基于PI3K/AKT信号通路研究新加归肾丸促卵泡发育的分子机制
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摘要
实验一：雷公藤多苷致卵泡发育障碍大鼠模型的构建
目的：在前期研究的基础之上,复制雷公藤多苷致卵泡发育障碍大鼠模型,用于本课题的实验研究。
方法：参照前期的实验研究,将符合实验条件的SD大鼠行阴道脱落细胞涂片,根据阴道涂片结果选出动情周期正常的16只为受试对象,随机分为2组,即模型和正常对照组,每组8只。其中模型组以雷公藤多苷40mg/kg·d剂量连续灌胃10周造模,正常对照组每日灌服等量的蒸馏水。造模第8周始行阴道脱落细胞涂片检查以判断动情周期变化,当所有大鼠出现动情周期延长或者紊乱时确定造模成功。股动脉采血检测性激素水平；剖腹取出双侧卵巢称重,并计算卵巢指数：卵巢组织切片HE染色,光学显微镜下观察卵泡的生长情况和闭锁情况,进一步验证模型是否成功。
结果：经持续雷公藤多苷40mg/kg·d灌胃10周后,与正常对照组相比较,模型组动物表现为动情周期延长、紊乱,差异具有统计学意义(P<0.01)；血清E2、P降低,差异具有统计学意义(P<0.01),血清FSH、LH差异无统计学意义(P>0.05)；卵巢湿重和指数均降低,差异具有统计学意义(P<0.05或P<0.01)；生长卵泡数目及黄体数目均减少,闭锁卵泡数目增多,差异具有统计学意义(P<0.05或P<0.01)。这些表现均与人类的卵泡发育障碍性疾病相类似,确定造模成功。
结论：雷公藤多苷40mg/kg·d剂量连续灌胃10周,可成功建造卵泡发育障碍大鼠模型。
实验二：新加归肾丸对雷公藤多苷致卵泡发育障碍模型大鼠卵巢PI3K/AKT信号通路的影响
目的：在实验一基础之上,以与卵泡发育密切相关的PI3K/AKT信号通路为切入点,再次复制雷公藤多苷致卵泡发育障碍大鼠模型,并以此为实验对象,研究雷公藤多苷抑制卵泡发育以及补肾活血中药复方新加归肾丸促卵泡发育与卵巢PI3K/AKT信号通路的关系,从细胞增殖和细胞凋亡两方面深入探讨补肾活血中药复方新加归肾丸促卵泡发育的分子机制。
方法：参照实验一的造模方法,建立45只卵泡发育障碍的大鼠模型,随机分为5组,每组9只,分别为：模型对照组、西药对照组、中药高剂量组、中药中剂量组、中药低剂量组,加上造模前随机取出的9只(正常对照组),共6组。中药各剂量组分别给予补肾活血中药复方新加归肾丸高、中、低剂量治疗；西药对照组给予戊酸雌二醇和醋酸甲羟孕酮序贯治疗；模型对照组和正常对照组给予等量的蒸馏水灌胃。所有动物均灌胃2周,灌胃期间行阴道脱落细胞涂片检查观察大鼠动情周期变化：灌胃结束后采血,检测血清E2、P、FSH、LH水平；采血后处死大鼠,称取体重,摘取双侧卵巢组织,称取卵巢湿重,计算卵巢指数；卵巢切片HE染色,光学显微镜下计数各级卵泡、闭锁卵泡及黄体的数目,并测量颗粒细胞的厚度；采用western-blot法分别检测卵巢组织中PI3K/AKT信号通路中的主要信号分子p-AKT、p-mTOR、p-p70S6K蛋白含量；采用RT-PCR去检测卵巢组织中cyclinD2mRNA、BaxmRNA的表达水平；采用免疫组化法检p-AKT、 cyclinD2、Caspase-3、PTEN在卵巢颗粒细胞中的表达。分别将cyclinD2、 Caspase-3、BaxmRNA与p-AKT在卵巢颗粒细胞中的表达进行相关性分析。
结果：①与正常对照组相比,模型对照组大鼠动情周期延长,体重、卵巢湿重、卵巢指数、血清E2、P水平均降低,各级生长卵泡数、黄体数目减少,闭锁卵泡数目增多,颗粒细胞厚度降低,卵巢p-AKT、p-mTOR、p-p70S6K、cyclinD2、 cyclinD2mRNA水平均降低,差异均具有统计学意义(P<0.05或P<0.01)；Caspase-3表达均升高,差异均具有统计学意义(P<0.01)：血清FSH、LH水平和卵巢颗粒细胞PTEN表达差异均无统计学意义(P>0.05)。②应用不同剂量的补肾活血中药复方新加归肾丸治疗2周后,其中中药高、中剂量组与模型对照组相比较,大鼠动情周期缩短,体重、卵巢湿重、卵巢指数、血清E2、P水平均升高,各级生长卵泡数、黄体数目增多,闭锁卵泡数目减少,颗粒细胞厚度增加,卵巢p-mTOR、p-p70S6K、cyclinD2mRNA水平均升高,差异均具有统计学意义(P<0.05或P<0.01); BaxmRNA、Caspase-3表达均降低,差异均具有统计学意义(P<0.05或P<0.01)；血清FSH、LH和卵巢PTEN差异均无统计学意义(P>0.05)。中药低剂量组大鼠体重和颗粒细胞Caspase-3表达较模型组升高,差异具有统计学意义(P<0.05或P<0.01),其他指标差异均无统计学意义(P>0.05)。③相关性分析结果：颗粒细胞中cyclinD2与p-AKT表达呈显著性正相关关系(r=0.833,P<0.01)；颗粒细胞中Caspase-3与p-AKT表达呈显著性负相关关系(r=-0.843,P<0.01)；卵巢中BaxmRNA与颗粒细胞中p-AKT的表达呈显著性负相关关系(r=-0.747,P<0.01)。
结论：雷公藤多苷可能通过抑制卵巢组织中PI3K/AKT信号通路中信号分子AKT的级联激活反应,一方面抑制其下游信号分子mTOR、p70S6K的活化,使有丝分裂信号传递受阻,颗粒细胞cyclinD2表达下调,阻碍细胞周期进程,抑制颗粒细胞增殖；另一方面上调促凋亡因子BaxmRNA、Caspase-3的表达,促使颗粒细胞过度凋亡,进而导致卵泡过度闭锁,卵泡生长、发育、成熟障碍,卵巢功能损伤。而补肾活血中药复方新加归肾丸可能通过上调p-AKT的表达,增强PI3K/AKT信号通路的活性而解除雷公藤多苷的抑制作用,一方面促使mTOR、p70S6K活化,下传有丝分裂信号,使颗粒细胞cyclinD2表达增多,促进细胞周期进程,颗粒细胞增殖；另一方面下调促凋亡因子BaxmRNA、Caspase-3的表达,抑制颗粒细胞过度凋亡,进而促使卵泡正常生长、发育、成熟,使受损卵巢功能得以修复。
OBJECTIVE:With follicular development is closely related to the PI3K/AKT signaling pathway,so in this experimental study we put this signaling pathway as the breakthrough point, reproduce rat model of follicular development disorder caused by GTW on the basis of previous research.To further explore the molecular mechanism of Compound Herbal of Xinjiaguishenwan with the functions of Reinforcing Kidney and Activating Blood on stimulating follicle development from two aspects of cell proliferation and apoptosis. Tries to reveal the connotation of traditional Chinese medicine to improve ovarian function.
METHODS:Reference to the pre-experimental study,the vaginal cytology smears of SD rats in child-bearing period were made every day. We selected54rats with normal estrous cycle as subjects according to the results of vaginal smears. Nine of them were randomly selected as normal control control group, the remaining45rats were were all given intra-gastric administration with GTW40mg/(kg-d) for10weeks to make the required models,and the blank control group rats were given intra-gastric administration with distilled water.Monitored the exchanges of estrous cycles of rats through the variation of vaginal cytology smears from the eighth week.When all the rats showed irregular estrous cycle,we believed the models were successfully made.Then we divided the45rats into5groups randomly-the model control group, the western medicine control group, three different dosages (high, medium and low) of herbal compound Xinjiaduishenwan groups, with the normal control group,there were6groups altogether in this experiment, every group with9rats. The normal control group and model control groups were filled daily with distilled water; The western medicine control group were treated with estrogen and progestin sequential through intra-gastric administration; three different dosages of herbal compound Xinjiaduishenwan group were given different dose of Chinese medicine in the same way. All groups were treated for2weeks ontinuously. Vaginal exfoliated cells smears were operated during the treatment to observe oestrous cycle of the rats.24hours after the last treatment,all the rats were killed after taken femoral artery blood. Then the rats'abdominal cavity were opened and both ovaries were taken out. We taken the weight of the ovary and calculated the ovary index, detected the concentration of serum "E2","P","FSH"and"LH" with RIA and ELISA. Stained the paraffin sections of ovarian with HE, counted primordial, scondary, antral, atresia follicles and corpus luteum under optical microscope,measured the thickness of the granulosa cells of secondary and antral follicles. Detected the expression of "p-AKT""p-mTOR"and "p-p70S6K" in ovarian with Western blotting; cyclinD2mRNA and BaxmRNA with RT-PCR;"PTEN","cyclinD2","p-AKT","Caspase-3" with immunohistochemical.
RESULTS:
GTW40mg/(kg-d) gavage for10weeks can result in follicular development disorder of the child-bearing period SD rats. They showed the disorders of estrous cycle, the decreasing of ovarian indexes, level of serum E2and P, thickness of the granulosa cells, number of growing follicles, number of corpus luteum, expression of p-AKT, p-mTOR, p-p70S6K, mRNA levels of cyclinD2in rat ovary;the increasing of number of atresia follicles, expression of Caspase-3, mRNA levels of Bax in ovary.The above indicators had significant difference compared with the normal control group (P<0.05or P<0.01).No changes of serum FSH, serum LH, PTEN, The above indicators had no significant difference (p>0.05).
After treatment with Xinjiaguishenwan for two weeks,the model rats had obvious improvement in their general conditions. They showed normal or close to the normal estrous cycles, the increasing of ovarian indexes, level of serum E2, P, thickness of granular cell, number of growing follicles and corpus luteum, expression of p-AKT, p-mTOR p-p70S6K, mRNA levels of cyclinD2, and obvious decreasing of the number of atresia follicles, expression of Caspase-3, mRNA levels of Bax in ovary. The above indicators had significant difference compared with the model control group (P<0.05or P<0.01).The effect of high and medium dosages of herbal compound group were more obvious than low dosage.
CONCLUSION:
1. GTW40mg/(kg-d) gavage for10weeks can result in follicular development disorder of the child-bearing period SD rats.
2. GTW probably inhibit the cascade activation reaction of AKT in the PI3K/AKT signaling pathway.On the one hand inhibit the activation of downstream signaling molecules-mTOR and p70S6K, block the mitosis signal transmission and down-regulate the expression of cyclinD2in granulosa cells.eventrually inhibit the proliferation of granulosa cells by blocking the cell cycle.On the other hand promote the apoptosis of granulosa cells by up-regulating the expression of Bax and Caspase-3.Therefore disorder the follicle's normal development and damage the function of ovarian.
3.Compound Herbal of Xinjiaguishenwan with the functions Reinforcing Kidney and Activating Blood can restore the activity of PI3K/AKT signaling pathway through up-regulating the expression of p-AKT.On the one hand activate mTOR and p70S6K,downlink the mitosis signal,up-regulate the expression of cyclinD2in granulosa cells.Eventrually promote the proliferation of granulosa cells by accelerating cell cycle process.On the other hand inhibite the apoptosis of granulosa cells by down-regulating the expression of Bax and Caspase-3.Therefore resume the development of follicle, renovate the damaged function of the ovaries.
目的：在前期研究的基础之上,复制雷公藤多苷致卵泡发育障碍大鼠模型,用于本课题的实验研究。
方法：参照前期的实验研究,将符合实验条件的SD大鼠行阴道脱落细胞涂片,根据阴道涂片结果选出动情周期正常的16只为受试对象,随机分为2组,即模型和正常对照组,每组8只。其中模型组以雷公藤多苷40mg/kg·d剂量连续灌胃10周造模,正常对照组每日灌服等量的蒸馏水。造模第8周始行阴道脱落细胞涂片检查以判断动情周期变化,当所有大鼠出现动情周期延长或者紊乱时确定造模成功。股动脉采血检测性激素水平；剖腹取出双侧卵巢称重,并计算卵巢指数：卵巢组织切片HE染色,光学显微镜下观察卵泡的生长情况和闭锁情况,进一步验证模型是否成功。
结果：经持续雷公藤多苷40mg/kg·d灌胃10周后,与正常对照组相比较,模型组动物表现为动情周期延长、紊乱,差异具有统计学意义(P<0.01)；血清E2、P降低,差异具有统计学意义(P<0.01),血清FSH、LH差异无统计学意义(P>0.05)；卵巢湿重和指数均降低,差异具有统计学意义(P<0.05或P<0.01)；生长卵泡数目及黄体数目均减少,闭锁卵泡数目增多,差异具有统计学意义(P<0.05或P<0.01)。这些表现均与人类的卵泡发育障碍性疾病相类似,确定造模成功。
结论：雷公藤多苷40mg/kg·d剂量连续灌胃10周,可成功建造卵泡发育障碍大鼠模型。
实验二：新加归肾丸对雷公藤多苷致卵泡发育障碍模型大鼠卵巢PI3K/AKT信号通路的影响
目的：在实验一基础之上,以与卵泡发育密切相关的PI3K/AKT信号通路为切入点,再次复制雷公藤多苷致卵泡发育障碍大鼠模型,并以此为实验对象,研究雷公藤多苷抑制卵泡发育以及补肾活血中药复方新加归肾丸促卵泡发育与卵巢PI3K/AKT信号通路的关系,从细胞增殖和细胞凋亡两方面深入探讨补肾活血中药复方新加归肾丸促卵泡发育的分子机制。
方法：参照实验一的造模方法,建立45只卵泡发育障碍的大鼠模型,随机分为5组,每组9只,分别为：模型对照组、西药对照组、中药高剂量组、中药中剂量组、中药低剂量组,加上造模前随机取出的9只(正常对照组),共6组。中药各剂量组分别给予补肾活血中药复方新加归肾丸高、中、低剂量治疗；西药对照组给予戊酸雌二醇和醋酸甲羟孕酮序贯治疗；模型对照组和正常对照组给予等量的蒸馏水灌胃。所有动物均灌胃2周,灌胃期间行阴道脱落细胞涂片检查观察大鼠动情周期变化：灌胃结束后采血,检测血清E2、P、FSH、LH水平；采血后处死大鼠,称取体重,摘取双侧卵巢组织,称取卵巢湿重,计算卵巢指数；卵巢切片HE染色,光学显微镜下计数各级卵泡、闭锁卵泡及黄体的数目,并测量颗粒细胞的厚度；采用western-blot法分别检测卵巢组织中PI3K/AKT信号通路中的主要信号分子p-AKT、p-mTOR、p-p70S6K蛋白含量；采用RT-PCR去检测卵巢组织中cyclinD2mRNA、BaxmRNA的表达水平；采用免疫组化法检p-AKT、 cyclinD2、Caspase-3、PTEN在卵巢颗粒细胞中的表达。分别将cyclinD2、 Caspase-3、BaxmRNA与p-AKT在卵巢颗粒细胞中的表达进行相关性分析。
结果：①与正常对照组相比,模型对照组大鼠动情周期延长,体重、卵巢湿重、卵巢指数、血清E2、P水平均降低,各级生长卵泡数、黄体数目减少,闭锁卵泡数目增多,颗粒细胞厚度降低,卵巢p-AKT、p-mTOR、p-p70S6K、cyclinD2、 cyclinD2mRNA水平均降低,差异均具有统计学意义(P<0.05或P<0.01)；Caspase-3表达均升高,差异均具有统计学意义(P<0.01)：血清FSH、LH水平和卵巢颗粒细胞PTEN表达差异均无统计学意义(P>0.05)。②应用不同剂量的补肾活血中药复方新加归肾丸治疗2周后,其中中药高、中剂量组与模型对照组相比较,大鼠动情周期缩短,体重、卵巢湿重、卵巢指数、血清E2、P水平均升高,各级生长卵泡数、黄体数目增多,闭锁卵泡数目减少,颗粒细胞厚度增加,卵巢p-mTOR、p-p70S6K、cyclinD2mRNA水平均升高,差异均具有统计学意义(P<0.05或P<0.01); BaxmRNA、Caspase-3表达均降低,差异均具有统计学意义(P<0.05或P<0.01)；血清FSH、LH和卵巢PTEN差异均无统计学意义(P>0.05)。中药低剂量组大鼠体重和颗粒细胞Caspase-3表达较模型组升高,差异具有统计学意义(P<0.05或P<0.01),其他指标差异均无统计学意义(P>0.05)。③相关性分析结果：颗粒细胞中cyclinD2与p-AKT表达呈显著性正相关关系(r=0.833,P<0.01)；颗粒细胞中Caspase-3与p-AKT表达呈显著性负相关关系(r=-0.843,P<0.01)；卵巢中BaxmRNA与颗粒细胞中p-AKT的表达呈显著性负相关关系(r=-0.747,P<0.01)。
结论：雷公藤多苷可能通过抑制卵巢组织中PI3K/AKT信号通路中信号分子AKT的级联激活反应,一方面抑制其下游信号分子mTOR、p70S6K的活化,使有丝分裂信号传递受阻,颗粒细胞cyclinD2表达下调,阻碍细胞周期进程,抑制颗粒细胞增殖；另一方面上调促凋亡因子BaxmRNA、Caspase-3的表达,促使颗粒细胞过度凋亡,进而导致卵泡过度闭锁,卵泡生长、发育、成熟障碍,卵巢功能损伤。而补肾活血中药复方新加归肾丸可能通过上调p-AKT的表达,增强PI3K/AKT信号通路的活性而解除雷公藤多苷的抑制作用,一方面促使mTOR、p70S6K活化,下传有丝分裂信号,使颗粒细胞cyclinD2表达增多,促进细胞周期进程,颗粒细胞增殖；另一方面下调促凋亡因子BaxmRNA、Caspase-3的表达,抑制颗粒细胞过度凋亡,进而促使卵泡正常生长、发育、成熟,使受损卵巢功能得以修复。
OBJECTIVE:With follicular development is closely related to the PI3K/AKT signaling pathway,so in this experimental study we put this signaling pathway as the breakthrough point, reproduce rat model of follicular development disorder caused by GTW on the basis of previous research.To further explore the molecular mechanism of Compound Herbal of Xinjiaguishenwan with the functions of Reinforcing Kidney and Activating Blood on stimulating follicle development from two aspects of cell proliferation and apoptosis. Tries to reveal the connotation of traditional Chinese medicine to improve ovarian function.
METHODS:Reference to the pre-experimental study,the vaginal cytology smears of SD rats in child-bearing period were made every day. We selected54rats with normal estrous cycle as subjects according to the results of vaginal smears. Nine of them were randomly selected as normal control control group, the remaining45rats were were all given intra-gastric administration with GTW40mg/(kg-d) for10weeks to make the required models,and the blank control group rats were given intra-gastric administration with distilled water.Monitored the exchanges of estrous cycles of rats through the variation of vaginal cytology smears from the eighth week.When all the rats showed irregular estrous cycle,we believed the models were successfully made.Then we divided the45rats into5groups randomly-the model control group, the western medicine control group, three different dosages (high, medium and low) of herbal compound Xinjiaduishenwan groups, with the normal control group,there were6groups altogether in this experiment, every group with9rats. The normal control group and model control groups were filled daily with distilled water; The western medicine control group were treated with estrogen and progestin sequential through intra-gastric administration; three different dosages of herbal compound Xinjiaduishenwan group were given different dose of Chinese medicine in the same way. All groups were treated for2weeks ontinuously. Vaginal exfoliated cells smears were operated during the treatment to observe oestrous cycle of the rats.24hours after the last treatment,all the rats were killed after taken femoral artery blood. Then the rats'abdominal cavity were opened and both ovaries were taken out. We taken the weight of the ovary and calculated the ovary index, detected the concentration of serum "E2","P","FSH"and"LH" with RIA and ELISA. Stained the paraffin sections of ovarian with HE, counted primordial, scondary, antral, atresia follicles and corpus luteum under optical microscope,measured the thickness of the granulosa cells of secondary and antral follicles. Detected the expression of "p-AKT""p-mTOR"and "p-p70S6K" in ovarian with Western blotting; cyclinD2mRNA and BaxmRNA with RT-PCR;"PTEN","cyclinD2","p-AKT","Caspase-3" with immunohistochemical.
RESULTS:
GTW40mg/(kg-d) gavage for10weeks can result in follicular development disorder of the child-bearing period SD rats. They showed the disorders of estrous cycle, the decreasing of ovarian indexes, level of serum E2and P, thickness of the granulosa cells, number of growing follicles, number of corpus luteum, expression of p-AKT, p-mTOR, p-p70S6K, mRNA levels of cyclinD2in rat ovary;the increasing of number of atresia follicles, expression of Caspase-3, mRNA levels of Bax in ovary.The above indicators had significant difference compared with the normal control group (P<0.05or P<0.01).No changes of serum FSH, serum LH, PTEN, The above indicators had no significant difference (p>0.05).
After treatment with Xinjiaguishenwan for two weeks,the model rats had obvious improvement in their general conditions. They showed normal or close to the normal estrous cycles, the increasing of ovarian indexes, level of serum E2, P, thickness of granular cell, number of growing follicles and corpus luteum, expression of p-AKT, p-mTOR p-p70S6K, mRNA levels of cyclinD2, and obvious decreasing of the number of atresia follicles, expression of Caspase-3, mRNA levels of Bax in ovary. The above indicators had significant difference compared with the model control group (P<0.05or P<0.01).The effect of high and medium dosages of herbal compound group were more obvious than low dosage.
CONCLUSION:
1. GTW40mg/(kg-d) gavage for10weeks can result in follicular development disorder of the child-bearing period SD rats.
2. GTW probably inhibit the cascade activation reaction of AKT in the PI3K/AKT signaling pathway.On the one hand inhibit the activation of downstream signaling molecules-mTOR and p70S6K, block the mitosis signal transmission and down-regulate the expression of cyclinD2in granulosa cells.eventrually inhibit the proliferation of granulosa cells by blocking the cell cycle.On the other hand promote the apoptosis of granulosa cells by up-regulating the expression of Bax and Caspase-3.Therefore disorder the follicle's normal development and damage the function of ovarian.
3.Compound Herbal of Xinjiaguishenwan with the functions Reinforcing Kidney and Activating Blood can restore the activity of PI3K/AKT signaling pathway through up-regulating the expression of p-AKT.On the one hand activate mTOR and p70S6K,downlink the mitosis signal,up-regulate the expression of cyclinD2in granulosa cells.Eventrually promote the proliferation of granulosa cells by accelerating cell cycle process.On the other hand inhibite the apoptosis of granulosa cells by down-regulating the expression of Bax and Caspase-3.Therefore resume the development of follicle, renovate the damaged function of the ovaries.
引文
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