Comparative Study Of The Effect About Bushen Treatment And Shugan Treatment On The Anovulatory Model Rats HPOA And People Luteinized Granulose Cells Secretory Function

Part one: Comparative study of the effect about Bushen treatment and Shugan treatment on the anovulatory model rats HPOAObjective: Through the study of comparative Bushen method and Shugan method anovulation similarities and differences between the impact of rats HPOA to explore the Bushen method and Shugan method Treatment of ovulation disorders mechanism of action in order to enrich the theory of reproductive medicine and promote the development of traditional Chinese medicine gynecology .Methods: 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 testosterone 1.25mg/only (0.05ml/only) at nucha. All these 204 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 170 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 Bushentiaojing Recipe high-dose group, the Bushentiaojing 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 Bushentiaojing 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 take blood to determination E₂, P, FSH, LH, T, PRL, take out the part of the pituitary, ovarian and uterus tissues from those rats by Aseptic caesarean, soaking up blood, after weighing, fixed them in LM liquid.Results: 1 Two models results: (1) Triptolide-induced ovulation disorder model reproductive hormones in serum compared with normal group, estrogen before and after E₂ decreased (all P<0.05), androgen cause anovulation model before and after PRL decrease (P<0.05, P<0.01), estrogen before and after LH increased (all P<0.01) metaestrus T increased (P<0.05), proestrus P increased (P<0.01). Ovarian and Uterus mass index did not change compared with normal group (all P>0.05). Pituitary morphology, ovarian morphology, uterine shape are mild injury. (2) Triptolide-induced ovulation disorder model in serum compared with normal group, estrogen before and after FSH decreased (all P<0.01), proestrus LH lower (P <0.05), estrogen before and after the PRL increase (all P<0.01), estrogen before and after T increased (both P<0.01), estrogen before and after E₂ increased (all P<0.01). Estrogen before and after ovarian index, estrogen before and after uterine index lower compared with the normal group (all P <0.01) on the pituitary morphology, ovarian morphology, uterine injury patterns.2 The effect of Bushenfa Recipe on ovulation disorder HPOA of rats: (1) Triptolide-induced ovulation disorder model group, compared the model group with Bushentiaojing Recipe low group would enable model rats metaestrus increased E₂ metaestrus (P<0.05), restored to the normal level (P> 0.05). Proestrus LH decreased significantly (P<0.01), restored to the normal level (P>0.05). The pituitary, ovary, uterus damage can back to normal. Bushentiaojing Recipe high group would enable proestrus E₂ model rats increased significantly (P<0.01), restored to the normal level (P>0.05). Metaestrus E₂ significantly increased (P <0.01), higher than normal (P<0.05). Metaestrus PRL increased (P<0.05), restored to the normal level (P>0.05). Proestrus P significantly decreased (P <0.01), restored to the normal level (P> 0.05). Estrus before and after the LH lower (P<0.01, P<0.05), restored to the normal level (all P>0.05). Metaestrus T reduced (P<0.05), restored to the normal level (P>0.05). The damaged pituitary, ovarian, uterine morphology can returned to normal. (2) Androgen-induced ovulation model group with Bushentiaojing Recipe, compared with model group, Bushentiaojing Recipe low group would enable to regulate menstruation model rats with esturs before and after the FSH increased (P<0.01, P<0.05), restored to the the normal level (all P>0.05). Metaestrus ovarian index increased significantly (P<0.01), restored to the normal level (P>0.05). Metaestrus uterine index increased significantly (P <0.01), restored to the normal level (P>0.05). Estrus before and after E₂ decreased (all P<0.05), restored to the normal level (all P>0.05). Estrus before and after PRL decreased significantly (all P<0.01), restored to the normal level (P>0.05). Estrus before and after the T decreased significantly (all P <0.01), restored to the normal level (all P>0.05) and can improve the damaged ovaries, uterus and pituitary. Bushengtiaojing Recipe high group would enable model rats estrus before and after the FSH increased (P<0.01, P<0.05), restored to the normal level (all P>0.05). Proestrus LH significantly increased (P<0.01), restored to the normal level (P>0.05). Estrus before and after ovarian index was significantly increased (all P<0.01), restored to the normal level (all P>0.05). Estrus before and after uterine index was significantly increased (all P<0.01), restored to the normal level (all P> 0.05). Estrus before and after E₂ decreased significantly (all P<0.01), restored to the normal level (all P>0.05). Estrus before and after PRL decreased significantly (all P<0.01), restored to the normal level (all P> 0.05). Estrus before and after the T decreased significantly (all P<0.01), restored to the normal level (all P>0.05). The damaged ovaries, uterus, pituitary basically can back to normal.3 The effect of Shuganfa on HPOA of ovulation disorder rats: (1) Triptolide-induced ovulation disorder model group after shuganfa treatment, compared with model group, the Xiaoyao pill low group would enable model rats'PRL of estrus before and after increased significantly (all P<0.01), restored to the normal level (all P>0.05). Proestrus LH lower (P<0.01), restored to the normal level (P>0.05). The damaged pituitary, ovarian, uterine morphology can returned to normal. Xiaoyao pill high group would enable model rats metaestrus E₂ increased (P<0.05), restored to the normal level (P> 0.05). Estrus before and after PRL increased (both P<0.01), restored to the normal level (all P>0.05). Reduced model rats proestrus P to a normal level (P>0.05). Estrus before and after the LH decreased significantly (all P<0.01), restored to the normal level (all P>0.05). The damaged pituitary, ovarian, uterine morphology can returned to normal. (2) Androgen-induced ovulation model group with Shuganfa, compared with model group, the Xiaoyao pill low group would enable model rats estrus before and after FSH increased (P <0.01, P <0.05), restored to the normal level (all P>0.05). Increase in model rats proestrus LH to normal (P>0.05). Estrus before and after PRL decreased significantly (all P<0.01), restored to the normal level (all P>0.05). Estrus before and after the T decreased significantly (all P<0.01), restored to the normal level (all P>0.05) and can improve the damaged ovaries, uterus, pituitary gland shape. Xiaoyao pill high group would enable model rats proestrus FSH increased significantly (P<0.01), higher than normal (P<0.05). Metaestrus FSH increased (P<0.05), restored to the normal level (P>0.05). Increase in model rats proestrus LH higher than normal (P<0.05). Increase in model rats with estrus before and after the uterine index to a normal level (all P>0.05). Metaestrus E₂ reduced (P<0.05), restored to the normal level (all P> 0.05). Estrus before and after PRL decreased significantly (all P<0.01), restored to the normal level (all P>0.05). Estrus before and after the T decreased significantly (all P <0.01), restored to the normal level (all P>0.05) and can improve the damaged ovaries, uterus, pituitary gland shape.4 Comparison of the effects of Bushenfa and Shuganfa on gonad axis in rats with ovulation disorder: (1) Triptolide-induced ovulation disorder model group, the Bushentiaojing Recipe high group to regulate model rats metaestrus E₂ higher than Xiaoyao pill group (P<0.05), proestrus PRL lower than Xiaoyao pill high group (P<0.05), the Bushentiaojing Recipe low group to regulate metaestrus E₂ was significantly higher than the Xiaoyao pill low group (P<0.01), higher than the Xiaoyao pill metaestrus LH the low group (P <0.05), proestrus PRL lower than Xiaoyao pill low group (P<0.05), metaestrus PRL lower than Xiaoyao pill low group (P<0.05).Improving in the ovarian morphology, the Bushentiaojing Recipe high and low group the number of follicles, mature follicle diameter, the number of GC storeys and level and dense arrangement better than Xiaoyao pill high and low groups. (2) Androgen-induced anovulation model group, the Bushentiaojing Recipe high group to regulate the ovarian index of estrus before and after was significantly higher than Xiaoyao pill high group (P<0.01), the metaestrus uterus index higher than Xiaoyao pill high group (P<0.05 ), the Bushentiaojing Recipe low group to regulate proestrus LH lower than Xiaoyao pill low group (P<0.05). Patterns to improve the pituitary, Xiaoyao pill high and low group of anterior pituitary gland cells, the number is greater than the Bushentiaojing Recipe high and low group. Improving in the ovarian morphology, the the Bushentiaojing Recipe high group have a higher rough profile than Xiaoyao pill low group, ovarian follicle cells thiner than the Xiaoyao pill high group, the number of follicles, follicle diameter and corpus luteum volume to be higher than Xiaoyao pill high group, the number of storeys, number and arrangement density of GC higher than Xiaoyao pill high group. Patterns in improving the uterus, the myometrial thickness, the number of glands, lining layer of columnar cells of continuity and integrity of the Bushentiaojing Recipe high and low group is superior to Xiaoyao pill high and low groups.Part two: Comparative study of the effect about Bushen treatment and Shugan treatment people luteinized granulose cells secretory functionObjective: To explore the Bushen and Shugan treatment of ovulation disorders in the mechanism, through the ovarian granulosa cell cultures to observe the two method of ovarian granulosa cell function, and to explore two methods of ovarian secretion system of self-study of ovulation barriers to the occurrence of ovarian granulosa cell function and the relationship between the Bushentiaojing Recipeand and Xiaoyao pill compare pairs of ovarian granulosa cell secretion function of the similarities and differences between, for treatment of Bushentiaojing Recipeand and Xiaoyao pill ovulation disorders provide experimental basis for clinical application.Methods: 21 6-week-old healthy female rats were randomly divided into 7 groups, and control group were fed daily with normal saline 2ml/100g; Bushentiaojing Recipeand group of low, middle and high-dose group and Xiaoyao pill high, middle and low-dose group were given Bushentiaojing Recipeand and Xiaoyao pill. In each group were taken at 1 hour after the last administration of rat femoral artery blood. Put it aside at room temperature 2h, 3000r/min centrifugal 10min, take the supernatant, 56℃water bath 30 min inactivated complement, 0.22μm sterile filter filtration sterilization, sub-loaded, -20℃to save backup.Select the age of 20-30 years of age, physical health and treatment of infertility due to ovulatory dysfunction, Hebei Medical University School of Medicine clinics in the female volunteers 4. B- super-monitoring no mature follicle, and the exclusion of ovarian, uterine organic diseases, bilateral tubal patency imaging results showed that as the last in March did not take hormones. Each side oral Bushentiaojing Recipeand and Xiaoyao pill. Continuous medication 3 days, the first 4 days early morning empty stomach medication (medication before the fasting 12h), one taking a day dose, at 1 hour after the last administration venous blood taken 5ml, put it aside at room temperature 2h, 3000r/min centrifugal 10min, taking on the Ching, 56℃water bath 30 min inactivated complement, 0.22μm sterile filter filtration sterilization, sub-installed. Preparation of blank serum: the age of 20-30 years of age, physical health, menstrual laws, B super-monitoring of mature follicles, and no ovaries, uterus organic diseases of female volunteers, an early morning fasting venous blood 5ml, the rest of my colleagues Preparation of drug-containing serum. -20℃to save backup.From the Second Affiliated Hospital of Hebei Province, assisted reproductive branches fetch the follicular fluid containing granulosa cells by 2000r/min centrifuge, discard supernatant, precipitation granulosa cells, after washing with DMEM medium by density gradient 0.5Percoll suspension 2500 r / min centrifugation to remove red blood cells separated 30min. Adding 0.25% trypsin, 37℃to digest 5-10min or so, to join with 10% fetal bovine serum and the 1640 termination of digestion. PBS washing, centrifugation. After trypan blue staining cells in 85% -90% survival, with 1640 containing 10% fetal bovine serum and the concentration of granule cell culture medium will be adjusted to 2×105 single-cell suspension, single-cell suspension into a 24-hole of the culture plate, each plate 1ml, at 5% CO2, 37℃incubation box training. 24h of cell adhesion. Were added to Bushentiaojing recipeand and Xiaoyao pill containing human serum and rat serum is made with 6%, 12%, 18%, high, medium and low-dose-treated cells 48h. Canadian-based normal and control rat serum were blank.Results: 1 Bushentiaojing Recipeand containing serum on human luteinized granulosa cell function: (1) Human containing serum groups:â‘ E₂, P, cAMP levels of impact: compared with the blank group, Bushentiaojing high, medium, Low group E₂ are higher than control group (P<0.05, P<0.01, P<0.01). Bushentiaojing medium group P is significantly lower than control group (P<0.01). Bushentiaojing middle and high cAMP group is significantly higher than control group (all P<0.01). Pairwise comparison, Bushentiaojing high group E₂ is higher than Bushentiaojing medium, low group (P <0.05, P<0.01), Bushentiaojing medium group E₂ is higher than the low group (P<0.01). Bushentiaojing high and low group P is higher than medium group (P<0.05, P<0.01). Bushentiaojing middle and high group cAMP are significantly higher than low group (P<0.01).â‘¡FSHR expression: Protein blotting: Bushentiaojing high, medium and low groups FSHR expression are higher than control group, Bushentiaojing high, medium group of FSHR expression are higher than low group. RT-PCR method: comparison with the control group, Bushentiaojing middle and high group FSHR expression are significantly higher than control group (all P<0.01). Pairwise comparison, Bushentiaojing medium group FSHR is significantly higher than high, low group (all P<0.01), Bushentiaojing high group FSHR is significantly higher than low group (P<0.01).â‘¢P450, PPARγexpression: compared with the control group, kidney regulate menstruation, the high expression group of P450 is higher than the control group (P<0.01, P<0.05). Bushentiaojing high, medium and low groups PPARγexpression are lower than control group (all P <0.01). Pairwise comparison, Bushentiaojing high group of P450 expression is significantly higher than that low group (all P<0.01). Bushentiaojing low, medium group of PPARγexpression are significantly lower than the Bushentiaojing high group (all P<0.01). (2) rats containing serum groupâ‘ E₂, P, cAMP levels of impact: compared with the control group, Bushentiaojing high, medium group E₂ are higher than control group (all P<0.01). Bushentiaojing medium group P is significantly lower than control group (P<0.01). Bushentiaojing middle and high cAMP group are significantly higher than control group (P<0.05, P<0.01). Pairwise comparison, Bushentiaojing high, medium group E₂ are higher than Bushentiaojing low group (all P <0.01). Bushentiaojing medium group P is lower than low group (P<0.05, P <0.01). Bushentiaojing high group cAMP is higher than low group (P<0.05).â‘¡FSHR expression: Protein blotting: Bushentiaojing high, medium and low group FSHR expression are higher than control group. Bushentiaojing high, middle group are higher than low group. RT-PCR method: comparison with control group, Bushentiaojing middle and high groups FSHR expression are significantly higher than control group (all P<0.01). Pairwise comparison, Bushentiaojing medium, high group FSHR are sign ificantly higher than low group (all P<0.01).â‘¢P450, PPARγexpression: compared with control group, Bushentiaojing medium, high group P450 expression are higher than the control group (P<0.05, P<0.01). Bushentiaojing medium group PPARγexpression are significantly lower than control group (all P<0.01). Pairwise comparison, Bushentiaojing high group P450 expression is higher than low group (P<0.01, P<0.05). Bushentiaojing medium group PPARγexpression is lower than low and high group (P <0.05, P <0.01).2 Xiaoyao pill containing serum on human luteinized granulosa cell function .(1) Human containing serum groups:â‘ E₂, P, cAMP levels of impact: compared with control group, Xiaoyao pill high, medium and low groups E₂ are higher than control group (all P<0.01). Xiaoyao pill high group P is significantly lower than control group (P<0.05). Xiaoyao pill low, medium and high group cAMP are significantly higher than control group (all P<0.01). Pairwise comparison, Xiaoyao pill high group E₂ is higher than low group (P<0.01). Xiaoyao pill medium group cAMP significantly higher than high group and low group (all P<0.01).â‘¡FSHR expression: Protein blotting: Xiaoyao pill high, medium and low expression groups are higher than control group, Xiaoyao pill high group is higher than low, medium group, Xiaoyao pill medium group is higher than low group. RT-PCR method: comparison with control group, Xiaoyao pill high group FSHR expression is significantly higher than control group (all P<0.01). Pairwise comparison, Xiaoyao pill FSHR is higher than the high group and low group (all P<0.01).â‘¢P450, PPARγexpression: comparison with control group, Xiaoyao pill medium high group expression P450 expression is significantly higher than control group (P <0.01, P <0.05). Xiaoyao pill high, medium and low group PPARγexpression are lower than control group (P <0.01, P<0.01, P<0.05). Pairwise comparison, Xiaoyao pill high, medium group of P450 expression are significantly higher than the low group (all P<0.01). Xiaoyao pill low group PPARγexpression is lower than the medium,high group (P <0.01, P<0.05). (2) rats containing serum groups:â‘ pairs of E₂, P, cAMP levels of impact: compared with control group, Xiaoyao pill high, E₂ are higher than the control group (P<0.01, P<0.01, P<0.05) . Xiaoyao pill high group P is higher than control group (P<0.05). Xiaoyao pill high, medium group cAMP are significantly higher than control group (all P<0.01). Pairwise comparison, Xiaoyao pill high group E₂ is higher than low, medium group (all P<0.01), Xiaoyao pill medium group E₂ is significantly higher than low group (P<0.01). Xiaoyao pill medium, low group P are significantly lower than high group (all P<0.01). Xiaoyao pill high group cAMP is higher than medium, low group (P<0.05, P<0.01).â‘¡FSHR expression. Western blotting: Xiaoyao pill high, medium and low groups expression are higher than control group, Xiaoyao pill high group is higher than low, medium group, Xiaoyao pill high group is higher than low group. RT-PCR method: comparison with control group, Xiaoyao pill high group FSHR expression is significantly higher than control group (P<0.01). Pairwise comparison, Xiaoyao pill high group is higher than medium , low group (P<0.05, P<0.01).â‘¢P450, PPARγexpression: compared with control group, Xiaoyao pill high group P450 expression is higher than control group (P<0.05). Xiaoyao pill medium group PPARγexpression is significantly lower than that control group (P <0.01).3 Two kinds of drug-containing serum on human luteinized granulosa cell function affect the comparison of: (1) Human containing serum groups:â‘ E₂, P, cAMP levels of impact: Bushentiaojing low group E₂ is lower than Xiaoyao pill high group (P<0.01). Bushentiaojing medium group E₂ is higher than Xiaoyao pill low group (P<0.05). Bushentiaojing high group E₂ is higher than Xiaoyao pill high, medium and low group (all P<0.01). Bushentiaojing medium group P is lower than Xiaoyao pill low group (P<0.01). Bushentiaojing low group cAMP is lower than Xiaoyao pill high, medium group (all P<0.01). Bushentiaojing medium groups cAMP is higher than Xiaoyao pill low group (P<0.05), lower than Xiaoyao pill high group (P <0.01). Bushentiaojing high group cAMP is lower than Xiaoyao pill high group (P<0.01).â‘¡FSHR. Western blotting: Bushentiaojing high, medium and low groups expression of FSHR are higher than Xiaoyao pill high, medium and low groups, and Bushentiaojing low group FSHR expression is higher Xiaoyao pill high group. RT-PCR method: Bushentiaojing low group FSHR is lower than Xiaoyao pill high, medium group (P<0.05,P<0.01). Bushentiaojing medium group FSHR is higher than Xiaoyao pill high, medium and low group (all P <0.01). Bushentiaojing high group FSHR is higher than Xiaoyao pill low, medium group (all P<0.01).â‘¢P450, PPARγexpression: Bushentiaojing low group P450 is lower than Xiaoyao pill medium, high group (P <0.01, P <0.05). Bushentiaojing medium group P450 is higher than Xiaoyao pill low group (P<0.01). Bushentiaojing medium group P450 expression is higher than Xiaoyao pill high, low group (P<0.05). Bushentiaojing medium group PPARγis higher than Xiaoyao pill low group (P<0.05). Bushentiaojing medium group PPARγis higher than Xiaoyao pill low group (P<0.05), lower than the Xiaoyao pill high group (P<0.01). Bushentiaojing high group PPARγis higher than Xiaoyao pill low group (P<0.01). (2) rats containing serum groups:â‘ E₂, P, cAMP levels of impact: Bushentiaojing low group E₂ is lower than Xiaoyao pill high, medium group (all P<0.01). Bushentiaojing medium group E₂ is higher than Xiaoyao pill medium, low group (P<0.05, P<0.01). Bushentiaojing high group E₂ is higher than Xiaoyao pill high ,low group (all P<0.01). Bushentiaojing low group P is lower than Xiaoyao pill high group (P<0.01). Bushentiaojing medium group P is lower than Xiaoyao pill medium, high group (P<0.05,P<0.01). Bushentiaojing high group P ia lower than Xiaoyao pill high group (P <0.01). Bushentiaojing low group cAMP is lower than Xiaoyao pill high, (all P<0.01). Bushentiaojing medium group cAMP is lower than Xiaoyao pill high group (P<0.01). Bushentiaojing high group cAMP is lower than Xiaoyao pill high group (P <0.01).â‘¡FSHR expression. Western blotting: Bushentiaojing middle, low groups FSHR are higher than Xiaoyao pill high, low group. Bushentiaojing medium group is higher than Xiaoyao pill high, medium and low groups. RT-PCR method: Bushentiaojing FSHR low group is higher than Xiaoyao pill high group (P<0.05). Bushentiaojing medium group FSHR is higher than Xiaoyao pill medium, low group (P<0.05,P<0.01). Bushentiaojing high group FSHR is higher than Xiaoyao pill low group (P<0.01).â‘¢P450, PPARγexpression: Bushentiaojing low group P450 is lower than Xiaoyao pill medium, high group (P<0.01,P<0.05). Bushentiaojing medium group P450 is higher than Xiaoyao pill low group (P<0.01). Bushentiaojing high group P450 is higher than Xiaoyao pill low, medium and high group(all P<0.05). Bushentiaojing medium group PPARγis ower than Xiaoyao pill low, high group (all P<0.05). Bushentiaojing high group PPARγis lower than Xiaoyao pill medium group (P<0.05).Conclusions: 1 two kinds of ovulation disorder models are serum reproductive hormone disorders, pituitary, ovarian, uterine morphology are abnormal; Triptolide-induced ovulation disorder model rats ovaries, uterus weight index are lower, Androgen-induced ovulation model rats ovarian, uterine shape and weight index is more severe than Triptolide-induced ovulation disorder model. 2 Bushen and Shugan method both by improve the pituitary gland, ovarian, uterine shape, regulating reproductive endocrine hormone levels, increase ovary, uterus weight index, to play the promotion of follicular development and maturation, ovulation disorders treatment effect. Bushen method in improving the ovarian, uterine morphology, increased ovary, uterus weight index, regulation of ovarian secretion of reproductive hormones is better than Shugan, Shugan method improve pituitary morphology, regulation of pituitary secretion of reproductive hormones are better than Bushen. 3 Bushen, Shugan method can improve human luteinized granulosa cell function, self-regulation of ovarian secretion system, improve ovarian function. Bushen method in improve E₂, FSHR, reduce PPARγis better than Shugan, Sugan method improve cAMP is better than Bushen.