| Objective: To investigate the effective mechanism of Bushen Tiaojing Recipe and ease pill on treating anovulatory disease, to study the correlation between anovulatory disease and cytokine due to the change in ovarian cytokine of the anovulatory rats induced by androgen and tripterygium, to compare the effects of Bushen Tiaojing Recipe and ease pill acting on the cell factors in ovary, to provid experimental reference of clinical application of Bushen Tiaojing Recipe and ease pill promoting follicular development and ovulation, so as to promote the development of the treatment of TCM Gynecology.Method: 204 healthy female Wistar rats of 3 months old, weighing 180-250g were fed as well, Randomly 170 of these rats were improved as the methods of Zheng Jia Yun, Liang Wenbo Inc. TWP-chip 35mg/kg/d (a dose was about an adult dose for clinical use 20 times, weighing every week, adjusting the delivery quantity), Gastric lavage method for medicine once a day continued 57 days. Making module two consecutive smears of vaginal epithelial cells in cycles estrous cycles became irregular, mainly the cycle was extended and not complete, suggesting that triptolide-induced rat models of dysfunction ovulation were made successfully. the remaining 34 rats were treated with the distilled water as a normal control group.204 healthy female SD rats of 9-day age from the same batch, whose weight was about 15g, were adopted in our research. 170 of those rats were made as models by subcutaneous injection of testoserone 1.25mg/only (0.05ml/only) at nucha. All these 170 rats were weaned when they were 22-day old, which were raised by Special manager under free feeding and others same conditions. When they were 70-day old, their vaginals debouched. We began to make the smear of the vaginal epithelial cells for 2 successive sexual cycle for the model group, the Vaginal epithelial cells showed continuously cutinization, which suggested us that models of anovulatory had been set up successfully. The remaining 34 rats were chosen randomly as normal group, each of them was injected 0.05ml neutral tea oil subcutaneously at nucha. Following the methods of Yu Jin etc.The two batchs of model rats were randomly divided into five groups: the model group, the Bushen Tiaojing Recipe high-dose group, the Bushen Tiaojing Recipe low-dose group, the Xiaoyao pill high-dose group, the Xiaoyao pill low-dose group, each group was made up of 34 rats; the treatment groups were drenched with Bushen Tiaojing Recipe and Xiaoyao pill, model groups and normal groups were drenched with distilled water once a day. Treatments continued for 5 weeks, and made the smear of vaginal epithelial cells for 2 successive sexual cycle. Prohibited feeding them within 8 hours after administration, each group were decapitated at proestrus and estrus periods, We took out the left of the ovarian tissues from those rats by Aseptic caesarean, soaking up blood, after weighing, fixed them in 4% paraformaldehyde solution, made them conventional paraffin-embedded in order to get 5μm thick serial sections and respectively, had them immunohistochemical stainined to observe the ovarian morphological changes and organizational IL-1β, TNF-α, TGF-β1, TGF-βR1 protein expression intensity. In addition, placed the right ovarian tissues in the freezing tubes of DEPC treatment to preserve by liquid nitrogen cryopreservation, during which we used real-time fluorescent quantitative PCR to detect IL-1β, TNF-α, TGF-β1, TGF-βR1 mRNA expression.Results:1 The effect of Bushen Tiaojing Recipe on the two pictchs of anovulatory model rats in ovarian cell factors1.1 Protein expressions of IL-1β, TNF-α, TGF-β1, TGF-βR1 in ovary tissueIn the rats, whose ovulation dysfunction was induced by testosterone and androgen, whether it was at proestrus or at metaestrus, compared with the normal group, the expressions of IL-1βin ovarian granular cells and oocyte increased, the expressions of TNF-αin the oocytes and the macrophage-like cells in interstitium and corpus luteum, and the expressions of TGF-β1 and TGF-βR1 in the granular cells and the interstitial cells all increased in the model group (all P <0.01).In the rats, whose ovulation dysfunction was induced by testosterone, Compared with the model group, the expressions of IL-1βof the high-dose BuShen and low-dose BuShen groups in ovarian granular cells and oocytes decreased(all P<0.01), and close to normal (all P>0.05); the expressions of TNF-αof the high-dose BuShen and low-dose BuShen groups in the oocytes and macrophage-like cells of interstitium and corpus luteum decreased (all P<0.01), during the metaestrus, the expressions of low-dose BuShen group close to normal (P>0.05); the expressions of TGF-β1, TGF-βR1 of the high-dose BuShen and low-dose BuShen groups in the granular cells and interstitial cells decreased (all P<0.01), the expressions of TGF-βR1 close to normal (both P>0.05);In the rats, whose ovulation dysfunction was induced by androgen, compared with the model group, the expressions of IL-1βof the high-dose BuShen and low-dose BuShen groups in ovarian granular cells and oocytes, the expressions of TNF-αof the high-dose BuShen and low-dose BuShen groups in the oocytes and macrophage-like cells of interstitium and corpus luteum, the expressions of TGF-β1, TGF-βR1of the high-dose BuShen and low-dose BuShen groups in the granular cells and interstitial cells decreased (all P<0.01), and close to normal (all P>0.05).1.2 Expressions of IL-1β, TNF-α, TGF-β1, TGF-βR1 mRNA in ovary tissueIn the rats, whose ovulation dysfunction was induced by testosterone and androgen, whether it was at proestrus or at metaestrus, compared with the normal group, the expressions of ovarian IL-1β, TNF-α, TGF -β1, TGF-βR1 mRNA all increased in the model group (all P<0.01).In the rats, whose ovulation dysfunction was induced by testosterone, compared with the model group, the expressions of IL-1β, TNF-α, TGF -β1, TGF-βR1 mRNA of the high-dose BuShen and low-dose BuShen groups of rats both decreased (all P<0.01), during the proestrus or metaestrus of the rats, the expressions of IL-1β, TGF-βR1mRNA of the high-dose BuShen and low-dose BuShen groups, during the metaestrus of the rats, the expressions of TNF-α, TGF-β1 mRNA of the low-dose BuShen groups close to normal (all P>0.05).In the rats, whose ovulation dysfunction was induced by androgen, compared with the model group, the expressions of IL-1β, TNF-α, TGF -β1, TGF-βR1mRNA of the high-dose BuShen and low-dose BuShen groups of rats all decreased (all P<0.01), and close to normal (all P>0.05).2 The effect of XiaoYao pill on the two pictchs of anovulatory model rats in ovarian cell factors2.1 Protein expressions of IL-1β, TNF-α, TGF-β1, TGF-βR1 in ovary tissueIn the rats, whose ovulation dysfunction was induced by testosterone and androgen, whether it was at proestrus or at metaestrus, compared with the normal group, the expressions of IL-1βin ovarian granular cells and oocyte increased, the expressions of TNF-αin the oocytes and the macrophage-like cells in interstitium and corpus luteum, the expressions of TGF-β1 and TGF-βR1 in the granular cells and the interstitial cells all increased in the model group (all P <0.01).In the rats, whose ovulation dysfunction was induced by testosterone, Compared with the model group, the expressions of IL-1βof the high-dose XiaoYao pill and low-dose XiaoYao pill groups in ovarian granular cells and oocytes decreased(all P<0.01), and close to normal(all P>0.05); the expressions of TNF-αof the high-dose XiaoYao pill and low-dose XiaoYao pill groups in the oocytes and macrophage-like cells of interstitium and corpus luteum decreased(both P<0.01), during the proestrus or metaestrus, the expression of high -dose XiaoYao pill group close to normal(P>0.05); the expressions of TGF-β1, TGF-βR1of the high-dose XiaoYao pill and low-dose XiaoYao pill groups in the granular cells and interstitial cells decreased (all P <0.01), during the proestrus, the expression of low-dose XiaoYao pill groups haven't closed to normal (P<0.05 or P <0.01).In the rats, whose ovulation dysfunction was induced by androgen, compared with the model group, the expressions of IL-1βof the high-dose XiaoYao pill and low-dose XiaoYao pill groups in ovarian granular cells and oocytes, the expressions of TNF-αof the high-dose XiaoYao pill and low-dose XiaoYao pill groups in the oocytes and macrophage-like cells of interstitium and corpus luteum, the expressions of TGF-β1, TGF-βR1of the high-dose XiaoYao pill and low-dose XiaoYao pill groups in the granular cells and interstitial cells decreased (all P <0.01), and close to normal (all P>0.05).2.2 Expressions of IL-1β, TNF-α, TGF-β1, TGF-βR1 mRNA in ovary tissueIn the rats, whose ovulation dysfunction was induced by testosterone and androgen, whether it was at proestrus or at metaestrus, compared with the normal group, compared with the normal group, the expressions of ovarian IL-1β, TNF-α, TGF -β1, TGF-βR1mRNA all increased in the model group (all P<0.01).In the rats, whose ovulation dysfunction was induced by testosterone, compared with the model group, the expressions of IL-1β, TNF-α, TGF -β1, TGF-βR1mRNA of the high-dose XiaoYao pill and low-dose XiaoYao pill groups of rats all decreased (all P<0.01), during the proestrus or metaestrus of the rats, the expressions of TNF-αmRNA of the low-dose XiaoYao pill group, during the metaestrus of the rats, the expressions of TGF-β1, TGF-βR1 mRNA of the low-dose XiaoYao pill groups haven't close to normal (all P<0.01).In the rats, whose ovulation dysfunction was induced by androgen, compared with the model group, the expressions of IL-1β, TNF-α, TGF -β1, TGF-βR1mRNA of the high-dose XiaoYao pill and low-dose XiaoYao pill groups of rats all decreased (all P<0.01), and close to normal (all P>0.05).3 Comparision of the effect of Bushen Tiaojing Recipe and XiaoYao pill for the two pictchs of anovulatory model rats in ovarian cell factors3.1 Protein expressions of IL-1β, TNF-α, TGF-β1, TGF-βR1 in ovary tissueIn the rats, whose ovulation dysfunction was induced by Tripterygium, There was no significant difference among the BuShen high-dose group, the BuShen low-dose group, the high-dose group and XiaoYao pill low-dose group on the expression of IL-1βin ovarian granular cells and oocytes(all P>0.05); during the proestrus of those rats, the expressions of TNF-αin oocytes and macrophage-like cells of interstitium and luteum of the BuShen high-dose group was higher than that of the BuShen low-dose group, Xiao Yao pill low-dose group and the Xiao Yao pill high-dose group (all P<0.05), the expressions of the BuShen low-dose group and Xiao Yao pill low-dose group were higher than that of the Xiao Yao pill high-dose group (all P<0.01); during the metaestrus of the rats, the expression of the high-dose BuShen group was higher than that of low-dose BuShen group and high-dose Xiao Yao pill group (all P<0.01), the expression of the low-dose Xiao Yao pill group was higher than that of the low-dose BuShen group and high-dose Xiao Yao pill group (all P<0.01); During the proestrus or metaestrus of the rats, the expressions of TGF-β1 in granular cells and interstitial cells of the high-dose BuShen group were higher than that of the low-dose BuShen group, the low-dose Xiao Yao pill group and high-dose Xiao Yao pill group (all P<0.01), the expressions of the low-dose BuShen group and low-dose Xiao Yao pill group were higher than that of the high-dose Xiao Yao pill group (all P<0.01); During the proestrus of those rats, the expression of TGF-βR1 in granular cells and interstitial cells was no difference among the high-dose Xiao Yao pill group, high-dose BuShen group and low-dose BuShen group (all P<0.01), but the expression of these groups was lower than that of the low-dose Xiao Yao pill group; during the metaestrus of the rats, there was no significant difference on the expression of TGF-βR1 between the high-dose BuShen group, the low-dose BuShen group, low-dose XiaoYao pill group and the high-dose XiaoYao pill group(all P>0.05) .In the rats, whose ovulation dysfunction was induced by androgen, whether the rats were at their proestrus or at metaestrus, it was no significant difference for them on the expressions of IL-1βin ovarian granular cells and oocyte, the expressions of TNF-αin the oocytes and the macrophage-like cells in interstitium and luteum, the expressions of TGF-β1 and TGF-βR1 in the granular cells and the interstitial cells among the high-dose BuShen group, the low-dose BuShen group, the high-dose Xiao Yao pill group and the low-dose Xiao Yao pill group(all P>0.05).3.2 Expressions of IL-1β, TNF-α, TGF-β1, TGF-βR1 mRNA in ovary tissueIn the rats, whose ovulation dysfunction was induced by testosterone, There was no significant difference on the expression of IL-1βmRNA in ovaries among the high-dose BuShen group, the low-dose BuShen group, the high-dose Xiao Yao pill group and the low-dose Xiao Yao pill group(all P>0.05); during the proestrus, the expression of TNF-αmRNA in ovaries of the high-dose BuShen group was higher than that of the low-dose BuShen group and the high-dose Xiao Yao pill group, the low-dose Xiao Yao pill group(all P<0.01), the expressions of the low-dose BuShen group and the low-dose Xiao Yao pill group were higher than that of the high-dose Xiao Yao pill group(all P>0.05); during the metaestrus, the expression of the high-dose BuShen group was higher than that of the low-dose BuShen group and the high-dose Xiao Yao pill group(all P<0.01), the expression of the low-dose Xiao Yao pill group was higher than that of the low-dose BuShen group and the high-dose Xiao Yao pill group (all P<0.01).During the proestrus, the expression of TGF-β1mRNA in ovaries of the high-dose BuShen group was higher than that of the low-dose BuShen group and the high-dose Xiao Yao pill group, the low-dose Xiao Yao pill group(all P<0.01), the expression of the low-dose BuShen group and the low-dose Xiao Yao pill group was higher than that of the high-dose Xiao Yao pill group(all P<0.01); during the metaestrus, the expressions of the low-dose Xiao Yao pill group and the high-dose Xiao Yao pill group were not obviously different from that of the low-dose BuShen group(all P>0.05), but lower than that of the high-dose BuShen group(all P<0.01); during the proestrus, the expression of TGF-βR1mRNA in ovaries of the high-dose Xiao Yao pill group was not different from that of the high-dose BuShen group and that of the low-dose BuShen group(all P>0.05), but lower than that of the low-dose Xiao Yao pill group(all P<0.01); during the metaestrus, there was no significant difference on the expressions of TGF-βR1mRNA in ovaries among the high-dose BuShen group, the low-dose BuShen group and the high-dose Xiao Yao pill group, the low-dose Xiao Yao pill group(all P>0.05).In the rats, whose ovulation dysfunction was induced by androgen, whether it was at proestrus or metaestrus, it was no significant difference on the expressions of IL-1β, TNF-α, TGF-β1, TGF-βR1 mRNA among the high-dose BuShen group, the low-dose BuShen group and the high-dose Xiao Yao pill group, the low-dose Xiao Yao pill group(all P>0.05).Conclusion:1 In the rats, whose ovulation dysfunction was induced by testosterone and androgen, the protein and gene levels of ovarian IL-1β, TNF-α, TGF-β1 and TGF-βR1 in the model group were significantly increased than that in normal group. The IL-1β, TNF-α, TGF-β1 and TGF-βR1 in ovarian and ovulation are closely linked.2 Bushen Tiaojing Recipe and XiaoYao pill can both obviously regulate the proteic and genic levels of ovarian IL-1β, TNF-α, TGF-β1 and TGF-βR1 for anovulatory rats, maintain a stable environment within the ovary, make granular cells proliferation and speed oocytes maturation up, Thereby promoting the development of the follicles mature.3 There is no difference on regulating the proteic and genic levels of ovarian IL-1β, TNF-α, TGF-β1 and TGF-βR1 by using Bushen Tiaojing Recipe and XiaoYao pill for anovulatory rats induced by induced by androgen. XiaoYao pill is better than Bushen Tiaojing Recipe on regulating the proteic and genic levels of ovarian TNF-α, TGF-β1 and TGF-βR1 for anovulatory rats induced by testosterone.
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