拆方肾气丸对肾阳虚雌鼠靶腺轴调控的实验及临床研究
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摘要
目的：
动物实验：观察氢化可的松法所致肾阳虚雌性大鼠下丘脑一垂体—肾上腺轴(HPA轴)及下丘脑一垂体一卵巢轴(HPO轴)功能变化,探讨金匮肾气丸及其拆方所代表的不同补肾法(补肾阳、补肾阴、阴阳双补)对肾阳虚雌鼠HPA、HPO轴功能的改善情况,对其治疗肾阳虚的作用机制进行初步探讨,为中医“肾主生殖”及“阴中求阳”基本理论提供现代科学依据。
临床研究：观察金匮肾气丸结合中医周期疗法对肾阳虚月经后期女性一般状况、月经状况、临床症状积分、基础体温(BBT)、生殖内分泌激素水平的改善情况,为临床治疗肾阳虚引起的月经后期及其他月经病、排卵障碍性不孕等生殖内分泌疾病提供依据。
方法：
动物实验：肾气丸“阴中求阳”配伍对肾阳虚雌性大鼠HPA、HPO轴调控的实验研究
将“阴中求阳”代表方金匮肾气丸进行原方与拆方的配伍研究,分为正常组、模型组及拆方肾气丸各组：补阳组(附子、桂枝+茯苓、泽泻、丹皮)、补阴组(干地黄、山茱英、山药+茯苓、泽泻、丹皮)、阴阳双补组(即肾气丸全方组：附子、桂枝、干地黄、山茱英、山药、茯苓、泽泻、牡丹皮)。将SPF级雌性SD大鼠55只,随机分为5组：正常组、模型组、补阳组、补阴组、阴阳双补组,每组11只。正常光照,自由摄食、饮水,适应性喂养1周。除正常组对照外,其余各组大鼠以氧化可的松法复制肾阳虚雌性大鼠模型：每¨日大腿内侧肌肉丰厚处消毒注射氢化可的松25mg／(kg·d),连续12天。造模后第13天开始灌胃给药,每天1次,连续干预15天。正常组、模型组按lml／100g体重灌服等体积生理盐水,每日一次,连续十预15天。造模第1天,灌胃第1天、第21天称量各组大鼠体重。造模第11天,灌胃第10天、第20天测量各组大鼠肛温。造模第12天、灌胃第20记录各组大鼠自主活动情况。造模第10天,灌胃第19天记录各组大鼠负重游泳时间。灌胃第21天上午麻醉大鼠,眼眶取血,静置,离心取上层血清冷藏待用。观察各组动物造膜前后、灌胃前后一般情况的改善,观察肾气丸各拆方组对氢化可的松所致肾阳虚型模型动物灌胃后体重、下丘脑、垂体、肾上腺重量,卵巢、子宫脏器重量指数,肛门温度,自主活动情况,抗疲劳试验(计算负重游泳时间)情况。采用光镜、酶联免疫分析、免疫组织化学分析、反转录聚合酶链反应(RT-PCR)等方法,对各组大鼠进行肾上腺、子宫内膜、卵巢组织的形态学及超微结构观察,计算各级卵泡总数、病理性囊性扩张卵泡总数,测定血清HPA轴(促肾上腺皮质激素ACTH、皮质醇COR)及HPO轴激素(卵泡刺激素FSH、黄体生成素LH、雌二醇E2、泌乳素PRL、孕酮P、睾酮T)水平,测定卵泡细胞凋亡途径相关因子(TNF-α肿瘤坏死因子-a、Bcl-2凋亡抑制基因、Bax促凋亡基因)表达、卵巢组织FSHRmRNA(卵泡刺激素受体mRNA)表达测定,探讨金匮肾气丸“阴中求阳”配伍结构对肾阳虚雌性大鼠HPA轴和HPO轴功能的调控。
临床疗效观察：金匮肾气丸结合中医周期疗法对肾阳虚月经后期患者的疗效观察
以中医辨证论治理论为指导,将临床收集的肾阳虚型月经后期女性患者给予金匮肾气丸口服治疗,同时结合中医周期疗法加减用药。采用病历跟踪、酶联免疫分析等方法,比较肾阳虚月经后期女性用药三个月前后一般状况,月经状况,基础体温(BBT),临床症状积分情况,血清内分泌激素水平：卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E2)、泌乳素(PRL)、睾酮(T)等,观察金匮肾气丸配合中医周期疗法改善肾阳虚月经后期女性生殖机能的临床疗效及肾阳虚证候疗效情况,为临床治疗提供科学依据。
结果：
动物实验显示：1、通过对动物肛温、一般状况、自主活动情况等的观察,表明成功复制肾阳虚大鼠模型；2、肾阳虚雌性大鼠肛温下降,自主活动减少,负重游泳时间缩短,体重、下丘脑、垂体增重减慢,卵巢、子宫脏器指数增长减慢。金匮肾气丸各给药组(补阳组、补阴组、阴阳双补组)均能一定程度上改善肾阳虚模型大鼠-般状况、提升肛温、增加自主活动时间、增加负重游泳时间、增加体重及下丘脑、垂体、肾上腺重量、增加子宫、卵巢等脏器指数(P0.05)。5、凋亡基因TNF-α、Bcl-2、Bax在卵泡的闭锁中起到重要的作用。肾阳虚雌性大鼠卵巢颗粒细胞凋亡相关蛋白Bc1-2表达明显降低(P<0.01), TNF-α、Bax表达明显升高(P 临床疗效观察显示：肾阳虚证与下丘脑-垂体-卵巢轴(HPO轴)功能的紊乱密切相关。金匮肾气丸配合中医周期疗法对肾阳虚月经后期患者的临床疗效总有效率为75%,肾阳虚证候疗效总有效率为75%,可明显降低肾阳虚型月经后期患者临床症状积分(P 结论：
1、氢化可的松法所复制肾阳虚雌性大鼠呈现肛温下降,自主活动减少,抗疲劳能力减弱,体重及脏器增重减慢等一系列肾阳虚症状外,出现下HPA、HPO轴功能紊乱,相关激素水平含量异常；肾上腺、卵巢及子宫内膜组织形态和超微结构被破坏；卵巢颗粒细胞凋亡因子及卵泡刺激素受体mRNA表达异常,影响雌性大鼠卵巢功能。
2、肾气丸“阴中求阳”配伍主要出大量补肾生精、滋补肝肾药物配伍少量温阳药物而成,以少火生气,微微生火达到阴生阳长、温补肾阳之效。金匮肾气丸及其拆方所代表的不同补肾法(补肾阳、补肾阴、阴阳双补法)均能不同程度改善肾阳虚雌性大鼠受损的卵巢功能,但单一的补阳法或补阴法效果不佳,阴阳双补法可提高疗效。纠正HPA、HPO轴激素水平紊乱,纠正细胞凋亡因子TNF-a、Bcl-2、Bax表达,增强卵巢颗粒细胞FSHRmRNA的表达,都可能是肾气丸“阴中求阳”治疗肾阳虚证的配伍机制。
3、以中医辨证论治理论为指导,金匮肾气丸结合中医周期疗法在治疗肾阳虚月经后期中起到良好疗效。温补肾阳名方配合中医周期疗法可以发挥整体调节之优势,改善卵巢机能微环境,重建生殖内分泌的生理平衡,达到调经种子之功效。
Objective
Animal 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).
Methods
Animal 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.
Results
The 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 (P0.05).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).
Conclusion
1. 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.
动物实验：观察氢化可的松法所致肾阳虚雌性大鼠下丘脑一垂体—肾上腺轴(HPA轴)及下丘脑一垂体一卵巢轴(HPO轴)功能变化,探讨金匮肾气丸及其拆方所代表的不同补肾法(补肾阳、补肾阴、阴阳双补)对肾阳虚雌鼠HPA、HPO轴功能的改善情况,对其治疗肾阳虚的作用机制进行初步探讨,为中医“肾主生殖”及“阴中求阳”基本理论提供现代科学依据。
临床研究：观察金匮肾气丸结合中医周期疗法对肾阳虚月经后期女性一般状况、月经状况、临床症状积分、基础体温(BBT)、生殖内分泌激素水平的改善情况,为临床治疗肾阳虚引起的月经后期及其他月经病、排卵障碍性不孕等生殖内分泌疾病提供依据。
方法：
动物实验：肾气丸“阴中求阳”配伍对肾阳虚雌性大鼠HPA、HPO轴调控的实验研究
将“阴中求阳”代表方金匮肾气丸进行原方与拆方的配伍研究,分为正常组、模型组及拆方肾气丸各组：补阳组(附子、桂枝+茯苓、泽泻、丹皮)、补阴组(干地黄、山茱英、山药+茯苓、泽泻、丹皮)、阴阳双补组(即肾气丸全方组：附子、桂枝、干地黄、山茱英、山药、茯苓、泽泻、牡丹皮)。将SPF级雌性SD大鼠55只,随机分为5组：正常组、模型组、补阳组、补阴组、阴阳双补组,每组11只。正常光照,自由摄食、饮水,适应性喂养1周。除正常组对照外,其余各组大鼠以氧化可的松法复制肾阳虚雌性大鼠模型：每¨日大腿内侧肌肉丰厚处消毒注射氢化可的松25mg／(kg·d),连续12天。造模后第13天开始灌胃给药,每天1次,连续干预15天。正常组、模型组按lml／100g体重灌服等体积生理盐水,每日一次,连续十预15天。造模第1天,灌胃第1天、第21天称量各组大鼠体重。造模第11天,灌胃第10天、第20天测量各组大鼠肛温。造模第12天、灌胃第20记录各组大鼠自主活动情况。造模第10天,灌胃第19天记录各组大鼠负重游泳时间。灌胃第21天上午麻醉大鼠,眼眶取血,静置,离心取上层血清冷藏待用。观察各组动物造膜前后、灌胃前后一般情况的改善,观察肾气丸各拆方组对氢化可的松所致肾阳虚型模型动物灌胃后体重、下丘脑、垂体、肾上腺重量,卵巢、子宫脏器重量指数,肛门温度,自主活动情况,抗疲劳试验(计算负重游泳时间)情况。采用光镜、酶联免疫分析、免疫组织化学分析、反转录聚合酶链反应(RT-PCR)等方法,对各组大鼠进行肾上腺、子宫内膜、卵巢组织的形态学及超微结构观察,计算各级卵泡总数、病理性囊性扩张卵泡总数,测定血清HPA轴(促肾上腺皮质激素ACTH、皮质醇COR)及HPO轴激素(卵泡刺激素FSH、黄体生成素LH、雌二醇E2、泌乳素PRL、孕酮P、睾酮T)水平,测定卵泡细胞凋亡途径相关因子(TNF-α肿瘤坏死因子-a、Bcl-2凋亡抑制基因、Bax促凋亡基因)表达、卵巢组织FSHRmRNA(卵泡刺激素受体mRNA)表达测定,探讨金匮肾气丸“阴中求阳”配伍结构对肾阳虚雌性大鼠HPA轴和HPO轴功能的调控。
临床疗效观察：金匮肾气丸结合中医周期疗法对肾阳虚月经后期患者的疗效观察
以中医辨证论治理论为指导,将临床收集的肾阳虚型月经后期女性患者给予金匮肾气丸口服治疗,同时结合中医周期疗法加减用药。采用病历跟踪、酶联免疫分析等方法,比较肾阳虚月经后期女性用药三个月前后一般状况,月经状况,基础体温(BBT),临床症状积分情况,血清内分泌激素水平：卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E2)、泌乳素(PRL)、睾酮(T)等,观察金匮肾气丸配合中医周期疗法改善肾阳虚月经后期女性生殖机能的临床疗效及肾阳虚证候疗效情况,为临床治疗提供科学依据。
结果：
动物实验显示：1、通过对动物肛温、一般状况、自主活动情况等的观察,表明成功复制肾阳虚大鼠模型；2、肾阳虚雌性大鼠肛温下降,自主活动减少,负重游泳时间缩短,体重、下丘脑、垂体增重减慢,卵巢、子宫脏器指数增长减慢。金匮肾气丸各给药组(补阳组、补阴组、阴阳双补组)均能一定程度上改善肾阳虚模型大鼠-般状况、提升肛温、增加自主活动时间、增加负重游泳时间、增加体重及下丘脑、垂体、肾上腺重量、增加子宫、卵巢等脏器指数(P
1、氢化可的松法所复制肾阳虚雌性大鼠呈现肛温下降,自主活动减少,抗疲劳能力减弱,体重及脏器增重减慢等一系列肾阳虚症状外,出现下HPA、HPO轴功能紊乱,相关激素水平含量异常；肾上腺、卵巢及子宫内膜组织形态和超微结构被破坏；卵巢颗粒细胞凋亡因子及卵泡刺激素受体mRNA表达异常,影响雌性大鼠卵巢功能。
2、肾气丸“阴中求阳”配伍主要出大量补肾生精、滋补肝肾药物配伍少量温阳药物而成,以少火生气,微微生火达到阴生阳长、温补肾阳之效。金匮肾气丸及其拆方所代表的不同补肾法(补肾阳、补肾阴、阴阳双补法)均能不同程度改善肾阳虚雌性大鼠受损的卵巢功能,但单一的补阳法或补阴法效果不佳,阴阳双补法可提高疗效。纠正HPA、HPO轴激素水平紊乱,纠正细胞凋亡因子TNF-a、Bcl-2、Bax表达,增强卵巢颗粒细胞FSHRmRNA的表达,都可能是肾气丸“阴中求阳”治疗肾阳虚证的配伍机制。
3、以中医辨证论治理论为指导,金匮肾气丸结合中医周期疗法在治疗肾阳虚月经后期中起到良好疗效。温补肾阳名方配合中医周期疗法可以发挥整体调节之优势,改善卵巢机能微环境,重建生殖内分泌的生理平衡,达到调经种子之功效。
Objective
Animal 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).
Methods
Animal 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.
Results
The 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
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).
Conclusion
1. 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.
引文
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