| Objective:To observe the effect of the Bushen Tiaojing Decoction on PCOS model rats during estrous cycle,serum anti-mullerian hormone(AMH)levels,and the effect on Ovarian histomorphology and the expression of anti-Mullerian hormone receptor type 2(AMHR?)in ovarian tissue;To investigate the pathogenesis of PCOS and the functional mechanism of Bushen Tiaojing Decoction.Method:Forty-eight female SD rats aged 70 days(average weight 270.56±2.13g)were randomly divided into experimental group(n = 36)and normal control group(n = 12)at a ratio of 3:1.Then use the Poretsky modeling method of experimental rats by subcutaneous injection of insulin(INS)and human chorionic gonadotropin(HCG),with 5% glucose solution by gavage,to establish the model of PCOS rats model.After building the model,6 rats in the experimental group were randomly selected in the 3:1 ratio,with 2 rats in normal control group.Then to smear vaginal exfoliative cell,measure androstenedione(ASD)and luteinizing hormone(LH)levels through enzyme linked immunosorbent assay,gauge the weight and the major diameter of one side of ovaries,and to observe the morphological changes of ovarian tissue,to verify the success of modeling according to the test results.The rats in the experimental group were randomly divided into three groups(n = 10): the model control group,Chinese medicine group and western medicine group.Then the rats of the traditional Chinese medicine group were treated with Bushen Tiaojing Decoction.The rats in the western medicine group were treated with ethinyl estradiol cyproterone.The rats in the normal control group and the model control group were treated with normal saline.Treatment was administered for 4 days for a course,every 4 days to stop 1 day,a total of 15 days(3 cycles),light microscope observation of vaginal smears of rats for 10 days,the rats were sacrificed and the level of AMH in serum were measured by enzyme-linked immunosorbent assay(ELISA),the ovarian tissue morphological changes were observed under light microscope,the expression of AMHR II of ovarian tissue in rats was detected by the application of Western method in Blot.Results:1.To verify the success of modeling according to the test results: The rats in the normal control group were good in general situation at the aspects of spirit,cibation,activity and responsived quickly when confronting external stimuli.After the completion of the modeling,the rats in experimental group were subcutaneously injected with INS and HCG showed body obese,action slow,fatigue lethargy,less activity,fur color dull gray,occasionally hair removal,etc.Then 6 rats in the experimental group were randomly selected in the 3:1 ratio,with 2 rats in normal control group.(1)Smearing rat vaginal exfoliative cell: rats in the normal control group showed a regular estrous cycle of vaginal exfoliative cell smear;the rats in the experimental group were subcutaneously injected with INS and HCG to complete the vaginal exfoliation of the cells were prompted to have estrous cycle disorders,prolongation or stagnation,and especially during the estrous cycle.(2)Measureing androstenedione(ASD)and luteinizing hormone(LH)levels through enzyme linked immunosorbent assay: the serum ASD and LH levels in the experimental group was higher than that in the normal control group.(3)Gauging the weight and the major diameter of one side of ovaries:the experimental group was heavier and longer than that in the normal control group.(4)Observing the morphology of ovarian tissue in rats: rats in normal control group were observed under microscope in each stage of development of ovarian follicle and corpus luteum,cortex and medulla structure is clear,the majority of the growth of follicle cells is large,the structure is normal,follicular fluid content,more granular cell layers,luteal volume is large,interstitial inflammatory cell infiltration and fibrosis.6 rats in the experimental group were randomly selected from the ovary after the completion,under the microscope were seen in cystic dilatation of more follicles,follicular number and developmental stage of the corpus luteum significantly reduced,oocytes and radiata follicles disappeared,granular cell layers decreased or disappeared,theca cells and interstitial cells hyperplasia,abnormal follicular development and ovulation disorder.Based on the above results,verifing the success of modeling.2.Drug intervention,smearing rat vaginal exfoliative cell:after the western medicine group and traditional Chinese medicine drug intervention treatment,rats with estrous cycle disorder has improved,the performance of the estrous cycle,estrus interval gradually restored,tend to normal.But in the model control group,the vaginal exfoliation of the cells were also prompted to have estrous cycle disorders,prolongation or stagnation,and especially during the estrous cycle.3.Drug intervention,observing the morphology of ovarian tissue in rats: western medicine group and Chinese medicine group rat ovarian microscopically in different developmental stages of follicles and luteal multiple follicles,granule cell reproduced and appeared several layers,tips Follicular development and ovulation gradually returned to normal.But in the model control group,were also prompted to abnormal follicular development and ovulation disorder.4.Drug intervention,the serum AMH levels in rats: The serum AMH level in the model control group was higher than that in the normal control group,significant difference(P<0.05);compared with the model control group,the serum AMH content of the western medicine group and the traditional Chinese medicine group were decreased,but the difference was not significant(P > 0.05).5.Expression of AMHR? in rat ovarian tissue: The expression of AMHR? in ovariectomized rats in model control group was significantly higher than that in the normal control group,the difference was statistically significant(P < 0.05);compared with the model control group,Western medicine group and traditional Chinese medicine group were lower in ovarian AMHR? expression,the difference was statistically significant(P < 0.05);The expression of ovarian AMHR? in traditional Chinese medicine group was lower than that in western medicine group,but the difference was not significant(P > 0.05).Conclusion:1.Subcutaneous injection of INS and Poretsky by HCG method can successfully establish the PCOS model rats;2.Both Bushen Tiaojing Decoction and Ethinylestradiol & Ciprofloxacin have the tendency to reduce serum AMH content in PCOS model rats;3.Bushen Tiaojing Decoction and Ethinylestradiol & Ciprofloxacin can lower the expression of AMHR II in ovarian tissue of PCOS model rats;4.The pathogenesis of PCOS may be related to AMH signaling,Bushen Tiaojing Decoction could effectively improve the links of primordial follicle development and ovulation in ovarian tissue of PCOS model rats,which has therapeutic effects on PCOS. |
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