| Objective: To establish a rat polycystic ovary syndrome(PCOS) model for observingthe effect of a sequential method of nourishing Yin and tonifying Yang on the ovarianfunctions of ovulation and secretion and to explore the mechanisms of expression of steroidhormone synthetase (3β-HSD、P450arom), local ovarian factor (IGF-Ⅰ) and hormonereceptor (AR). Through these experiments, we seek to provide evidence for clinicalapplication of the sequential method of nourishing Yin and tonifying Yang, to explain thepath and target for treating PCOS patients under traditional Chinese medicine, and toprovide experimental evidence for regulating reproductive rhythm and establish a newconnotation for the theory of traditional Chinese medicine on regulating menstrual cycle.Methods:A total of 70 female SD rats age 21 days were randomly divided into sevengroups (10 rats in each group): control, model, nourishing-Yin, tonifying-Yang, sequentialmethod,western medicine and combined medication. Beginning at 23 days of age,the ratswere subcutaneously(s.c.) injected daily for up to 20 days with DHEA (6mg/100g BW)dissolved in 0.2 ml sesame oil for all groups, except control, who were injected with anequal amout of sesame oil.Each group was then treated with a different medicine on the following day of ’modelfinished’. Rats in the control and model groups were administrated Sodium Chloride. Ratsin nourishing Yin group were drenched with yin-nourishing formula, tonifying Yang grouprats were drenched with Yang-tonifying formula, sequential method group rats weredrenched with yin-nourishing formula for two days at first, and then were treated withcombined yin-nourishing formula and Yang-tonifying formula for two days, westernmedicine group rats were drenched with metformin and rats in the combined medicationgroup were treated with sequential method and metformin. All groups were successivelydrenched for twelve days. On the next day of the final treatment, all rats were weighed, blood was collected formeasuring serum hormone level, and the serum and red cells were separated bycentrifugation. The serum was stored at -20℃for subsequent analysis. Testosterone andestradiol were measured by chemoluminescence technique and androstenedione, FSH, LH,INS and IGF-Ⅰwere measured by RIA. Rats were killed by decapitation and the ovarieswere dissected, trimmed and weighed. One ovary from each rat was observed by lightmicroscope, and some were observed by transmission electron microscope. Thecontra-lateral ovaries were measured for the expression of 3β-HSD, P450arom, IGF-ⅠandAR by RT-PCR.Results: 1.The results on ovarian gross anotomy: the color of ovaries in the modelgroup were more pale than those of the normal group, and the volume of the model groupovaries was bigger than that in normal group. The ovarian weight in the model group washigher than that of the normal group(P＜0.01),and the ovarian weight in the sequentialmethod group was lighter than that in the model group(P＜0.01).2.The results of light microscope: the ovaries in model group showed multiplefollicular cysts,and the quantity of corepus luteum in the model group was less than that ofthe normal group. The diameter of cystic follicles in every group, except in the tonifyingYang group, was smaller than that in the model group(P＜0.05, P＜0.01). The quantity ofcorepus luteums in the sequential method group and combined the medication group wasmore than that in the model group.3.The results of transmission electron microscope: More mitochondria, endoplasmicreticlum, fat drops an so on existed in the model group than those in the normal group.Mitochondria, endoplasmic reticlum and fat drops were clearly decreased afer treatment bysequential nourishing Yin and tonifying Yang method.4.Hormone levels in serum results: Concentrations of the serum androstenedione andtestosterone were significantly higher in the model group than those in the normal group (P＜0.01), and their levels were clearly decreased after treatment with medicine. The serumandrostenedione level was significantly lower in the sequential method group than thatin the nourishing Yin group and tonifying Yang group (P＜0.01), but there was nosignificant differenence compared to that in the western medicine group and the combinedmedication group (P＞0.05). The serum testosterone level was significantly lower in thesequential method group than that in the tonifying Yang group and western medicine group(P＜0.01, P＜0.05), but there was no significant deviation compared to that in the nourishing Yin group and combined medication group (P＞0.05). The serum estradiolconcentratrion was significantly higher in the model group than that in the normal group (P＜0.05), and the level was clearly decreased for all groups except in the nourishing Yingroup (P＜0.05, P＜0.01).There was no significant deviation for the serum FSH and LH levels in the modelgroup compared to those in the normal group.The serum insulin concentratrion in the model group was significantly higher thanthat in the normal group (P＜0.05), and the insulin levels in the sequential method group,western medicine group and combined medication group were significantly lower than thatin the model group (P＜0.01, P＜0.05). There were no significant deviations for theserum insulin levels in the nourishing Yin group and tonifying Yang group compared tothat in the model group (P＞0.05). The serum insulin concentratrion in the sequentialmethod group significantly was lower than that in the model group (P＜0.01),but there wasno significant deviation compared to those of the western medicine group and combinedmedication group (P＞0.05).Serum level of insulin-like growth factor-1 in the model rats was significantly higherthan that in normal rats(P＜0.05). The levels in the tonifying Yang group, sequentialmethod group and combined medication group were significantly lower than that in themodel rats(P＜0.01), but there was no significant deviation for the Serum level ofinsulin-like growth factor-1 in the model group compared to that of the nourishing Yingroup (P＞0.05).The level of insulin-like growth factor-1 in the sequential method groupwas significantly lower than that in the nourishing Yin group (P＜0.05), but there was nosignificant deviation in the sequential method group compared to that of the tonifying Yanggroup, western medicine group and combined medication group (P＞0.05).5. The expressions of 3β-HSD, P450arom,IGF-Ⅰand AR mRNA in rat ovaries: It wasfound that the expressions of 3β-HSD, P450arom,IGF-Ⅰand AR mRNA in the model groupwere significantly higher than those of the normal group(P＜0.05, P＜0.01),however, theexpressions were decreased after treatment by the sequential method of nourishing Yin andtonifying Yang.Conclusions: 1. The dehydroepiandrosterone(DHEA) induced rat PCOS model issuccessfully established, and the morphologic and hormonal changes in animal model aresimilar to those observed in patients with PCOS, and same as hyperinsulinemia is observed.This animal model is, therefore, an ideal model for studying PCOS. 2. The sequential method of nourishing Yin and tonifying Yang can improve ovarianpolycystic change and ovulation failure. This method also can decrease the abnormallyincreasing amounts of mitochondria, endoplasmic reticlum and fat drops in granule cellsand theca cells seen in rats with PCOS, and also show that the sequential method candecrease the level of serum androgen by affecting ultrastructures of ovary cells in rats withPCOS.3. The sequential method of nourishing Yin and tonifying Yang can decrease thelevels of serum androstenedione, testosterone and estradiol and improve endocrinedisturbance in PCOS rats.4. The sequential method of nourishing Yin and tonifying Yang can decrease thelevels of serum androstenedione, testosterone and estradiol, through the mechanism ofdecreasing the levels of serum insulin and insulin-like growth factor-1.5. The sequential method of nourishing Yin and tonifying Yang can decrease theexpressions of 3β-HSD、P450arom mRNA, which then decreased the levels of serumandrostenedione, testosterone and estradiol by suppressing the expression of steroidhormone synthetase.6. The sequential method of nourishing Yin and tonifying Yang could decrease theexpression of IGF-ⅠmRNA, showing that one of mechanisms of decreasing the synthesisof androgen is due to diminishing the expression of ovary local factor—IGF-Ⅰ..7. The sequential method of nourishing Yin and tonifying Yang can decrease theexpression of AR mRNA of ovaries in rats with PCOS, showing that this method improveovary polycystic change, and inhibite the synthesis of ovarian hormone by down regulatingthe expression of ovarian AR, suppressing the hyperfunction of autocrine and paracrinemediated by local ovarian AR, and lessening the effect of exorbitant androgen on the ovary.8. The sequential method of nourishing Yin and tonifying Yang more greatlyimproved ovarian functions mediated by increasing the quantity of corepus luteum anddecreasing the levels of serum androstenedione、testosterone and estradiol, over either thenourishing Yin or tonifying Yang methods alone. |
PDF Full Text Request