[Adverse effect of environmental endocrine disruptors on gonadal development of prepubertal male rats and therapeutic effect of bushen tianjing recipe on it].

OBJECTIVE: To verify the antagonistic effect of Bushen Tianjing Recipe (BTR) on environmental endocrine disruptors (EEDs) induced gonadal dysgenesis (GD) Sprague-Dawley (SD) male rat model. METHODS: Totally 70 3-week-old male SD rats were randomly divided into seven groups, i.e., the control group (fed with corn oil), the model A group [di-2-ethylhexyl-phthalate (DEHP) 500 mg/kg], the CM A group (fed with DEHP 500 mg/kg + BTR 40 mL/kg), the exposed group B (fed with CYP 80 mg/kg), the CM B group (fed with CYP 80 mg/kg + BTR 40 mL/kg), the model C group [fed with DEHP 500 mg/kg + CYP 80 mL/kg], the CM C group (DEHP 500 mg/kg + CYP 80 mg/kg + BTR 40 mL/kg), respectively, 10 in each group. All were administered with corresponding medication by gastrogavage, once daily, for total 30 days. Rats were killed 24 h after the last administration, and their body weight and wet testis weight were weighed. The coefficient of testis was calculated. The serum testosterone (T) level was measured by chemiluminescent immunoassay. The histopathologic tissue was prepared. The ultrastructural changes of genital cells were observed by electron microscope. RESULTS: Compared with the control group, there was no statistical difference in the body weight increase among all groups (P > 0.05). The time of testicular descent and preputial separation were significantly delayed in each exposed group (P < 0.01). In the exposed group A and the exposed group C, the wet weight of the testes was reduced and serum T level decreased (P < 0.01). The coefficient of testis significantly decreased in the exposed group A (P < 0.01). Compared with corresponding model group, the time of testicular descent and preputial separation were significantly fore-laid in each corresponding CM group (P < 0.01). The weight of the testes, the coefficient of testis, and the serum T level increased in the CM A group (P < 0.01). The serum T level obviously increased in the CM B group (P < 0.05). CONCLUSIONS: The GD rat model was successfully duplicated by using DEHP. EEDs were proved to have significant anti-androgen activities. BTR was verified to have significant antagonistic to its anti-androgen effect.

[The establishment of environmental endocrine disruptors induced precocious puberty Macaca fascicularis model and the intervention of yin nourishing fire purging Chinese materia medica].

OBJECTIVE: To establish the environmental endocrine disruptors (EEDs) induced precocious puberty macaca fascicularis model, thus verifying the antagonism of yin nourishing fire purging Chinese materia medica (YNFPCMM) against the estrogenic effects. METHODS: (1) The female prepubescent monkeys at different months old were fed with high dose and low dose EEDs. By observing EEDs' estrogenic effects, the optimal age, the optimal exposure dose, and the optimal exposure time were determined. (2) Fifteen female prepubescent monkeys were randomly divided into the exposure group, the treatment group, and the control group, 5 in each group. Those in the exposure group were fed with EEDs such as nonylphenol (4-NP) and bisphenol A (BPA). Those in the treatment group were fed with EEDs and YNFPCMM. Those in the control group were fed with dissolvent such as corn oil. The therapeutic course was 4 weeks. By the end of the treatment, the serum estrogen level was measured and the maturation index of the vaginal keratinocytes was calculated. The uterus was extracted to measure the wet weight, the endometrial thickness, the ring-shaped myometrium thickness, the endometrial epithelial cells, and the glandular epithelium height. RESULTS: (1) Twenty month was the optimal exposure age. The low dose EEDs was the best exposure dose, and 4 weeks was the optimal exposure time. (2) Compared with the control group, the serum estrogen level obviously increased, the maturation index of the vaginal keratinocytes significantly increased, the wet uterus weight, the endometrial thickness, the ring-shaped myometrium thickness, the endometrial epithelial cells, and the glandular epithelium height significantly increased in the exposure group (P<0.05). Compared with the exposure group, the aforesaid indices significantly decreased in the treatment group (P<0.05). CONCLUSIONS: The EEDs induced precocious puberty macaca fascicularis model was successfully established in this study. The EEDs were proved to have estrogenic-like activities. YNFPC-MM was proved to have significant antagonism like estrogenic activities.

Oral arginine improves linear growth of long bones and the neuroendocrine mechanism.

OBJECTIVE: To investigate the effect of oral administration of arginine on linear growth of long bones in male pubertal rats and the underlying mechanisms, focusing on expression of genes related to the hypothalamus-pituitary growth axis and the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. METHODS: Rats were randomly divided into control and intervention groups. In the intervention group, arginine was solved in water (0.045 g L-arginine was mixed with 1 mL water) and administered in rats (10 mL/kg) through gastric perfusion once per day, for totally 28 d. Rats in the control group received normal saline treatment. Bone histomorphometry analysis was used to measure growth plate width and mineral apposition rate of the tibia, as well as trabecular bone volume fraction, osteoblast surface and osteoclast surface of the femur. Serum growth hormone (GH) concentration was determined by radioimmunoassay. Real-time PCR was used to measure the expression of neuronal nitric oxide synthase (nNOS), soluble guanylyl cyclases (sGCα1 and sGCß1), growth hormone-releasing hormone (Ghrh) and somatostatin (SS) in hypothalamus, as well as Gh in pituitary. Western blot was used to detect the protein levels of nNOS, sGCα1 and sGCß1 in hypothalamus. RESULTS: After treatment with arginine, the growth plate width of tibia and osteoblast surface of femur were increased (P < 0.05), and serum GH concentration was elevated (P < 0.05). Besides, mRNA and protein levels of nNOS and sGCα1 (P < 0.05), as well as the expression of Gh mRNA (P < 0.01), were significantly up-regulated, while the expression of SS mRNA was down-regulated (P < 0.05). CONCLUSION: Oral administration of arginine could improve linear growth of long bones by regulating mRNA expression of SS and Gh and inducing GH secretion, possibly via nNOS-NO-sGC-cGMP signal transduction pathway.

[Effect of Chinese herbs for nourishing shen-yin and removing xiang-fire on estrogen receptor expression in reproductive organ of rats contaminated with environmental endocrine disruptor].

OBJECTIVE: To investigate the effect of Chinese herbs for nourishing Shen-yin and removing Xiang-fire (NYRF) on estrogen receptor (ER) expression in uterus and ovary of rats contaminated with nonylphenol (NP) or its bisphenol A (BPA) mixture, for exploring the action mechanism of NYRF in antagonizing the estrogen-mimetic activity of environmental endocrine disruptors (EEDs). METHODS: EEDs contaminated female SD rats, 3-week old, were divided into two groups, the treated group fed with NYRF and the control group with corn oil during the same period of contaminating for 15 days. The wet weight (WW) and organ coefficient (OC) of uterus in rats, as well as the ER protein and mRNA expressions in rat's uterus and ovary were detected and compared. RESULTS: As compared with normal range, WW and OC increased significantly in the contaminated rats of the control group, with significantly down-regulated ER protein expression in uterus, and expressions of ER alpha and ER beta gene and protein in ovary (P<0.05). While in the treated group, the above-mentioned abnormalities of various indicators were markedly reversed to a certain extent (P<0.05). CONCLUSION: EEDs show estrogenic-mimetic action on productive organs, which could be antagonized by NYRF, resulting in the down-regulated mRNA and protein expressions of ER in reproductive organs, so as to reduce the sensibility of reproductive organs to EEDs, which is probably one of the acting mechanisms of NYRF.

[Regulative effects of Chinese herbs for nourishing yin and removing fire on gene expressions of estrogen receptor alpha, insulin-like growth factor-1 receptor, epithelial growth factor receptor and protein synthesis in epiphyseal growth plate of female pubertal rats].

OBJECTIVE: To investigate the effect of Chinese herbs for nourishing yin and removing fire (NYRF) on gene expressions of estrogen receptor alpha (ER alpha), insulin-like growth factor-1 receptor (IGF-1R) and epithelial growth factor receptor (EGFR) in the epiphyseal growth plate of the female pubertal rats. METHODS: The rats were randomly divided into the control group and the intervened group. Immunohistochemistry and realtime-PCR methods were used to measure the gene expression of ER alpha, IGF-1R and EGFR and their protein synthesis in epiphyseal growth plate. RESULTS: After being intervened with NYRF, the gene expressions of ER alpha and IGF-1R were down-regulated and their protein synthesis markedly reduced, while those of EGFR were unchanged. CONCLUSION: NYRF can modulate the development and maturation of bone by regulating the expressions of ER alpha and IGF-1R in the epiphyseal growth plate.

[Determination of four kinds of environmental endocrine disruptors in blood serum of precocious puberty patients by high performance liquid chromatography].

OBJECTIVE: This study was directed to the development of a simple and highly sensitive method for determination of four kinds of environmental endocrine discrupters (EEDs) in the serum of precocious puberty patients using reversed-phase HPLC. METHODS: The serum samples were extracted by n-hexane. The operating conditions were such as C18 column (4.6 mm x 250 mm 5 microm), methanol: H2O: trifuorocuetic acid (1000 : 50 : 1) as mobile phase at a flow rate of 1 ml/min. Using ultraviolet detector, DEHP and MEHP was determined with wavelength at 228 nm, p, p'-DDE was determined with wavelength at 238 nm. Fluorescence detector with the excitation and emission wavelength at 275 nm and 312 nm respectively were used to determine 4-NP. RESULTS: There was a good linear relationship between the concentrations of the analytes in serum and their peak areas in the range of for DEHP, for MEHP, for p, p'-DDE and for 4-NP. The detection limit of the method was 1 ng/ml for each of the analytes. Recoveries of serum samples were 96.84% - 103.44% for DEHP, 87.69% - 104.92% for MEHP, 87.6% - 109.89% for p, p'-DDE, 98.57% - 105.1% for 4-NP. CONCLUSION: The method is suitable for the determination of the four kinds of EEDs in serum. It is easy to dispose the samples. The volume of samples is decreased and the effect of detection is increased. The method is quicker and easier and could be widely used to determine these compounds. It is helpful for the clinical study of precocious puberty.

[Study on the level of environmental endocrine disruptors in serum of precocious puberty patients].

OBJECTIVE: To investigate the role of environmental endocrine disruptors (EEDs) in causing the precocious puberty. METHODS: The blood samples were collected from 79 cases of precocious puberty patients and 42 cases of normal children. The concentrations of 4-nonylphenol (4-NP), 1, 1-dichloro-2, 2, bis (p-chlorophenyl) ethylene (p, p'-DDE) and di-2-ethylhexyl phthalate (DEHP) in blood serum samples were measured by using reversed-phase high performance liquid chromatography (HPLC). The volume of uterus and ovary, the bone density, and the content of estradiol (E(2)) in serum were determined at the same time. The contents of EEDs in blood serums of precocious puberty and the indices of the target organs were analyzed by using of correlation and regression. RESULTS: In normal control group, p, p'-DDE was detected in all the blood samples (14.93 - 40.39 ng/ml), but 4-NP and DEHP were detected in some samples (ND -6.77 ng/ml, ND -17.61 ng/ml). The levels of 4-NP, p, p'-DDE and DEHP in blood serum in precocious puberty group were notably increased than that in control group (P < 0.01). In precocious puberty group, there was a positive correlations between the 4-NP in volume of uterus and the volume of ovary and the density of bone (r = 0.394, 0.286, 0.237, P < 0.01); p, p'-DDE and volume of uterus also showed a the positive correlation (r = 0.306, P < 0.01). The influencing extent of 4-NP was 1.3 times to that of the p, p'-DDE. CONCLUSION: The normal children and the children with precocious puberty should be all contaminated by EEDs, and the later be exposured to more EEDs. There might exist a close relationship between EEDs and the precocious puberty, and EEDs should be an important factor in causing the disease. Different kinds of EEDs might have different influencing extents to the target organs.

[Effects of Chinese herbal medicine on modulating the course of puberty development in children with precocious puberty].

OBJECTIVE: To explore the therapeutic effects of Ziyin Xiehuo Recipe (ZYXHR) for nourishing yin and lowering fire and Yishen Tianjing Recipe (YSTJR) for nourishing kidney and replenishing essence on regulating the gonadotrophic and somatotrophic functions of hypothalamic-pituitary axis, and to reveal the mechanisms of ZYXHR and YSTJR in modulating the course of pubertal development of children with precocious puberty. METHODS: The pubertal rats were fed with ZYXHR or YSTJR for 30 days, and the parameters of rats were monitored as the followings: The content of gonadotropin-releasing hormone (GnRH), the frequency and amplitude of GnRH impulse releasing, the releasing amounts of aminoacid neurotransmitters, and neuropeptide Y (NPY) and beta-endorphin (beta-END) in the gonadotrophic area of the hypothalamus were detected with neurobiological methods (push-pull perfusion, homogenate, incubation of brain slices, and immunohistochemical staining). The levels of gene and protein expressions of GnRH, growth hormone releasing hormone (GHRH) and somatostatin (SS) in hypothalamus, and follicular stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone (GH) in adenohypophysis as well as insulin-like growth factor I (IGF-I) in metaphysis were determined with real time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The ZYXHR could reduce the activity of GnRH neurons in hypothalamus through inhibiting the release of central exciting aminoacid neurotransmitters, whereas promoting the release of central inhibiting aminoacid neurotransmitters, NPY and beta-END in gonadotrophic area of hypothalamus. The expression levels of GnRH, FSH and LH mRNAs were down-regulated while the expression level of SS mRNA in hypothalamus was up-regulated in the ZYXHR-treated group. The GH mRNA in hypophysis and the IGF-I mRNA in metaphysis were both down-regulated by ZYXHR. The YSTJR could promote the activity of GnRH neurons in hypothalamus through inhibiting the release of NPY in gonadotrophic area of hypothalamus, up-regulating the expression levels of GnRH, FSH, LH and GH mRNAs in hypophysis, and IGF-I mRNA in metaphysis, while down-regulating the expression level of SS mRNA in hypothalamus. CONCLUSION: The ZYXHR and YSTJR could both regulate the gonadotrophic and somatotrophic functions of hypothalamic-pituitary axis through modulating the neuroendocrine regulation and the gene expressions of GnRH and SS in hypothalamus, GH, FSH and LH in hypophysis, and IGF-I in metaphysis. These may be the chief mechanisms of ZYXHR and YSTJR in modulating the course of pubertal development and ameliorating the skeletal development in children with precocious puberty.

[Study on the regulative rule of reinforcing shen principle on sexual precocity and senescence at the molecular level].

The imbalance of Shen-yin and Shen-yang is a common pathological basis of sexual precocity and senescence. Senescence can be regarded as physiological Shen-deficiency, which is mainly the deficiency of Shen-Yang, while the essence of sexual precocity is hyperactivity of Fire due to Yin-deficiency, and deficiency of Shen-Yin is the predominance in the imbalance. The process of growth, development, maturing and aging led by Shen-qi, is mainly involving the hypothalamus-pituitary-growth hormone axis and sexual gland axis. Early initiation of these two axes appears in sexual premature children, manifesting syndrome of Fire hyperactivity due to Yin-deficiency, while in senescence, the decline of the axes appears ahead of time and manifests syndrome of Yang qi consumption. It has been shown in experimental and clinical researches that significantly good effects could be obtained by applying Chinese recipe for warmly reinforcing Shen-Yang to postpone senescence and recipe for nourishing Yin and removing Fire to inhibit sexual precocity. It also been proved by determining syndrome and speculating the damaged site according to the effects of drug that the pathologic damage of sexual precocity and senescence is sited on the hypothalamus or higher center.

[Pattern of abnormal skeletal development in girls with idiopathic precocious puberty and therapeutic effect of Chinese herbs for nourishing yin and purging fire].

OBJECTIVE: To explore the pattern of abnormal skeletal development in girls with idiopathic precocious puberty (IPP) and the therapeutic effect of Chinese herbs for nourishing yin and purging fire (CH). METHODS: Measurement of bone age, bone mineral content (BMC) and bone density (BD) were performed and levels of serum gla-protein (BGP), insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP3) were determined in girls with IPP at various stages of pubertal development. Data were compared with those collected from matched girls, and the correlativity between various parameters and the extent of the disease were analyzed to explore the pattern. Thirty-eight girls out of them were treated with CH and the therapeutic effect was observed. RESULTS: The bone age of the IPP girls was ahead of time obviously (P < 0.05), levels of BMC, BD and serum BGP, IGF-1 were significantly higher than the levels in the healthy girls of matched age (P < 0.05). And the degree of these changes were markedly positive correlated with the severity of disease (P < 0.05). After being remitted with CH treatment, the advanced bone age was apparently alleviated, and above-mentioned abnormal criteria were lowered significantly (P < 0.05). CONCLUSION: The skeletal development of the girls with IPP was accelerated and the skeletal maturity were ahead of time, the more serious the condition of disease, the more obvious the acceleration and the advanced bone age. CH could decelerate skeletal development and delay the skeletal maturity of IPP patients.

[Effects of kidney-tonifying Chinese herbal medicine on hypothalamic-pituitary gonadotrophic function].

OBJECTIVE: To observe the effects of kidney-tonifying Chinese herbal medicine on the synthesis and secretion of gonadotropin releasing hormone (GnRH) and the related neurotransmitters and neuropeptides, and to explore the mechanism of the regulative effect of Chinese herbal medicine on the hypothalamic-pituitary gonadotrophic function. METHODS: Female Sprague-Dawley rats during the period of normal adolescent initiation (160-180 g, 1.5 months) were randomly divided into three groups. The control group was fed with normal saline and the two experimental groups were fed with Chinese herbal medicine for nourishing yin to reduce fire (Zi Yin Xie Huo, ZYXH) or nourishing kidney to replenish essence (Yi Shen Tian Jing, YSTJ) respectively. The dosage was 5 ml/d for 30 days by gastric gavage. Integrated optic densities of the GnRH and neuropeptide Y (NPY) in medial preoptic area (MPOA), arcuate nucleus (ARC) and median eminence (ME) of hypothalamus were determined by immunohistochemistry method and image processing. The content of GnRH in homogenate of hypothalamus preoptic area was determined by radioimmunoassay (RIA). The releasing amount of monoamine neurotransmitters from medial basal hypothalamus (MBH) was determined by brain slices incubation and high pressure liquid chromatography (HPLC). The releasing amount of GnRH and NPY from POA was determined by push-pull perfusion and RIA, and the releasing amount of monoamine neurotransmitters from this site was determined by HPLC. RESULTS: ZYXH could inhibit the synthesis and secretion of GnRH from periodic and tonic secretory centers of GnRH, while YSTJ could stimulate the synthesis and secretion of GnRH from the both secretory centers of GnRH. ZYXH could inhibit the activity of GnRH neurons via diminishing the releasing of norepinephrine (NE) from tonic secretory center of GnRH, increasing the releasing of dopamine (DA) in periodic secretory center of GnRH and increasing the synthesis and releasing of NPY from the both secretory centers of GnRH, it hence inhibited the hypothalamic-pituitary gonadotrophic function. YSTJ could stimulate the activity of GnRH neurons via diminishing the synthesis and releasing of NPY from tonic secretory center of GnRH, it hence promoted the hypothalamic-pituitary gonadotrophic function. CONCLUSION: Chinese herbal medicine for tonifying the kidney could modulate hypothalamic-pituitary gonadotrophic function via regulating the synthesis and secretion of GnRH and the related neurotransmitters (NE, DA) and neuropeptides (NPY).