| ObjectiveAnimal experiment:To observe changes in the functions of HPA and HPO of female rat models with hydrocortisone-caused kidney-Yang deficiency, and to explore the mechanism of JinKui Shenqi Pill and its diferent combinative drugs (tonifying kidney-Yang, tonifying kidney-Yin and nourishing both kidney-Yang and kidney-Yin) in treating kidney-Yang deficiency by investigating the functional improvement made by the formulas in HPA and HPO of female rat models with kidney-Yang deficiency so as to provide scientific basis for the basic theory of "kidney dominating reproduction" and "reinforcing Yang from Yin in traditional Chinese medicine (TCM).Clinical research:To observe the improvement made by JinKui Shenqi Pill and TCM period ic therapy in the general condition, the menstruat ion condit ion, the clinical symptom integrals, the basal body temperature (BBT) and the level of reproductive endocrine hormone of females with delayed menorrhea due to kidney-Yang deficiency so as to offer basis for the clinical treatment of reproductive endocrine diseases caused by kidney-Yang deficiency (e.g. dysfunctional ovulatory sterility, menorrhea and other kinds of menopathy).MethodsAnimal experiment:the experimental study on the regulation of HPA and HPO of female rat models with kidney-Yang deficiency by the compatibility of "reinforcing Yang from Yin" of JinKui Shenqi Pill.Research was conducted on the compatibility of JinKui Shenqi Pill,which is a typical formula of "reinforcing Yang from Yin ", and the different combinative drugs with it. The formulas were divided into the normal group, the model group and the groups with different combinative drugs with JinKui Shenqi Pill as follows:the Yang-tonifying group (aconite, cassia twig, poria cocos, rhizoma alismatis and moutan bark), the Yin-tonifying group (dried rehmannia, asiatic cornel ian cherry fruit, Chinese yam, poria cocos, rhizoma alismatis and moutan bark) and the Yin-and-Yang-nourishing group (aconite, cassia twig, dried rehmannia,asiatic cornelian cherry fruit,Chinese yam, poria cocos, rhizoma alismatis and moutan bark).55female SD rats at the SPF level were randomly divided into five groups:normal, model, tonifying Yang, the tonifying Yin and nourishing both Yin and Yang with11rats in each group. The rats fed themselves and consumed water voluntarily under normal illumination. The adaptive feeding lasted for one week. The female rat model with kidney-Yang deficiency was induced by administering hydrocortisone to the rats apart from those in the normal group.25mg/(kg·d) hydrocortisone was injected into the strong inner thigh muscles of these rats after sterilization per day, which lasted for12days. The rats were gavaged with the formulas once per day on the13th day since the creation of the model, which lasted for15days. As for the rats in the normal group and the model one, they were gavaged with the same volume of normal saline in accordance with the ratio of volume to body weight as1ml/100g once per day, which lasted for15days. The body weight of the rats in all the groups were measured on the day when the model was created and on the1st and the21st day of intragastric administ ration. The anal temperature of the rats in all the groups were measured on the11th day since the creation of the model and on the10th and the20th day of intragastric administration. The autonomic activity of the rats in all the groups was recorded on the12th day since the creation of the model and on the20th day of intragastric administration. The weight-bearing swimming time of the rats in all the groups was recorded on the10th day since the creation of the model and on the19th day of intragastric administration. On the morning of the21st day of i ntragastric administration, blood was drawn from the rats’orbit after anesthesia and was kept stationary. The upper I ayer of serum was collected by centrifugation and was ref r i gerated for later use. Observation was made of the general condition of the rats before and after the creation of the model and intragastric administration. Inspection was conducted of the effects of the groups of different combinative drugs with JinKui Shenq i Pill on the body weight, the weight of hypothalamus, pituitary gland and adrenal gland, the ovary and uterus visceral indexes, the anal temperature, the autonomic activity and the anti-fatigue test (calculation of weight-bearing swimming time) of the rat models with hydrocortisone-caused kidney-Yang deficiency after intragastric administration. Inspection was carried out of the morphology and the ultra-microstructure of the adrenal gland, the endometr ium, the ovar ian tissue of the ratsin all the groups by means of light microscope, enzyme-linked immuno sorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). Precisely, calculation was made of the total number of follicles at different levels and follicles with pathologic cystic expansion. Measurement was performed of the hormones of HPA of serum, namely adrenocorticotrophic hormone (ACTH) and cortisol (COR) as well as those of HPO including follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), prolact in (PRL), progesterone (P) and testosterone (T). Measurement was also carried out of the expression of correlation factors of the apoptotic pathway of follicular cells, namely tumor nectosis factor-alphagene (TNF-α), apoptosis-inhibit ing gene Bcl-2and B apoptosis cell matrixgene (Bax) as well as the expression of follicle-stimulating hormone receptor (FSHR) mRNA in ovarian tissues. In this way, investigation was made into the regulation of the functions of HPA and HPO in female rat models with kidney-Yang deficiency by the compatibility structure of "reinforcing Yang from Yin" of JinKui Shenqi Pill.Observation of clinical effects:Observation of the effects of JinKui Shenqi Pill integrated with TCM periodic therapy on patients with delayed menorrhea due to kidney-Yang deficiency.Under the guidance of treatment based on syndrome differentiation of TCM theory, oral doses of JinKui Shenqi Pill were administered to the clinical patients with delayed menorrhea due to kidney-Yang deficiency. Meanwhile, the doses were modified according to TCM periodic therapy. Comparison was made between the condition of the female patients with delayed menorrhea due to kidney-Yang deficiency before and after three months of treatment in terms of the general condition, the menstruation condit ion, BBT, clinical symptom integrals, the levels of endocrine hormones in serum namely FSH, LH, E2, PRL, T and so on. Observation was made of the clinical effects of JinKui Shenqi Pill combined with TCM periodic therapy on the reproductive function of female patients with delayed menorrhea due to kidney-Yang deficiency and the kidney-deficiency syndrome so as to provide scientific basis for clinical treatment.ResultsThe animal experiment demonstrated:1.The anal temperature, the general condition and the autonomic activity of the rats proved successful creation of rat models with kidney-Yang deficiency.2. The anal temperature of the female rat models with kidney-Yang deficiency declined. Their autonomic activity and weight-bearing swimming time reduced. The increase in the weight of their bodies, hypothalamus, hypophysis slowed, and so did the increase in the ovary and uterus visceral indexes. To some extent, in the groups in which JinKui Shenqi Pill and the different comb inative drugs with it (ton ify i ng kidney-Yang, tonifying kidney-Yin and nourishing both kidney-Yang and kidney-Yin) were administered, the general condition of the rat models with kidney-Yang deficiency improved. Besides, there was a rise in their anal temperature, autonom i c act iv ity t i me, weight-bear ing swimming time, body weight, the weight of hypothalamus, hypophysis, adrenal gland, the visceral index such as that of uterus and ovary (P<0.01,P<0.05). Among all the groups, the effects in the Yin-and-Yang-nourishing group were the most evident.3. Abnormal ultra-microstructure and d ifferent degrees of pathol ogic changes such as organ atrophy occurred in the adrenal gland, the endometrium and the ovary of the female rat models with kidney-Yang deficiency. To a certain degree, JinKui Shenqi Pill and the different combinative drugs with it including the Yang-tonifying formula and the Yin-tonifying one could inhibit the atrophy of the organs such as adrenal gland, uterus and ovary as well as improve the morphology of pathologic organs in the female rat models with kidney-Yang deficiency. The formulas, to some extent, could repair the abnormality in the visceral ultra-microstructure caused by the drugs used in the creation of the model. Specifically, they could increase the total number of follicles at various levels and reduce the follicles with pathologic cystic expansion. Efficacy was the best in the Yin-and-Yang-nourishing group (P<0.01,0.05).4. Kidney-Yang deficiency syndrome was closely linked with the functional disorder of HPA and HPO. There was a significant decline in the level of ACTH, COR, FSH, LH, E2and P (P<0.01, P<0.05) and a substantial rise in the level of PRL and T (P<O.01, P<O.05) in the serum of the female rat models with kidney-Yang deficiency. To some degree, all formulas with the different combinative drugs with JinKui Shenqi Pill could enhance the level of FSH, LH, E2and P in the serum of the rat models with kidney-Yang deficiency (P<0.01, P<0.05). Efficacy was the most apparent in the Yin-and-Yang-nourishing group while it was not evident in the Yang-tonifying group or the Yin-tonifying one. All the formulas with the different combinative drugs with JinKui Shenqi Pill, to some extent, could reduce the level of PRL in the serum of the model rats. The reduction was the most significant in the Yin-and-Yang-nourishi ng group whereas it was not apparent in the Yang-tonifying group or the Yin-tonifying one. All the formulas with the different combinative drugs with JinKui Shenqi Pill failed to significantly reduce the level of T in the serum of the rat models with kidney-Yang deficiency (P>0.05).5. The apoptosis genes, namely TNF-α, Bcl-2, Bax, played a crucial role in follicular atresia. In the rat models with kidney-Yang deficiency, there was a considerable decline in the expression of Bcl-2, which is a protein related to the apoptosis of ovarian granular cells (P<0.01). In contrast, the expression of TNF-α and Bax rose significantly (P<0.01,P<0.05). To a certain degree, all the formulas with the different combinative drugs with JinKui Shenqi Pill could raise the expression level of Bcl-2(P<0.01, P<0.05) and reduce that of TNF-a and Bax (P<0.01,P<0.05). The efficacy was the most noticeable in the Yin-and-Yang-nourishing group.6.The expression of ovarian FSHRmRNA in the female rat models with kidney-Yang deficiency was much less than that in the normal group (P<0.01). All the formulas with the different combinative drugs with JinKui Shenqi Pill coul d enhance the express ion level of ovarian FSHRmRNA in the female rat models with kidney-Yang deficiency (P<0.01). Such efficacy was the most noticeable in the Yin-and-Yang-nourishing groups. Investigation was conducted on the mechanism of the different combinative drugs with JinKui Shenqi Pill for treating kidney-Yang deficiency through observation of their regulation of HPA and HPO of the female rat models with kidney-Yang deficiency. The above exploration provided experimental basis for the TCM basic theory of "kidney dominating reproduction" and "reinforcing Yang from Yin"Observation of clinical effects manifested that kidney-deficiency syndrome had a close link with the functional disorder of HPO. The total efficacy rates of JinKui Shenqi Pill combined with TCM periodic therapy in treating kidney-Yang deficiency syndrome and delayed menorrhea due to kidney-Yang deficiency amounted to75%. The therapies could significantly reduce the clinical symptom integrals (P<0.01) and improve BBT (P<0.01) of the patients with delayed menorrhea due to kidney-Yang deficiency. In terms of their effects on the sex hormone level in the serum, the formulas could substantially reduce the level of FSH, LH and T (P<0.01, P<0.05) while raising that of E2so that the patients’menstruation was improved. The effects of the formulas shown in the experiment provided basis for the clinical treatment of reproductive endocrine diseases caused by kidney-Yang deficiency (e.g. dysfunctional ovulatory sterility, menorrhea and other kinds of menopathy).Conclusion1. The female rat models with hydrocort i sone-caused kidney-Yang def ic iency displayed a series of symptoms of kidney-Yang deficiency including a decline in anal temperature, autonomic activity and anti-fatigue ability as well as a slowdown in the increase of body and visceral weight. They also exhibited functional disorder of HPA and HPO as well as abnormal levels of relevant hormones. The morphology and ultra-microstructure of their adrenal glands, ovaries and endometrium were damaged.In addition, the expression of apoptosis factors in ovarian granular cells and FSHRmRNA was abnormal, which influenced the function of the female rats’ovaries.2. The compatibility of "reinforcing Yang from Yin" i n J inKui Shenqi Pill was composed of large amounts of kidney-and-liver-nourishing, essence-generating medicine as well as a small amount of medicine that invigorated Yang. The pills contributed to generation of qi by junior fire so as to boost both Yin and Yang and nourish kidney-Yang. JinKui Shenqi Pill and the different kidney-tonifying formulas represented by the different combinative drugs with it (tonifying kidney-Yang, tonifying kidney-Yin and nourishing both kidney-Yang and kidney-Yin) could, to various degrees, improve the impaired ovarian function of the female rat models with kidney-Yang deficiency. However, the effects of the Yang-tonifying formula or the Yin-tonifying one were not satisfactory whereas the Yin-and-Yang-nourishing formula could improve the curative effect. Treating the hormonal disorder of HPA and HPO as well as normalizing the expression of TNF-α, Bcl-2and Bax were probably the compatibility mechanism of "reinforcing Yang from Yin" of JinKui Shenqi Pill in treating kidney-Yang deficiency syndrome. 3. Under the guidance of treatment based on syndrome differentiation of traditional Chinese medicine theory, JinKui Shenqi Pill integrated with TCM periodic therapy had a good curative effect in treating delayed menorrhea due to kidney-Yang deficiency. The well-known kidney-Yang-tonifying formula combined with TCM periodic therapy had the advantage of holistic regulation. They could improve the microenvironment of ovarian functions and restore the physical balance in the reproductive endocrine system. |
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