| Objective: Bushen Tiaojing Recipeand was used as representative recipeand to adding kidney function; Xiaoyao side was used as as the traditional prescription to regulating liver function. Based on the experimental evidence of Bushen and Shugan in improving the ovarian function, we study the comparation of the effect about Tiaojing Recipeand and Xiaoyao Pill on ovarian oxidative damage and apoptosis of granular in anovulatory model rats induced by androgen and tripterygium. To investigate the mechanism and role of Bushen and Shugan in improving the ovarian function and enrich Traditional Chinese Medical theory about"Kidney plays an important role in the reproductive"and"the liver is the innate in women", to promote the development of Chinese medicine of 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 70mg/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 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 part 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 4μm thick serial sections and respectively, had them immunohistochemical stainined to observe the ovarian morphological changes and organizational Caspase-3 protein expression intensity and observe apoptosis of granular by Tunel and FCM .Results:1 The effect of Bushen Tiaojing Recipe to oxidative injury and apoptosis of granulosa cells of the two kinds of rats with ovarian anovulation1.1 The impact of Bushen Tiaojing Recipe to ovary oxidative damage of the two kinds of ovulation disorder model rat1.1.1 The impact of Bushen Tiaojing Recipe to bilirubin in serum and ovarian of the two kinds of ovulation disorder model rats Anovulatory rats induced by androgen and tripterygium fed with Bushen Tiaojing Recipe, compared with model group: serum T-AOC in normal group, high-dose group, low dose in proestrus and post-estrous was significantly increased (both P<0.05), High-and low-dose group with a significant difference between (P<0.05), were not returned to normal (both P<0.05); serum MDA in normal group, high-dose group proestrus and post-estrous was significantly lower (all P<0.05), high-dose group in proestrus and post-estrus was returned to normal (both P>0.05); The normal group, high-dose group, low dose group proestrus and post-estrus in ovarian tissue T-AOC was significantly higher (all P<0.05), the difference between the high and low dose groups was significantly (P<0.05), high-dose proestrus and post-estrus and low-dose proestrus were returned to normal (all P>0.05). The normal group, high-dose group, low dose group proestrus and post-estrus in ovarian tissue MDA were significantly decreased (all P<0.05), high and low doses of proestrus and post-estrus were no significant differences between each part (all P>0.05), high and low-dose before and metaestrus were all returned to normal (all P>0.05).Androgen cause no ovulation model rats fed with Bushen Tiaojing Recipe, compared with the model group: serum Tbil in normal group, high-dose group, low-dose group, both before and metaestrus was significantly increased(all P<0.05), high and low-dose group between before and metaestrus were significantly different (all P<0.05), high-dose group before and metaestrus were returned to normal (all P>0.05); serum Dbil between each group had no significant difference (all P>0.05); the normal group, high-dose group before and metaestrus of the serum Dbil in ovarian tissue were significantly increased (all P<0.05), high-dose both early and late estrogen returned to normal (both P>0.05).1.1.2 The effect of Bushen Tiaojing Recipe on the plasma T-AOC, MDA in ovulation and ovarian tissue of the two anovulatory rats modelsAnovulatory rats induced by androgen and tripterygium with Bushen Tiaojing Recipe, compared with the model group: normal group, high-dose group, low dose group before and metaestrus in serum T-AOC were significantly increased (all P<0.05), high and low-dose group with a significant difference (P<0.05), were not returned to normal (both P<0.05); the normal group, high-dose group before and metaestrus of serum MDA were significantly lower (all P<0.05), high dose group before and metaestrus was returned to normal (both P>0.05); the normal group, high-dose group, low dose group before and metaestrus of ovarian tissue T-AOC was significantly higher (P<0.05), the difference between the high and low dose groups was significantly(P<0.05), high-dose before and metaestrus and low-dose proestrus were returned to normal (all P>0.05); the normal group, high-dose group, low dose group before and metaestrus of MDA in ovarian tissue were significantly decreased (all P<0.05), high and low doses before and metaestrus were no significant differences (all P>0.05), high and low-dose before and metaestrus were returned to normal (all P>0.05).Androgen cause no ovulation model rats fed with Bushen Tiaojing Recipe, compared with the model group: normal group, high-dose group, low dose group before and metaestrus of serum T-AOC were significantly increased (both P<0.05), high and low-dose group before and metaestrus were significantly different (all P<0.05), high-dose group before and metaestrus was returned to normal (both P>0.05), low-dose group proestrus was returned to normal (P>0.05); the normal group, high-dose group, low dose group before and metaestrus of serum MDA were significantly decreased (all P<0.05), proestrus of the high and low-dose group were significant difference (P<0.05), high-dose group was higher than the low dose group. The latter part of high and low dose of estrogen was no significant difference (P>0.05), high and low dose before and metaestrus were returned to normal (all P>0.05); the normal group, high-dose group, low dose group before and metaestrus of T-AOC in ovarian tissue were significantly increased (all P<0.05), high and low dose group before and metaestrus were significant difference (all P<0.05), high-dose before and metaestrus was returned to normal (both P>0.05), low-dose metaestrus was returned to normal (P>0.05), the normal group, high-dose group, low-dose group before and metaestrus of MDA in ovarian tissue were significantly decreased (all P<0.05), high and low doses before and metaestrus were no significant difference (all P>0.05), high and low-dose before and metaestrus were returned to normal (all P>0.05).1.2 The Effect of Bushen Tiaojing Recipe on rats with two kinds of ovulation disorder on ovarian granulosa cell apoptosisTriptolide-induced ovulation disorder rats fed with Bushen Tiaojing Recipe, immunohistochemistry results of Caspase-3 and TUNEL each group showed the order of the size of the average gray scale as follows: model group0.05), high and low-dose before and metaestrus were significant differences (all P<0.05), high-dose was superior to low-dose group. The anovulatory rats induced by androgen and tripterygium with Bushen Tiaojing Recipe, immunohistochemistry results of Caspase-3 and TUNEL showed the size of the average gray scale as follows: model group 0.05); high and low-dose group was significant difference (P<0.05), high-dose was superior to low-dose group.2 The impact of Xiaoyao Pill to two kinds of anovulation model rats with oxidative damage and ovarian granulosa cell apoptosis2.1 The effect of Xiaoyao Pill to two kinds of anovulation rat model with ovary oxidative damage2.1.1 The effect of Xiaoyao Pill to two kinds of anovulation rat model on serum and bilirubin of ovarianTriptolide-induced ovulation disorder rats fed with Xiaoyao Pill, the comparison with model group: serum Tbil in normal group, high-dose group proestrus and post-estrus was significantly increased (all P<0.05), proestrus and post-estrus of high-dose group were returned to normal (all P>0.05); serum Dbil in the normal group and high-dose group with Xiaoyao Pill proestrus and post-estrus was significantly different (all P<0.05), high dose group with Xiaoyao Pill proestrus and post-estrus was returned to normal (both P>0.05); Tbil in ovarian tissue of each group was no significant difference (all P>0.05).Model of androgen-induced anovulation rats after gavaging Xiaoyao Pill, compared with model group: normal group, high-dose group, low dose group proestrus and post-estrus in serum T-AOC were significantly increased (all P<0.05), high and low-dose group had no significant difference (P>0.05), proestrus and post-estrus in high-dose group and proestrus in low-dose were returned to normal (all P>0.05); serum MDA in the normal group, high-dose group, low dose group proestrus and post-estrus was significantly lower (both P<0.05), high and low-dose group had no significant difference (both P>0.05), before and metaestrus of high-dose group were returned to normal (all P>0.05); ovarian tissue T-AOC in the normal group, high-dose group, low-dose group proestrus and post-estrus was significantly increased (all P<0.05), high and low-dose was no significant difference (both P>0.05), high and low-dose proestrus did not meet the normal (both P<0.05), post-estrogen was returned to normal (P>0.05), ovarian tissue MDA in the normal group, high-dose group proestrus and post-estrus was significantly decreased (all P<0.05), high-dose both early and late emotional was returned to normal (all P>0.05).2.1.2 The effect of Xiaoyao Pill on plasma and ovarian tissue T-AOC, MDA of two kinds of anovulation model ratsTriptolide-induced ovulation disorder rats fed with Xiaoyao Pill, compared with the model group: normal group, high-dose group, low dose group proestrus and post-estrus in serum T-AOC were significantly increased (all P<0.05), the difference between high and Low-dose before and metaestrus was significant (both P<0.05), high and low-dose before and metaestrus were not returned to normal (all P<0.01); serum MDA of each group was no significant difference(P>0.05); ovarian tissue T-AOC of the normal group, high-dose group, low dose group proestrus and post-estrus was significantly increased (all P>0.05), high and low-dose was no significant difference (both P>0.05), high and low-dose estrogen was close to normal (both P>0.05), post-estrus was not returned to normal (both P<0.05); ovarian tissue MDA in normal group, high-dose group, low-dose group proestrus and post-estrus was significantly decreased (all P<0.05), high and low-dose had no significant difference (both P>0.05), high and low-dose before and metaestrus were returned to normal (all P>0.05).Model of androgen-induced anovulation rats after gavaging Xiaoyao Pill, compared with model group: normal group, high-dose group, low dose group proestrus and post-estrus in serum T-AOC were significantly increased (both P<0.05), high and low-dose group had no significant difference (P>0.05), proestrus and post-estrus in high-dose group and proestrus in low-dose were returned to normal (all P>0.05); serum MDA in the normal group, high-dose group, low dose group proestrus and post-estrus was significantly lower (both P<0.05), high and low-dose group had no significant difference (both P>0.05), before and metaestrus high-dose group were returned to normal (both P>0.05); ovarian tissue T-AOC in the normal group, high-dose group, low-dose group proestrus and post-estrus was significantly increased (all P<0.05 ), high and low-dose was no significant difference(both P>0.05), proestrus high and low-dose did not meet the normal (both P<0.05), post-estrogen was returned to normal (P>0.05); ovarian tissue MDA in the normal group, high-dose group proestrus and post-estrus was significantly decreased (all P<0.05), high-dose in both early and late emotional was returned to normal (all P>0.05).2.2 The effect of Xiaoyao Pill to ovarian granulosa cell apoptosis on two kinds of anovulation ratsTriptolide-induced ovulation disorder rats fed with Xiaoyao Pill, immunohistochemistry results of Caspase-3 and TUNEL in each group showed the average sequence of gray as follows: model group0.05).3 The differences between the effect of Bushen Tiaojing Recipe and Xiaoyao Pill to ovary oxidative damage and ovarian granulosa cell apoptosis of two kinds of anovulation model rats3.1 The differences between the effect of Bushen Tiaojing Recipe and Xiaoyao Pill to ovary oxidative damage of two kinds of anovulation model rats3.1.1 The effect of Bushen Tiaojing Recipe and Xiaoyao Pill to plasma and ovarian tissue T-AOC, MDA of two kinds of anovulation model ratsTriptolide-induced ovulation disorder rats fed with Bushen Tiaojing Recipe and Xiaoyao Pill, high-dose of Bushen Tiaojing Recipe, and low dose of Xiaoyao Pill proestrus had significant difference (P<0.05), other groups proestrus and metaestrus had no significant difference (all P> 0.05). Proestrus and post-estrus of Bushen Tiaojing Recipe and Xiaoyao Pill had no significant difference (all P>0.05). Ovarian tissue Tbil in each group proestrus and post-estrus had no significant difference (all P> 0.05).Androgen cause no ovulation model rats fed with Bushen Tiaojing Recipe and Xiaoyao Pill, high-dose of Bushen Tiaojing Recipe and low dose of Xiaoyao Pill proestrus had significant differences (all P<0.05); high-dose of Bushen Tiaojing Recipe and high and low dose of Xiaoyao Pill had significantly different in metaestrus(all P<0.05). Bushen Tiaojing Recipe group and Xiaoyao Pill group proestrus and post-estrus had no significant difference (all P>0.05). Ovarian tissue Tbil in each group proestrus and post-estrus had no significant difference (all P>0.05).3.1.2 The differences between the effect of Bushen Tiaojing Recipe and Xiaoyao Pill on plasma and ovarian tissue T-AOC, MDA of two kinds of anovulation model ratsTriptolide-induced ovulation disorder rats fed with Bushen Tiaojing Recipe and Xiaoyao Pill, serum T-AOC in high-dose of Bushen Tiaojing Recipe and high-and low-dose of Xiaoyao Pill group was significant differences (all P<0.05); serum MDA of Bushen Tiaojing Recipe and Xiaoyao Pill proestrus and post estrus had no significant difference (all P> 0.05). Ovarian tissue T-AOC in high-dose of Bushen Tiaojing Recipe and high and low-dose of Xiaoyao Pill had significant differences (all P<0.05); ovarian tissue MDA of Bushen Tiaojing Recipe and Xiaoyao Pill group proestrus and post-estrus had no significant differences (all P>0.05).Anovulation in androgen- rats fed with Bushen Tiaojing Recipe and Xiaoyao Pill, serum T-AOC in high and low-dose of Bushen Tiaojing Recipe and low-dose of Xiaoyao Pill proestrus and post-estrus had significant differences(P<0.05); proestrus high-dose of Bushen Tiaojing Recipe and low-dose of Xiaoyao Pill was significantly different (P<0.05). Ovarian tissue T-AOC in high-dose of Bushen Tiaojing Recipe and high and low-dose of Xiaoyao Pill had significant differences (all P<0.05); ovarian tissue MDA in Bushen Tiaojing Recipe and Xiaoyao Pill proestrus and post-estrus was no significant difference (P>0.05).3.2 The differences between the effect of Bushen Tiaojing Recipe and XiaoyaoPill on ovarian granulosa cell apoptosis of two kinds of anovulation model rats Triptolide-induced ovulation disorder rats fed with Bushen Tiaojing Recipe and Xiaoyao Pill, immunohistochemistry results ofåµå·¢é¢—粒细胞Caspase-3 and TUNEL showed the average gray scale as follows: model group0.05).Triptolide-induced ovulation disorder rats fed with Bushen Tiaojing Recipe and Xiaoyao Pill, immunohistochemistry results of Caspase-3 and TUNEL showed the size of the average gray scale as follows: model group0.05). High-dose group was better than low-dose group, Bushen Tiaojing Recipe was better than Xiaoyao Pill with high-dose. Granulosa cell apoptosis rate in High-dose of Bushen Tiaojing Recipe and high-and low-dose of Xiao-Yao Pill proestrus and post-estrus was significantly different (all P<0.05), apoptosis of high-dose of Bushen Tiaojing Recipe was lower than Xiaoyao Pill with high-and low-dose group.Conclusions: (1) Bushen Tiaojing Recipe and Xiaoyao Pill can both improve oxidative damage and ovarian granulosa cells apoptosis of ovulation disorder model rat to improve the ovarian function.(2) Bushen Tiaojing Recipe and Xiaoyao Pill had no deference in the regulation to tripterygium wilfordi cause low reproductive rats with ovary oxidative damage and ovarian granulosa cell apoptosis. Bushen Tiaojing Recipe was superior to Xiaoyao Pill in androgen cause no ovulation model rats with ovary oxidative damage and ovarian granulosa cell apoptosis.
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