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Experimental Study on Regulation of Warming Kidney Yang and Descending Ministerial Fire Methods on Reproductive Function of Female Rats

Author: LiuYanXia
Tutor: JinZhe
School: Beijing University of Traditional Chinese Medicine
Course: Traditional Chinese Medicine
Keywords: warm kidney yang descend ministerial fire HPG granulose cell
CLC: R242
Type: PhD thesis
Year: 2008
Downloads: 238
Quote: 0
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Abstract


Large fundamental and clinical researches in integration of traditional Chinese and Western medicine proved that the function of HPOA was similar to the kidney function of dominating reproduction.It was a two-way regulation in the treatment of tonifing kidney to the genital gland ax, which could encourage the function of HPOA. Instead of replacing the effect of hormone, there was parahormonelike effect in traditional Chinese medicines.This might exactly be the feature and significance of reproductive endocrine adjusting effect of traditional Chinese medicine.When trimming literatures of ancient times, we found that besides yin-blood was sufficient, ancient people extraordinary emphasized the effect of yang qi in pregnant and breeding cognition. Ancient people thought that fire (qi) was ahead of essence, innate fire of life gate was motivity in forming embryo. Therefore, the formulae of promoting gestation were mostly warming and tonifing, and this proved the theory of“warm generate and transformate all things, cold kill and inhibit all things”.To investigate the theory of“warm generate and transformate all things, cold kill and inhibit all things”was scientific further, we chose Yougui Pills which was the representative formula of warming kidney yang,and Zhibai dihuang Pills which was the representative formula of nourishing yin and descending ministerial fire to investigate the effect and mechanism of warming kidney yang and descending ministerial fire method on reproductive function of female rats from intact and cellular level.Which enrich and develop reproductive physiological theory of traditional Chinese gynecology, and provide evidence of medication scientifically.Main work and results were as follow:1 Literature studyThrough sorting and studying the literatures and the medication regulation of promoting gestation formulae of ancient times, conclusions we found was as follow. 1.1 Foundation of pregnance and breedEssence from father and blood from mother were the foundation of pregnance and breed.It might be influenced if people chose time to aphrodisia while ovulating.And it was emphersized that what was proper and what should be avoid during conception.Many factors could influence pregnance and breed,such as weather,environment,emotion, and so on.1.2 Condition of pregnance and breedExuberance of kidney qi,beginning of sex-stimulating essence, harmonious coexistence of yin and yang, prosperity of qi and blood, kidney-yang meets,essence collect and fetus to shape.And people shoule pay attention to avoid sexual overindulgence during the year of conception.1.3 Factors influencing pregnance and breedMany factors could influence pregnance and breed,such as Kidney insufficiency,cold in uterus,deficiency of both qi and blood, phlegm-damp obliteration,stagnation of liver qi,blood stasis, hyperactivity of ministerial fire,and so on.1.4 Methods of promoting gestationIncluding regulating menstruation before promoting gestation, offspring generated while menstruation was disciplinarian; regulating both yin and yang,then all things generated and transformated; warm generating and transformating all things, do not depress kidney-yang,and so on.1.5 Medication regulation of promoting gestation formulae of ancient timesEmphasized the close relationship between kidney and reproduction therapeutically,mainly using medicine that could reach kidney meridian. Harmonizing and tonifing both qi and blood was the main treating methods, and emphasizing the association between regulating qi and blood and conception.Most drugs were sweet and pungent in flavor and warm in property, recuperation with herbs warm in property mainly while medication which was influenced by the point of“warm generating and transformating all things, cold kill and inhibit all things”that consistent with the point of correspondence between man and universe, relevant adaptation of human body to natural environment.2 Experimental study2.1 Effect of warming kidney yang and descending ministerial fire methods on HPOA of female rats 2.1.1 Effect of warming kidney yang and descending ministerial fire methods on histomorphology of uterus and ovaryTo investigate the effect of warming kidney yang and descending ministerial fire method on the thickness of endometrium and the number of follicles of female rats. 62 rats (10-12 months of age)were devided into 5 groups at random: the control group, group of Yougui pills LD,group of Yougui pills HD, group of Zhibai dihuang Pills LD, group of Zhibai dihuang Pills HD.After feeded the rats with traditional Chinese medicines or NS for 8 weeks, we used the method of histopathology to measure the thickness of endometrium and the number of follicles in bilateral ovaria. Results: The thickness of endometrium in group of Yougui pills HD was thickened than that in the control group and Zhibai dihuang Pills HD、LD groups, there was a significant difference.(P<0.05)The number of follicles in bilateral ovaria was increased in group of Yougui pills HD than that in the control group and Zhibai dihuang Pills HD、LD groups, there was a significant difference.(P<0.05)The number of follicles in group of Yougui pills LD was increased than that in the group of Zhibai dihuang Pills HD, there was a significant difference.(P<0.05)The number of follicles in bilateral ovaria was decreased in group of Zhibai dihuang Pills HD than that in the control group, there was a significant difference.(P<0.05)2.1.2 Effect of warming kidney yang and descending ministerial fire methods on pituitary-ovarian gonadotropin and sex hormoneTo investigate the effect of warming kidney yang and descending ministerial fire method on pituitary gland secreting LH、FSH and ovary secreting E2、P、T.We used radioimmunitical method to detect the E2、P、T、LH、FSH levels of the rats of every group. Results: The levels of serum E2 in group of Zhibai dihuang Pills HD were decreased than those in the control group and Yougui pills HD、LD groups, there was a significant difference.(P<0.05)The levels of serum E2 in group of Zhibai dihuang Pills LD were decreased than those in Yougui pills HD、LD groups, there was a significant difference.(P<0.05)The levels of serum E2 in groups of Yougui pills HD、LD were increased than those in the control group,but it was a quiet difference.(P>0.05)The levels of serum P in group of Zhibai dihuang Pills HD were decreased strikingly.(P<0.05)There was no significant deviation in every group of the levels of serum T. The levels of serum FSH in group of Yougui pills HD were increased than those in the control group、groups of Yougui pillsLD and Zhibai dihuang Pills HD、LD, there was a significant difference.(P<0.05)There was no significant deviation in every group of the levels of serum LH.2.1.3 Effect of warming kidney yang and descending ministerial fire methods on the GnRH contents of hypothalamusWe used immunohistochemical method to detect the gray values and optical density of GnRH of hypothalamus. Results: The gray values of GnRH of hypothalamus in group of Yougui pills HD compare with those in the control group, there was no significant deviation.(P>0.05)The gray values of GnRH of hypothalamus in groups of Zhibai dihuang Pills LD were increased than those in the control group, there was a significant difference.(P<0.05)The gray values of GnRH of hypothalamus in groups of Zhibai dihuang Pills HD were increased than those in the control group, there was a extraordinary significant difference.(P<0.001)The gray values of GnRH of hypothalamus in groups of Zhibai dihuang Pills HD、LD were increased than those in groups of Yougui pills HD、LD, there was a extraordinary significant difference.(P<0.001)The optical density of GnRH of hypothalamus in Yougui pills LD groups was increased than that in the control group, there was a significant difference.(P<0.05)The optical density of GnRH of hypothalamus in Yougui pills HD groups was increased than that in the control group, there was a extraordinary significant difference.(P<0.001)The optical density of GnRH of hypothalamus in Zhibai dihuang Pills HD、LD groups was decreased than that in the control group, there was a extraordinary significant difference.(P<0.001)The optical density of GnRH of hypothalamus in Yougui pills HD、LD groups was increased than that in Zhibai dihuang Pills HD、LD groups, there was a extraordinary significant difference.(P<0.001)2.1.4 Effect of warming kidney yang and descending ministerial fire methods on the express ERαof endometriumWe used immunohistochemical method to detect the gray values and optical density of uterus. There was no significant deviation in every group of the gray values and optical density of ERαfrom endometrial glandular epithelial cells.(P>0.05) 2.2 Effect of warming kidney yang and descending ministerial fire methods on proliferative and secretary function of ovarian granule cells in rats 2.2.1 Effect of warming kidney yang and descending ministerial fire methods on proliferation of ovarian granule cells in vitro of ratsAfter isolated, the ovarian granule cells of rats were cultured in vitro for 24 hours in aqueous extract of Yougui pills or Zhibai dihuang Pills which were prepare to 7 concentration gradients: 5、10、20、40、80、160、320μg /100μl.Then detected the effect of defferent concentrations of drugs on granule cells,using MTT method. Results: The aqueous extract of Yougui pills enhanced the growth of granule cells in vitro.The effect was more dominant as the concentration increased.The aqueous extract of Zhibai dihuang Pills inhibited the growth of granule cells in vitro.The effect was more dominant as the concentration increased.2.2.2 Effect of warming kidney yang and descending ministerial fire methods on secretion of ovarian granule cells in vitro of ratsAfter isolated, the ovarian granule cells of rats were cultured in vitro for 24 hours in aqueous extract of Yougui pills or Zhibai dihuang Pills which were prepare to 4 concentration gradients: 5、10、20、40μg /100μl. Then detected the levels of E2、P in cells supernatant , using RIA. Results: The levels of E2 in granule cells supernatant of Yougui pills HD group was the highest, compared with those in the control group, there was a significant difference.(P<0.05)The levels of E2 of Zhibai dihuang pills 10μg /100μl、40μg/100μl groups was the lowest, compared with those in the control group, there was a significant difference.(P<0.05)The levels of P of Yougui pills 10μg /100μl、20μg/100μl and 40μg/100μl groups was the highest, compared with those in the control group, there was a significant difference.(P<0.05)The levels of P of Zhibai dihuang pills 5μg /100μl、40μg/100μl groups was the lowest, compared with those in the control group, there was a significant difference.(P<0.05)Conclusion:(1)Warming kidney yang method could increased the thickness of endometrium and the number of follicles obviously; descending ministerial fire method could decreased the number of follicles.(2)Warming kidney yang method could increased the levels of serum FSH, and there was a increased tendency of serum E2. Descending ministerial fire method could decreased the levels of serum E2、P.(3)Warming kidney yang method could enhance the secretation of hypothalamic GnRH; descending ministerial fire method could inhibit the secretation of hypothalamic GnRH.(4)There no effect of warming kidney yang and descending ministerial fire methods on the express ERαof endometrium(5)Warming kidney yang method could enhanced proliferative and secretary function of ovarian granule cells in vitro; descending ministerial fire method could inhibited proliferative and secretary function of ovarian granule cells in vitro.In a word,using the experiment we come to the conclusion that warming kidney yang method had a up-regulation effect on HPOA, while descending ministerial fire method had a down-regulation effect on HPOA.Furthermore, warming kidney yang method enhanced proliferative and secretary function of ovarian granule cells in vitro,and the encouraged ovum to grow,and had favourable effect on reproduction. Descending ministerial fire method probably inhibited proliferative and secretary function of ovarian granule cells in vitro,and interfered ovum to grow,and had disadvantage effect on reproduction.This experiment verificated the theory of“warm generate and transformate all things, cold kill and inhibit all things”was scientific.

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