景岳归肾丸对肾虚排卵障碍大鼠模型生殖系统的影响
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摘要
目的：
在前期的研究基础上,通过随机对照的研究方法,研究景岳归肾丸对肾虚排卵障碍大鼠模型的激素水平及子宫内膜形态的影响。客观观察评价景岳归肾丸对肾虚排卵障碍大鼠激素水平及子宫内膜形态的疗效及其安全性,为中医药防治排卵障碍性疾病的临床研究及其新药开发建立必备的评估系统和提供严谨的科研资料。
方法：
研究对象为72只雌性大鼠,随机分为阴性对照组、模型组、克罗米芬组、归肾丸高、中、低剂量组,12只/组。造模：除阴性对照组外,其他各组大鼠肌注氢化可的松注射液5ml·kg-1·d-1,阴性对照组大鼠肌注等量的生理盐水,连续造模14d。给药：于造模第15d,各组大鼠10ml/kg体重给予相应药物,归肾丸低、中、高剂量组给予归肾丸0.57g生药/ml、1.14g生药/ml、2.28g生药/ml归肾丸溶液,阴性对照组、模型对照组给予蒸馏水,克罗米芬组给予0.54mg/ml枸橼酸氯米芬片药液(克罗米芬),连续15d。末次给药1h,乙醚麻醉眼眶静脉丛采血,颈椎脱臼法处死大鼠,解剖取样检测相应指标。
结果：
1.性周期：各给药组大鼠造模后第5天开始,除阴性对照组外,其余各组大鼠阴道脱落细胞性周期延长、不规则。第11d起,阴性对照组无明显周期改变,其余各组表现为周期紊乱,长短不一,甚至消失。
2.饲料消耗量：与阴性对照组比较,模型组大鼠在给药D1、D7的饲料消耗量有显著性减少(P<0.05)；与模型组比较,归肾丸高剂量组大鼠在给药D14、D21的饲料消耗量显著性减少(P<0.05),归肾丸低剂量组大鼠在给药D21的饲料消耗量显著性减少(P<005),其他各组大鼠在其他时间点的饲料消耗量组均无显著性差异(P>0.05)。
3.饮水消耗量：与模型组比较,归肾丸中、高剂量组大鼠在D14的饮水消耗量显著性增加(P<0.05),其他各组大鼠在其他时间点的饮水消耗量均无显著性差异(P>0.05)。
4.脏器系数：模型组大鼠肾上腺系数与阴性对照组比较显著性升高(P<0.05),克罗米芬组的子宫系数、卵巢系数与模型组比较有显著性下降(P<005)。归肾丸低、中、高剂量组大鼠的子宫、卵巢脏器系数与模型组比较均无显著性差异(P>0.05)。
5.激素水平：模型组大鼠血清雌二醇(E2)含量与阴性对照组比较均无显著性差异(P>0.05),各给药组大鼠的血清雌二醇(E2)含量与模型组大鼠比较均无显著性差异(P>0.05)；模型组大鼠血清孕激素(P)水平与阴性对照组比较显著性降低(P<0.05)；归肾丸低剂量组大鼠血清孕激素(P)与模型对照组比较有显著性增加(P<0.05)。其余各给药组大鼠的血清孕激素水平与模型组比较均无显著性差异(P>005)。
6.卵泡生长情况：阴性对照组卵巢可见各级卵泡,数量正常,黄体数目多,未见滤泡囊肿、血肿及肿物形成。模型组卵巢可见生长卵泡(初级卵泡和次级卵泡)、黄体数目均较阴性对照组明显减少,闭锁卵泡数目较阴性对照组明显增加。克罗米芬组卵巢可见生长卵泡、闭锁卵泡数目均较模型组明显增加。归肾丸高剂量组卵巢可见生长卵泡数目较模型组明显增加。归肾丸中剂量组、归肾丸低剂量组卵巢生长卵泡、成熟卵泡、闭锁卵泡、黄体数与模型组比较未见明显差别。
7.子宫内膜形态学变化：阴性对照组子宫壁各层结构清晰,子宫内膜较厚,子宫内膜粘膜上皮呈单层柱状排列,粘膜下固有膜结缔组织内有不分支的子宫腺,腺体数目多,子宫肌层和子宫外膜未见明显病理改变。模型组子宫腺体数、子宫内膜厚度较阴性对照组明显减少。克罗米芬组子宫腺体数较模型组明显减少,子宫内膜厚度较模型组明显减小。归肾丸高剂量组、归肾丸中剂量组、归肾丸低剂量组子宫内膜厚度、腺体数与模型组比较未见明显差别。
本研究发现,模型组卵巢生长卵泡(初级卵泡和次级卵泡)、黄体数目均较阴性对照组明显减少,闭锁卵泡数目较阴性对照组明显增加,模型组子宫腺体数较阴性对照组明显减少。克罗米芬组卵巢生长卵泡、闭锁卵泡数目均较模型组明显增加,子宫腺体数、子宫内膜厚度均较模型组明显减少。归肾丸高剂量组卵巢可见生长卵泡数目较模型组明显增加。实验结果提示归肾丸高剂量可促进肾虚模型SD大鼠卵巢原始卵泡向初级卵泡、次级卵泡发育。
结论：
1、以氢化可的松建立肾虚大鼠模型具有排卵抑制的表现,成功模拟肾虚排卵障碍的临床类型。
2、景岳归肾丸能够改善肾虚排卵障碍模型大鼠的卵泡发育,提高大鼠血清P激索水平
3、归肾丸具有促进卵巢卵泡发育的功能,可能通过调整肾—天癸—冲任—胞宫生殖轴,从而达到肾精渐盛,冲任气血通调,促进卵泡发育和排卵。
Objective
On the foundation of early researches, this randomized control study focuses on Jingyue's Kidney-invigorating Pill's effect on rat models' hormone levels and endometrial morphology who have ovulation difficulties due to kidney deficiency with the hope of giving objective evaluation of its curative effect and safety and providing necessary evaluation system and strict research material for the clinical research and new drug development in the prevention and treatment of ovulation difficulties by Chinese Medicine.
Methods
The study subjects were72female rats randomly divided into the Negative Control Group, the Model Group, Clomiphene Group and Kidney-invigorating Groups (high dose, medium dose and low dose) with12in each group. Modeling: Except the Negative Control Group's rats were given equal volume of intramuscular injection of normal saline, all the other groups' rats were given intramuscular injection of hydrocortisone injection5ml· kg-1· d-1. Conduct continuous modeling for14days. Administration:on the15th day of modeling, administer medicine at10ml/kg of the body weight of rats. The Kidney-invigorating High-, Medium-and Low-dose Groups are given Kidney-invigorating Pill solution at0.57g crude drug/ml,1.14g/ml,2.28g crude drug/ml. The Negative Control Group and the Model Group were given distilled water and the Clomiphene Group was given0.54mg/ml clomiphene citrate tablet liquid (clomiphene citrate) for15continuous days.1H after the last administration, etherize the orbital venous plexus for hemospasia. Kill rats with cervical dislocation method. Conduct anatomical sampling for the detection of corresponding indexes.
Results
1. Sexual Cycle:starting from the fifth day after modeling. Except the Negative Control Group, the rats in each group had prolonged and irregular vaginal exfoliate cell cycle. Since the11th day, the Negative Control Group had no obvious periodic change, while other groups showed irregularities in cycle and length, or even disappearance of the cycle.
2. Weight:Compared with the Negative Control Group, Model Group rats on modeling D1, D7, D14, D29lost weight significantly (P<0.05). Compared with the Model Group, Clomiphene Group rats, after administration of the drug21and29days, lost weight significantly (P<0.05); Rats in Kidney-invigorating Pill Groups of different dose have no significant difference (P>0.05) compared with the Model Group in body weight at different time points.
3. Feed consumption:Compared with the Negative Control Group, Model Group rats' feed consumption significantly reduced (P<0.05) on D1and D7after the administration. Compared with the Model Group, High-dose Group of Kidney-invigorating Pill rats'feed consumption significantly reduced (P <0.05) on D14, D21of administration. The Low-dose Group of Kidney-invigorating Pill rats'feed consumption significantly reduced (P <0.05) on D21of administration. The other groups'rats'feed consumption have no significant difference (P>0.05) at other time points.
4. Water consumption:Compared with the Model Group, Rats in the Medium and High-dose Group of Kidney-invigorating Pill on D14significantly increased water consumption (P<0.05). The other groups' rats at other time points showed no significant difference (P>0.05) in drinking water consumption.
5. Organ coefficient:Model Group rats' adrenal coefficients, compared with the Negative Control Group, significantly increased (P<0.05). Clomiphene Group rats' uterus coefficient and ovarian coefficient, compared with the Model Group, significantly decreased (P<0.05). Kidney-invigorating Pill High, Medium and Low-dose Groups, in terms of uterus and ovary organ coefficient, showed no significant difference (P>0.05) compared with Model Group.
6. Levels of the hormone:Model Group rats'serum estuarial (E2) content, compared with the Negative Control Group, showed no significant difference (P>0.05). Each administration group rats'serum estuarial (E2) content, compared with that of the rats in the Model Group, showed no significant difference (P>0.05). Model Group rats'serum progesterone (P) levels, compared with Negative Control Group, decreased significantly (P<0.05). Kidney-invigorating Pill Low-dose Group rats'serum progesterone (P), compared with the Model Group, significantly increased (P<0.05). The remainder of each group rats'serum progesterone levels, compared with the Model Group, had no significant difference (P>0.05).
7. Follicular Growth:Negative Control Group ovaries have visible follicles with normal number, large number of corpus luteum and without follicular cysts, hematoma or tumor. Model Group had visible ovarian follicles (primary and secondary follicles). Compared with the Negative Control Group, its corpus luteum obviously decreased in number and the atresic follicle increased significantly. The Clomiphene Group had visible growing follicles and follicular atresia which significantly increased compared with the Model Group. Kidney-invigorating Pill High-dose Group had visible ovarian follicle which increased significantly in number than the Model Group. Kidney-invigorating Pill Medium and Low-dose Groups, compared with Model Group, showed no significant difference in ovarian growing follicles, mature follicle, follicular atresia and number of corpus luteum.
8. Endometrial morphologic changes:In the Negative Control Group, the uterine wall had clear layer structure, endometrium was thick, endometrial epithelium had single-layer columnar arrangement, submucosal lamina propria connective tissue had nonbranching uterine glands, glands was in large number and the myometrium and perimetrium had no obvious pathological changes. The Model Group, compared with the Negative Control Group, decreased obviously in the number of uterine glands and endometrial thickness. Clomiphene Group decreased obviously in the number of uterine gland and the endometrium thickness than the Model Group. Compared with the Model Group, the Kidney-invigorating Pill Groups of high dose, medium dose and low dose, in terms of endometrial thickness and uterine gland number, showed no significant difference.
The study found that, compared with the Negative Control Group, the Model Group'ovarian growing follicle (primary follicle and secondary follicle) and corpus luteum number decreased obviously, atresic follicle number increased significantly and the number of uterine glands decreased obviously. Compared with the Model Group, Clomiphene Group's ovarian growing follicle and follicular atresia significantly increased in number and the number of uterine glands and endometrial thickness decreased obviously. Kidney-invigorating Pill High-dose Group had obviously increased ovarian growing follicles than the Model Group. The experimental results indicate Kidney-invigorating Pill with high dose can promote kidney-deficient SD rats' ovarian primordial follicle developing into primary follicle and secondary follicle.
Conclusion
1. The hydrocortisone established kidney-deficient rat models have ovulation inhibition performance. There's successful simulation of clinical types of ovulation difficulties due to deficiency of the kidney.
2. King Yue Kidney-invigorating Pill can improve the follicular development in rat model of ovulation difficulties due to kidney deficiency and, enhance rats' serum P levels.
3. Kidney-invigorating Pill had the function of promoting ovarian follicular development. With the adjustment of reproductive axis formed with kidney-menstruation-Chong and Ren Meridians-uterus. It helps to reinforce the kidney-essence, adjust qi and blood in Chong and Ren meridians and promote the follicular development and ovulation.
在前期的研究基础上,通过随机对照的研究方法,研究景岳归肾丸对肾虚排卵障碍大鼠模型的激素水平及子宫内膜形态的影响。客观观察评价景岳归肾丸对肾虚排卵障碍大鼠激素水平及子宫内膜形态的疗效及其安全性,为中医药防治排卵障碍性疾病的临床研究及其新药开发建立必备的评估系统和提供严谨的科研资料。
方法：
研究对象为72只雌性大鼠,随机分为阴性对照组、模型组、克罗米芬组、归肾丸高、中、低剂量组,12只/组。造模：除阴性对照组外,其他各组大鼠肌注氢化可的松注射液5ml·kg-1·d-1,阴性对照组大鼠肌注等量的生理盐水,连续造模14d。给药：于造模第15d,各组大鼠10ml/kg体重给予相应药物,归肾丸低、中、高剂量组给予归肾丸0.57g生药/ml、1.14g生药/ml、2.28g生药/ml归肾丸溶液,阴性对照组、模型对照组给予蒸馏水,克罗米芬组给予0.54mg/ml枸橼酸氯米芬片药液(克罗米芬),连续15d。末次给药1h,乙醚麻醉眼眶静脉丛采血,颈椎脱臼法处死大鼠,解剖取样检测相应指标。
结果：
1.性周期：各给药组大鼠造模后第5天开始,除阴性对照组外,其余各组大鼠阴道脱落细胞性周期延长、不规则。第11d起,阴性对照组无明显周期改变,其余各组表现为周期紊乱,长短不一,甚至消失。
2.饲料消耗量：与阴性对照组比较,模型组大鼠在给药D1、D7的饲料消耗量有显著性减少(P<0.05)；与模型组比较,归肾丸高剂量组大鼠在给药D14、D21的饲料消耗量显著性减少(P<0.05),归肾丸低剂量组大鼠在给药D21的饲料消耗量显著性减少(P<005),其他各组大鼠在其他时间点的饲料消耗量组均无显著性差异(P>0.05)。
3.饮水消耗量：与模型组比较,归肾丸中、高剂量组大鼠在D14的饮水消耗量显著性增加(P<0.05),其他各组大鼠在其他时间点的饮水消耗量均无显著性差异(P>0.05)。
4.脏器系数：模型组大鼠肾上腺系数与阴性对照组比较显著性升高(P<0.05),克罗米芬组的子宫系数、卵巢系数与模型组比较有显著性下降(P<005)。归肾丸低、中、高剂量组大鼠的子宫、卵巢脏器系数与模型组比较均无显著性差异(P>0.05)。
5.激素水平：模型组大鼠血清雌二醇(E2)含量与阴性对照组比较均无显著性差异(P>0.05),各给药组大鼠的血清雌二醇(E2)含量与模型组大鼠比较均无显著性差异(P>0.05)；模型组大鼠血清孕激素(P)水平与阴性对照组比较显著性降低(P<0.05)；归肾丸低剂量组大鼠血清孕激素(P)与模型对照组比较有显著性增加(P<0.05)。其余各给药组大鼠的血清孕激素水平与模型组比较均无显著性差异(P>005)。
6.卵泡生长情况：阴性对照组卵巢可见各级卵泡,数量正常,黄体数目多,未见滤泡囊肿、血肿及肿物形成。模型组卵巢可见生长卵泡(初级卵泡和次级卵泡)、黄体数目均较阴性对照组明显减少,闭锁卵泡数目较阴性对照组明显增加。克罗米芬组卵巢可见生长卵泡、闭锁卵泡数目均较模型组明显增加。归肾丸高剂量组卵巢可见生长卵泡数目较模型组明显增加。归肾丸中剂量组、归肾丸低剂量组卵巢生长卵泡、成熟卵泡、闭锁卵泡、黄体数与模型组比较未见明显差别。
7.子宫内膜形态学变化：阴性对照组子宫壁各层结构清晰,子宫内膜较厚,子宫内膜粘膜上皮呈单层柱状排列,粘膜下固有膜结缔组织内有不分支的子宫腺,腺体数目多,子宫肌层和子宫外膜未见明显病理改变。模型组子宫腺体数、子宫内膜厚度较阴性对照组明显减少。克罗米芬组子宫腺体数较模型组明显减少,子宫内膜厚度较模型组明显减小。归肾丸高剂量组、归肾丸中剂量组、归肾丸低剂量组子宫内膜厚度、腺体数与模型组比较未见明显差别。
本研究发现,模型组卵巢生长卵泡(初级卵泡和次级卵泡)、黄体数目均较阴性对照组明显减少,闭锁卵泡数目较阴性对照组明显增加,模型组子宫腺体数较阴性对照组明显减少。克罗米芬组卵巢生长卵泡、闭锁卵泡数目均较模型组明显增加,子宫腺体数、子宫内膜厚度均较模型组明显减少。归肾丸高剂量组卵巢可见生长卵泡数目较模型组明显增加。实验结果提示归肾丸高剂量可促进肾虚模型SD大鼠卵巢原始卵泡向初级卵泡、次级卵泡发育。
结论：
1、以氢化可的松建立肾虚大鼠模型具有排卵抑制的表现,成功模拟肾虚排卵障碍的临床类型。
2、景岳归肾丸能够改善肾虚排卵障碍模型大鼠的卵泡发育,提高大鼠血清P激索水平
3、归肾丸具有促进卵巢卵泡发育的功能,可能通过调整肾—天癸—冲任—胞宫生殖轴,从而达到肾精渐盛,冲任气血通调,促进卵泡发育和排卵。
Objective
On the foundation of early researches, this randomized control study focuses on Jingyue's Kidney-invigorating Pill's effect on rat models' hormone levels and endometrial morphology who have ovulation difficulties due to kidney deficiency with the hope of giving objective evaluation of its curative effect and safety and providing necessary evaluation system and strict research material for the clinical research and new drug development in the prevention and treatment of ovulation difficulties by Chinese Medicine.
Methods
The study subjects were72female rats randomly divided into the Negative Control Group, the Model Group, Clomiphene Group and Kidney-invigorating Groups (high dose, medium dose and low dose) with12in each group. Modeling: Except the Negative Control Group's rats were given equal volume of intramuscular injection of normal saline, all the other groups' rats were given intramuscular injection of hydrocortisone injection5ml· kg-1· d-1. Conduct continuous modeling for14days. Administration:on the15th day of modeling, administer medicine at10ml/kg of the body weight of rats. The Kidney-invigorating High-, Medium-and Low-dose Groups are given Kidney-invigorating Pill solution at0.57g crude drug/ml,1.14g/ml,2.28g crude drug/ml. The Negative Control Group and the Model Group were given distilled water and the Clomiphene Group was given0.54mg/ml clomiphene citrate tablet liquid (clomiphene citrate) for15continuous days.1H after the last administration, etherize the orbital venous plexus for hemospasia. Kill rats with cervical dislocation method. Conduct anatomical sampling for the detection of corresponding indexes.
Results
1. Sexual Cycle:starting from the fifth day after modeling. Except the Negative Control Group, the rats in each group had prolonged and irregular vaginal exfoliate cell cycle. Since the11th day, the Negative Control Group had no obvious periodic change, while other groups showed irregularities in cycle and length, or even disappearance of the cycle.
2. Weight:Compared with the Negative Control Group, Model Group rats on modeling D1, D7, D14, D29lost weight significantly (P<0.05). Compared with the Model Group, Clomiphene Group rats, after administration of the drug21and29days, lost weight significantly (P<0.05); Rats in Kidney-invigorating Pill Groups of different dose have no significant difference (P>0.05) compared with the Model Group in body weight at different time points.
3. Feed consumption:Compared with the Negative Control Group, Model Group rats' feed consumption significantly reduced (P<0.05) on D1and D7after the administration. Compared with the Model Group, High-dose Group of Kidney-invigorating Pill rats'feed consumption significantly reduced (P <0.05) on D14, D21of administration. The Low-dose Group of Kidney-invigorating Pill rats'feed consumption significantly reduced (P <0.05) on D21of administration. The other groups'rats'feed consumption have no significant difference (P>0.05) at other time points.
4. Water consumption:Compared with the Model Group, Rats in the Medium and High-dose Group of Kidney-invigorating Pill on D14significantly increased water consumption (P<0.05). The other groups' rats at other time points showed no significant difference (P>0.05) in drinking water consumption.
5. Organ coefficient:Model Group rats' adrenal coefficients, compared with the Negative Control Group, significantly increased (P<0.05). Clomiphene Group rats' uterus coefficient and ovarian coefficient, compared with the Model Group, significantly decreased (P<0.05). Kidney-invigorating Pill High, Medium and Low-dose Groups, in terms of uterus and ovary organ coefficient, showed no significant difference (P>0.05) compared with Model Group.
6. Levels of the hormone:Model Group rats'serum estuarial (E2) content, compared with the Negative Control Group, showed no significant difference (P>0.05). Each administration group rats'serum estuarial (E2) content, compared with that of the rats in the Model Group, showed no significant difference (P>0.05). Model Group rats'serum progesterone (P) levels, compared with Negative Control Group, decreased significantly (P<0.05). Kidney-invigorating Pill Low-dose Group rats'serum progesterone (P), compared with the Model Group, significantly increased (P<0.05). The remainder of each group rats'serum progesterone levels, compared with the Model Group, had no significant difference (P>0.05).
7. Follicular Growth:Negative Control Group ovaries have visible follicles with normal number, large number of corpus luteum and without follicular cysts, hematoma or tumor. Model Group had visible ovarian follicles (primary and secondary follicles). Compared with the Negative Control Group, its corpus luteum obviously decreased in number and the atresic follicle increased significantly. The Clomiphene Group had visible growing follicles and follicular atresia which significantly increased compared with the Model Group. Kidney-invigorating Pill High-dose Group had visible ovarian follicle which increased significantly in number than the Model Group. Kidney-invigorating Pill Medium and Low-dose Groups, compared with Model Group, showed no significant difference in ovarian growing follicles, mature follicle, follicular atresia and number of corpus luteum.
8. Endometrial morphologic changes:In the Negative Control Group, the uterine wall had clear layer structure, endometrium was thick, endometrial epithelium had single-layer columnar arrangement, submucosal lamina propria connective tissue had nonbranching uterine glands, glands was in large number and the myometrium and perimetrium had no obvious pathological changes. The Model Group, compared with the Negative Control Group, decreased obviously in the number of uterine glands and endometrial thickness. Clomiphene Group decreased obviously in the number of uterine gland and the endometrium thickness than the Model Group. Compared with the Model Group, the Kidney-invigorating Pill Groups of high dose, medium dose and low dose, in terms of endometrial thickness and uterine gland number, showed no significant difference.
The study found that, compared with the Negative Control Group, the Model Group'ovarian growing follicle (primary follicle and secondary follicle) and corpus luteum number decreased obviously, atresic follicle number increased significantly and the number of uterine glands decreased obviously. Compared with the Model Group, Clomiphene Group's ovarian growing follicle and follicular atresia significantly increased in number and the number of uterine glands and endometrial thickness decreased obviously. Kidney-invigorating Pill High-dose Group had obviously increased ovarian growing follicles than the Model Group. The experimental results indicate Kidney-invigorating Pill with high dose can promote kidney-deficient SD rats' ovarian primordial follicle developing into primary follicle and secondary follicle.
Conclusion
1. The hydrocortisone established kidney-deficient rat models have ovulation inhibition performance. There's successful simulation of clinical types of ovulation difficulties due to deficiency of the kidney.
2. King Yue Kidney-invigorating Pill can improve the follicular development in rat model of ovulation difficulties due to kidney deficiency and, enhance rats' serum P levels.
3. Kidney-invigorating Pill had the function of promoting ovarian follicular development. With the adjustment of reproductive axis formed with kidney-menstruation-Chong and Ren Meridians-uterus. It helps to reinforce the kidney-essence, adjust qi and blood in Chong and Ren meridians and promote the follicular development and ovulation.
引文
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