| Objective: Menoxenia, metrostaxis, amenorrhea and sterility, etc. owe to no ovulation or ovulatory disorders. Clinical use of kidney reinforcing, liver soothing treatment for Menopathy, infertility and other gynecological diseases have significant effect. Pre-and-recent experimental results show that: kidney reinforcing, liver soothing can greatly regulate hypothalamic-pituitary-ovarian axis of rats with anovulation, but to promote the recovery of ovarian function by kidney method, liver soothing successfully is based on improving the blood supply to the ovarian, and this needs further examination. Therefore, we have established two kinds of rat model of anovulation and used the application of kidney reinforcing to intervene with Xiaoyao pill prescriptions. Impacts of these two treatments on the two anovulation models of rat ovarian vasorelaxation-vasoconstriction factors and vascularization factors are observed. By comparing these two methods for promoting follicle growth and ovulation we find the similar and different mechanisms of them. In order to provide experimental basis for clinical applications, and improve the development of traditional Chinese medicine therapeutics gynecology we make many experiments.Part 1: Kidney Reinforcing Impact on the Ovarian Blood Supply of Rat with Ovulation DisorderMethods: Two kinds of ovulatory dysfunction in rats: 1. The establishment of androgen sterilized rat by reference to Yu Jin and other methods. That is, selecting 204 female rats of the same batch SD and their weight were about 15g, we selected 170 of those rats randomly and gave them injection of testosterone propionate 0.05ml / only on the neck subcutaneous when they were born the 9th day. On the 70th day after birth we smear their vaginal cell, and when the vaginal epithelial cells continue to be keratode it shows that the model is successful.The other rats were injected 0.05ml neutral tea oil subcutaneously at nucha as normal group. 2. The establishment of triptolide-induced rat model of reproductive system dysfunction, improving Zheng Jiarun, Liang Wenbo and other methods. 204 healthy female Wistar rats of 3 months old, weighing 180-250g were fed as well, we selected 170 of those rats randomly and gave them injection of TWP-chip 70mg/kg/d (20 times adult dose for clinical use, weigh and adjust the delivery volume weekly) to the stomach for 57 days. When estrous cycles become irregular,(the main cycle is not complete or cycle becomes longer), it indicates the model has been set up successfully. The remaining 34 rats were treated with the distilled water as normal group. 3. We divide the model rats randomly into two groups of treatment high-dose group, low-dose group, model group, and normal group, each group with about 34 rats. The first part treatment rat group will be fed with bushen tiaojing recipe, model group and normal group will be fed with distilled water once a day. After 5 weeks gavage, when vaginal cells cycle changes, we smear vagina cells for two cycles (a cycle of 4-5 days personality), the last two days smear every 2 hours, and record the rats estrous cycle of each group in detail. Don't give them water after the last prescription for 8 hours and to choose proestrus rats and postestrus in each group based on the results of vaginal smears and decapitated in groups of blood, prepare plasma, serum, and to cryopreservate -20℃for inspection preparation. Detect ET-1, cGMP levels, nitrate reductase assay of nitric oxide (NO) levels and at the same time perform the sterile operation immediately: remove the left side of the ovary in the ice tray quickly and gently put into a pre-cooling electron microscopy, fix light microscopy fixative in a small bottle, and by using immunohistochemical staining to observe the intensity of VEGF and MVD. Of the right ovarian tissue, we extract it's bloodstain and fter homogenizing the plasm we test ET-1, ATⅡand NO levels by using radioimmunoassay.Results:1 Kidney reinforing impact on vasomotor and shrink plasma levels of rats with ovulation disorder1.1 Triptolide-induced rat model of reproductive system dysfunction1.1.1 Impaction on the proestrus rats of modelCompared with normal group, the content of plasma ET-1 of model group increased, NO and cGMP content decreased; Compared with model group, the content of plasma ET-1 of Bushen high-dose and low-dose groups decreased, NO and cGMP content increased; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1 and cGMP, the content of NO in low-dose group was more than high-dose group.1.1.2 Impaction on the postestrus rats of modelCompared with normal group, the content of plasma ET-1 of model group increased, NO and cGMP content decreased; Compared with model group, the content of plasma ET-1 of Bushen high-dose and low-dose groups decreased, NO and cGMP content increased; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1, NO and cGMP.1.2 Androgen sterilized rat model1.2.1 Impaction on the proestrus rats of modelCompared with normal group, the content of plasma ET-1 of model group increased, NO and cGMP content decreased; Compared with model group, the content of plasma ET-1 of Bushen high-dose and low-dose groups decreased, there was no change in the content of plasma NO and cGMP of the two groups; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1, NO and cGMP.1.2.2 Impaction on the postestrus rats of modelCompared with normal group, the content of plasma ET-1 of model group increased, NO and cGMP content decreased; Compared with model group, the content of plasma ET-1 of Bushen high-dose and low-dose groups decreased, cGMP content increased,there was no change in the content of plasma NO of the two groups; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1, NO and cGMP.2 Kidney reinforing impact on vasomotor and shrink ovarian levels of rats with ovulation disorder2.1 Triptolide-induced rat model of reproductive system dysfunction2.1.1 Impaction on the proestrus rats of modelCompared with normal group, the content of ovarian Ang-Ⅱof model group increased, NO content decreased, ET-1 content was not changed; Compared with model group, the content of ovarian Ang-Ⅱand ET-1 of Bushen high-dose group decreased, the content of ovarian NO of Bushen low-dose group increased; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1, NO and Ang-Ⅱ.2.1.2 Impaction on the postestrus rats of modelCompared with normal group, the content of ovarian ET-1 of model group increased, NO content decreased, Ang-Ⅱcontent was not changed; Compared with model group, the content of ovarian NO of Bushen high-dose and low-dose groups increased, ET-1 content was not changed; the content of ovarian Ang-Ⅱof Bushen low-dose group decreased; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1, NO and Ang-Ⅱ.2.2 Androgen sterilized rat model2.2.1 Impaction on the proestrus rats of modelCompared with normal group, the content of ovarian ET-1 and Ang-Ⅱof model group increased, NO content decreased; Compared with model group, the content of ovarian NO and Ang-Ⅱof Bushen high-dose and low-dose groups was not changed, the content of ET-1 of Bushen high-dose group decreased; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1, NO and Ang-Ⅱ.2.2.2 Impaction on the postestrus rats of modelCompared with normal group, the content of ovarian ET-1 and Ang-Ⅱof model group increased, NO content was not changed; Compared with model group, the content of ovarian NO and Ang-Ⅱof Bushen high-dose and low-dose groups was not changed, the content of ET-1 of Bushen low-dose group decreased; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1, NO and Ang-Ⅱ.3 Kidney reinforing impact on ovarian vascularization factors of rats with ovulation disorder3.1 Triptolide-induced rat model of reproductive system dysfunction3.1.1 Impaction on the proestrus rats of modelCompared with normal group, the expression of ovarian VEGF and MVD decreased; Compared with model group, the expression of ovarian VEGF and MVD of Bushen high-dose and low-dose groups increased; The expression of ovarian VEGF of Bushen high-dose group was more than low-dose group, there was no obviously difference between the high-dose and low-dose groups in the expression of MVD.3.1.2 Impaction on the postestrus rats of modelCompared with normal group, the expression of ovarian VEGF and MVD decreased; Compared with model group, the expression of ovarian VEGF and MVD of Bushen high-dose and low-dose groups increased; The expression of ovarian VEGF of Bushen low -dose group was more than high -dose group, the expression of ovarian MVD of Bushen high-dose group was more than low-dose group.3.2 Androgen sterilized rat model3.2.1 Impaction on the proestrus rats of modelCompared with normal group, the expression of ovarian VEGF and MVD decreased; Compared with model group, the expression of ovarian VEGF and MVD of Bushen high-dose and low-dose groups increased; The expression of ovarian VEGF of Bushen high-dose group was more than low-dose group, there was no obviously difference between the high-dose and low-dose groups in the expression of MVD.3.2.2 Impaction on the postestrus rats of modelCompared with normal group, the expression of ovarian VEGF and MVD decreased; Compared with model group, the expression of ovarian VEGF and MVD of Bushen high-dose and low-dose groups increased; There was no obviously difference between the high-dose and low-dose groups in the expression of VEGF, the expression of ovarian MVD of Bushen high-dose group was more than low-dose group.Part 2 : Liver Soothing Impact on the Ovarian Blood Supply of Rat with Ovulation DisorderMethods: The treatment rat groups will be fed with xiaoyao pill, the others same with the first part.Results:1 Liver Soothing impact on vasomotor and shrink plasma levels of rats with ovulation disorder1.1 Triptolide-induced rat model of reproductive system dysfunction1.1.1 Impaction on the proestrus rats of modelCompared with normal group, the content of plasma ET-1 of model group increased, NO and cGMP content decreased; Compared with model group, the content of plasma ET-1 of Xiaoyao high-dose and low-dose groups decreased, NO and cGMP content increased; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1, NO and cGMP.1.1.2 Impaction on the postestrus rats of modelCompared with normal group, the content of plasma ET-1 of model group increased,NO and cGMP content decreased; Compared with model group, the content of plasma ET-1 of Xiaoyao high-dose and low-dose groups decreased, NO content increased, the content of plasma cGMP of Xiaoyao high-dose group increased; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1 and NO, the content of plasma cGMP of Xiaoyao high-dose group was more than low-dose group.1.2 Androgen sterilized rat model1.2.1 Impaction on the proestrus rats of modelCompared with normal group, the content of plasma ET-1 of model group increased, NO and cGMP content decreased; Compared with model group, the content of plasma ET-1 of Xiaoyao high-dose and low-dose groups decreased, cGMP content increased, the content of plasma NO of Xiaoyao low-dose group increased; There was NO obviously difference between the high-dose and low-dose groups in the content of ET-1, NO and cGMP.1.2.2 Impaction on the postestrus rats of modelCompared with normal group, the content of plasma ET-1 of model group increased, NO and cGMP content decreased; Compared with model group, the content of plasma ET-1 of Xiaoyao high-dose and low-dose groups decreased, cGMP content increased, the content of plasma NO of Xiaoyao low-dose group increased; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1 and cGMP, the content of plasma NO of Xiaoyao low -dose group was more than high -dose group.2 Liver Soothing impact on vasomotor and shrink ovarian levels of rats with ovulation disorder2.1 Triptolide-induced rat model of reproductive system dysfunction2.1.1 Impaction on the proestrus rats of modelCompared with normal group, the content of ovarian Ang-Ⅱof model group increased, NO content decreased, ET-1 content was not changed; Compared with model group, the content of ovarian ET-1 and Ang-Ⅱof Xiaoyao high-dose group decreased, the content of ovarian NO was not changed, the content of ovarian ET-1,Ang-Ⅱand NO of Xiaoyao low-dose group was not changed; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1,Ang-Ⅱand NO.2.1.2 Impaction on the postestrus rats of modelCompared with normal group, the content of ovarian ET-1 of model group increased, NO content decreased, Ang-Ⅱcontent was not changed; Compared with model group, the content of ovarian ET-1 and Ang-Ⅱof Xiaoyao high-dose and low-dose groups decreased, NO content increased; There was no obviously difference between the high-dose and low-dose groups in the content of ET-1,Ang-Ⅱand NO.2.2 Androgen sterilized rat model2.2.1 Impaction on the proestrus rats of modelCompared with normal group, the content of ovarian ET-1 and Ang-Ⅱof model group increased, NO content decreased; Compared with model group, the content of ovarian ET-1 of Xiaoyao low-dose group decreased, the content of ovarian Ang-Ⅱof Xiaoyao high-dose group decreased, the content of ovarian NO of Xiaoyao high-dose and low-dose groups was not changed; There was no obviously difference between the high-dose and low-dose groups in the content of Ang-Ⅱand NO, the content of ovarian ET-1 of Xiaoyao low-dose group was less than high -dose group.2.2.2 Impaction on the postestrus rats of modelCompared with normal group, the content of ovarian ET-1 and Ang-Ⅱof model group increased, NO content was not changed; Compared with model group, the content of ovarian ET-1 and Ang-Ⅱof Xiaoyao high-dose and low-dose groups decreased, NO content was not changed; There was no obviously difference between the high-dose and low-dose groups in the content of Ang-Ⅱand NO, the content of ovarian ET-1 of Xiaoyao high-dose group was less than low-dose group.3 Liver Soothing impact on ovarian vascularization factors of rats with ovulation disorder3.1 Triptolide-induced rat model of reproductive system dysfunction3.1.1 Impaction on the proestrus rats of modelCompared with normal group, the expression of ovarian VEGF and MVD decreased; Compared with model group, the expression of ovarian VEGF and MVD of Xiaoyao high-dose and low-dose groups increased; There was no obviously difference between the high-dose and low-dose groups in the expression of VEGF and MVD.3.1.2 Impaction on the postestrus rats of modelCompared with normal group, the expression of ovarian VEGF and MVD decreased; Compared with model group, the expression of ovarian VEGF and MVD of Xiaoyao high-dose and low-dose groups increased; The expression of ovarian VEGF of Xiaoyao high-dose group was more than low-dose group, there was no obviously difference between the high-dose and low-dose groups in the expression of MVD.3.2 Androgen sterilized rat model3.2.1 Impaction on the proestrus rats of modelCompared with normal group, the expression of ovarian VEGF and MVD decreased; Compared with model group, the expression of ovarian VEGF and MVD of Xiaoyao high-dose and low-dose groups increased; There was no obviously difference between the high-dose and low-dose groups in the expression of VEGF and MVD.3.2.2 Impaction on the postestrus rats of modelCompared with normal group, the expression of ovarian VEGF and MVD decreased; Compared with model group, the expression of ovarian VEGF and MVD of Xiaoyao high-dose and low-dose groups increased; There was no obviously difference between the high-dose and low-dose groups in the expression of VEGF and MVD.Part 3 : Comparison between Kidney Reinforcing and Liver Soothing impact on the Ovarian Blood Supply of Rat with Ovulation DisorderMethods: the same with part 1 and part 2 Results:1 Comparison of two methods impact on vasomotor and shrink plasma levels of rats with ovulation disorder1.1 Triptolide-induced rat model of reproductive system dysfunction1.1.1 Impaction on the proestrus rats of modelCompared with model group, the content of plasma ET-1 of Bushen high-dose,low-dose groups and Xiaoyao high-dose,low-dose groups decreased, the content of plasma NO and cGMP increased; There was no obviously difference between Xiaoyao high-dose,low-dose and Bushen high-dose ,low-dose groups in the content of plasma ET-1 and cGMP; The content of plasma NO of Xiaoyao high-dose ,low-dose group was more than Bushen high-dose group.1.1.2 Impaction on the postestrus rats of modelCompared with model group, the content of plasma ET-1 of Bushen high-dose,low-dose groups and Xiaoyao high-dose,low-dose groups decreased, the content of plasma NO increased, the content of plasma cGMP of Bushen high-dose,low-dose groups and Xiaoyao high-dose group increased;The content of plasma ET-1 of Xiaoyao low-dose group was less than Bushen high-dose ,low-dose groups; The content of plasma NO of Xiaoyao low-dose group was less than Bushen high-dose group;There was no obviously difference between Xiaoyao high-dose, low-dose and Bushen high-dose,low-dose groups in the content of plasma cGMP.1.2 Androgen sterilized rat model1.2.1 Impaction on the proestrus rats of modelCompared with model group, the content of plasma ET-1 of Bushen high-dose,low-dose groups decreased, NO content increased, the content of plasma cGMP of Bushen high-dose group increased, the content of plasma ET-1 of Xiaoyao high-dose,low-dose groups decreased, cGMP content increased, the content of plasma NO of Bushen low-dose group increased; There was no obviously difference between Xiaoyao high-dose, low-dose and Bushen high-dose ,low-dose groups in the content of plasma ET-1; The content of plasma NO of Xiaoyao low-dose group was more than Bushen high-dose group; The content of plasma cGMP of Xiaoyao high-dose ,low-dose group was more than Bushen high-dose ,low-dose group.1.2.2 Impaction on the postestrus rats of modelCompared with model group, the content of plasma ET-1 of Bushen high-dose,low-dose groups decreased, the content of NO and cGMP was not changed, the content of plasma ET-1 of Xiaoyao high-dose,low-dose groups decreased, cGMP content increased, the content of plasma NO of Bushen low-dose group increased; There was no obviously difference between Xiaoyao high-dose ,low-dose and Bushen high-dose ,low-dose groups in the content of plasma ET-1; The content of plasma NO of Xiaoyao low-dose group was more than Bushen low -dose group; The content of plasma cGMP of Xiaoyao high-dose, low-dose group was more than Bushen high-dose, low-dose group.2 Comparison of two methods impact on vasomotor and shrink ovarian levels of rats with ovulation disorder2.1 Triptolide-induced rat model of reproductive system dysfunction2.1.1 Impaction on the proestrus rats of modelCompared with model group, the content of ovarian ET-1 and Ang-Ⅱof Bushen high-dose group decreased, the content of NO of Bushen low-dose group increased, the content of ovarian ET-1 and Ang-Ⅱof Xiaoyao high-dose group decreased, the content of NO was NOt changed;There was no obviously difference between Xiaoyao high-dose, low-dose and Bushen high-dose, low-dose groups in the content of plasma ET-1, Ang-Ⅱand NO.2.1.2 Impaction on the postestrus rats of modelCompared with model group, the content of ovarian NO of Bushen high-dose, low-dose groups increased, ET-1 content was not changed , the content of ovarian Ang-Ⅱof Bushen low-dose group decreased, the content of ovarian ET-1 and Ang-Ⅱof Xiaoyao high-dose, low-dose groups decreased, NO content increased; There was no obviously difference between Xiaoyao high-dose, low-dose and Bushen high-dose, low-dose groups in the content of plasma ET-1, Ang-Ⅱand NO.2.2 Androgen sterilized rat model2.2.1 Impaction on the proestrus rats of modelCompared with model group, the content of ovarian NO and Ang-Ⅱof Bushen high-dose and low-dose groups was not changed, the content of ET-1 of Bushen high-dose group decreased, the content of ovarian ET-1 of Xiaoyao low-dose group decreased, the content of ovarian Ang-Ⅱof Xiaoyao high-dose group decreased, the content of ovarian NO of Xiaoyao high-dose and low-dose groups was not changed;There was no obviously difference between Xiaoyao high-dose ,low-dose and Bushen high-dose ,low-dose groups in the content of plasma Ang-Ⅱand NO; The content of plasma ET-1 of Xiaoyao low-dose was less than Bushen low-dose group.2.2.2 Impaction on the postestrus rats of modelCompared with model group, the content of ovarian NO and Ang-Ⅱof Bushen high-dose and low-dose groups was not changed, the content of ET-1 of Bushen low-dose group decreased, the content of ovarian ET-1 and Ang-Ⅱof Xiaoyao high-dose and low-dose groups decreased, NO conten was not changed; The content of plasma Ang-Ⅱof Xiaoyao high-dose, low-dose group was less than Bushen high-dose group; The content of plasma ET-1 of Xiaoyao high-dose group was less than Bushen high-dose, low-dose group; There was no obviously difference between Xiaoyao high-dose, low-dose and Bushen high-dose, low-dose groups in the content of plasma NO.3 Comparison of two methods impact on ovarian vascularization factors of rats with ovulation disorder3.1 Triptolide-induced rat model of reproductive system dysfunction3.1.1 Impaction on the proestrus rats of model The expression of ovarian VEGF and MVD of Bushen high-dose, low-dose and Xiaoyao high-dose, low-dose groups increased; The expression of ovarian VEGF of Xiaoyao high-dose,low-dose groups was more than Bushen high-dose, low-dose groups; The expression of ovarian MVD was more than Bushen low-dose group.3.1.2 Impaction on the postestrus rats of modelThe expression of ovarian VEGF and MVD of Bushen high-dose, low-dose and Xiaoyao high-dose, low-dose groups increased; The expression of ovarian VEGF of Xiaoyao high-dose,low-dose groups was more than Bushen high-dose, low-dose groups; The expression of ovarian MVD of Xiaoyao high-dose group was more than Bushen low-dose group.3.2 Androgen sterilized rat model3.2.1 Impaction on the proestrus rats of modelThe expression of ovarian VEGF and MVD of Bushen high-dose, low-dose and Xiaoyao high-dose, low-dose groups increased; The expression of ovarian VEGF and MVD of Xiaoyao high-dose, low-dose groups was more than Bushen low-dose group.3.2.2 Impaction on the postestrus rats of modelThe expression of ovarian VEGF and MVD of Bushen high-dose, low-dose and Xiaoyao high-dose, low-dose groups increased; There was no obviously difference between Xiaoyao high-dose ,low-dose and Bushen high-dose, low-dose groups in the expression of ovarian VEGF; The expression of ovarian MVD of Xiaoyao high-dose, low-dose groups was more than Bushen low-dose group.Conclusions:1 The function of vasorelaxation-vasoconstriction factors of two kinds of ovulatory dysfunction rats were disordered, obstructed in ovarian blood-supply, affected the fuction of ovary, induced reproductive system dysfunction. Bushen tiaojing recipe and Xiaoyao pill could increase the activity of NO and cGMP, could decrease the activity of ET-1 and Ang-Ⅱ, could regulate the function of vasomotion, improve the ovarian blood-supply and ovarian function, promote the development of follicular and ovulation.2 The expression of ovarian VEGF and MVD of two kinds of ovulatory dysfunction rats were decreased, dysfunction on vascularization. Bushen tiaojing recipe and Xiaoyao pill could increase the expression of ovarian VEGF and MVD, promote vascularization, improve the ovarian blood-supply, regulate the function of ovary.3 Soothing liver was better than Reinforcing kidney in regulate the function of vasomotion and vascularization, improve the ovarian blood-supply.
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