine. Gubenquyu Decoction is created by Prof. Li according to her clinical experience on PCOS. The clinical observation shows Gubenquyu Decoction may effectively decrease elevated plasma T, LH levels and LH/FSH, restore PCOS patients' menstruation, improve their ovulation and increase the pregnancy rate. We are meant to from organic-cellular-molecular levels, systemically research the therapeutic mechanism of Gubenquyu Decoction on the hypothalamus-pituitary-ovarian axis, on PCOS rat model induced by DHT, with molecular biology methods, in order to provide the scientific basis for its future promotion in clinical application.Methods:New born female Wistar rats, fed in 12h light/12h dark environment, till 21-day age, were randomly divided into 4 groups:①Control,②PCOS,③Flutamide,④Gubenquyu, each group 12 rats. Rats in PCOS, Flutamide and Gubenquyu groups implanted subcutaneously with dihydrostestosterone (DHT, 7.5mg,90-d continuous release pellets, equal to 1.66m/kg,83μg per day) to induce PCOS model. From 64-day age, Rats in Gubenquyu and Flutamide groups started to receive treatment. Gubenquyu Decoction was one kind of pure herbal extract made of 12 herbs. Flutamide was an anti-androgen hormone. Gubenquyu group and Flutamide group received treatment twice per day for 4 weeks.During treatment, the changes of weight, activity, diet, etc. of the rats in each group were observed once per week; changes of estrus cycle of the rats in each group were observed once per day. After treatment, tissue structures of ovaries, uterus, and pituitary were observed by histopathologic methods; plasma E2,T,P,LH and FSH levels were measured by RIA; expressions of GnRH-R in pituitary were measured by immunohistochemistry; expressions of GnRH, GnRH-R and AR at MPO and HDB in hypothalamic were measured by immunohistochemistry.Results:1. Changes of body weight:At 21-day age, the body weight of rats had no significant difference among each group (P> 0.05). At 63-day age, implanted with DHT for 6 weeks, the body weight of rats in 3 DHT model groups had very significant difference between in Control group (P<0.01), the body weight of rats in 3 DHT model groups were obviously higher than in Control group, which was similar to PCOS human metabolic changes. After 4 weeks'treatment, the body weight of rats in Gubenquyu group had very significant difference between in PCOS group (P<0.01), while had no significant difference between in both Control and Flutamide groups (P> 0.05), which indicated the weights of PCOS rats were obviously decreased treated by Gubenquyu Decoction for 4 weeks.2. Effects on estrus cycle:From the beginning of the treatment (namely at 64-day age), continuously recorded rats' estrus cycles of each group for 4 weeks until the experiment ended. We found that Control rats exhibited regular 4-5 days estrus cycle including diestrus, proestrus, estrus and metestrus stages; while PCOS rats exhibited no dynamic change in estrous cycle and were constantly in diestrus phase; meanwhile Gubenquyu rats exhibited recovery of regular estrus cycle after treatment, which indicated the recovery of PCOS rats' ovary functions gradually.3. Effects on organ coefficient:Ovarian coefficient of Gubenquyu rats had very significant, significant differences between of PCOS rats, Flutamide rats respectively (P<0.01,P<0.05), ovarian coefficient of Gubenquyu rats were lower than of PCOS and Flutamide rats; while had no significant difference between of Control rats(P> 0.05). Uterus and pituitary coefficients of rats had no significant difference among each group (P> 0.05)4. Effects on ovarian morphology:Ovary weight and size were higher in DHT rats than in control and Gubenquyu rats. Slices of Gubenquyu group exhibited different stages follicle, including primordial follicle, preantral follicle, primary follicle, zona pellucida, antral follicle, secondary follicle, provulatory follicle, and ovulation; diminished granulosa cell layer increased, thickened theca interna cell layer reduced, and thickened cystic wall decreased compared with PCOS rats. 5. Effects on plasma sex hormone levels:Plasma E2 level in Gubenquyu rats had very significant difference between in PCOS rats(P<0.01); while no significant difference between in both Control and flutamide rats (P> 0.05) Plasma T level in Gubenquyu rats had very significant difference between in PCOS rats(P<0.01); while no significant difference between in both Control and Flutamide rats (P>0.05). Plasma P level of rats had no significant difference among each group (P> 0.05). Plasma LH level in Gubenquyu rats had very significant difference between in PCOS rats(P<0.01); while had no significant difference between in both Control and Flutamide rats (P> 0.05) LH/FSH in Gubenquyu rats had very significant difference between in PCOS rats(P<0.01); while had no significant difference between in both Control and Flutamide rats (P>0.05). Plasma FSH level of rats had no significant difference among each group (P> 0.05)6. Effects on pituitary releasing LH level:Pituitary releasing LH level of 3 DHT implanted groups had very significant difference between Control group (P<0.01); Pituitary releasing LH level of Gubenquyu rats had very significant difference between of PCOS group (P<0.01), while of Flutamide rats had significant difference between of PCOS rats (P<0.05).7. Effects on GnRH-R expression in pituitary:The number of GnRH-R cells in pituitary of Gubenquyu rats had significant difference between of PCOS rats(P<0.05); while had no significant difference between of Control and Flutamide rats (P> 0.05)8. GnRH,GnRH-R,AR expression in MPO and HDB of hypothalamus:(1)GnRH:The number of GnRH cells of Gubenquyu rats had very significant, significant differences between of PCOS rats in MPO and HDB respectively(P<0.01,P<0.05); while had significant difference between of Flutamide rats in MPO (P<0.05); meanwhile had no significant difference between of Control rats in MPO and HDB (P>0.05) (2)GnRH-R:The number of GnRH-R cells of Gubenquyu rats had significant differences between of Control group in both MPO and HDB (P<0.05); while had no significant difference between of both PCOS and Fflutamide rats (P> 0.05)(3)AR:The number of AR cells of Gubenquyu rats had significant difference between of PCOS rats in MPO (P<0.05); while had no significant differences between of both Control and Flutamide rats (P> 0.05)Conclusions:1. Gubenquyu Decoction can significantly reduce PCOS increased weight; restore estrus cycle; decrease plasma T level and LH/FSH, etc.2. Gubenquyu Decoction can rectify pathologic changes of ovarian structures, decrease the increased ovarian weight and improve the ovulation.3. Gubenquyu Decoction can't rectify pituitary coefficient, but can modulate the function of pituitary:it can decrease pituitary releasing LH level, but can not restore normal level.4. Gubenquyu Decoction can effect the protein expression of GnRH-R in pituitary and GnRH,GnRH-R,AR expression in MPO and HDB of hypothalamus, to modulate increased GnRH pulse frequency, decrease sensitivity for GnRH in pituitary, reduce the frequency and amplitude of LH releasing, to improve ovulation.
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