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<p><b>OBJECTIVE</b>To investigate the mechanism of di-2-ethylhexyl phthalate (DEHP) and cypermethrin (CYP) inducing gonadal dysgenesis in prepubertal male rats.</p><p><b>METHODS</b>A total of 40 healthy 3-week-old specific pathogen-free male Sprague-Dawley rats were randomly and equally divided into four groups: control group (corn oil), DEHP group (500 mg/kg, dissolved in corn oil), CYP group (80 mg/kg, dissolved in corn oil), and combined exposure group (exposed to 500 mg/kg DEHP and 80 mg/kg CYP, dissolved in corn oil). Rats were treated by gavage administration once a day for 30 days. Twenty-four hours after the last exposure, the animals were sacrificed. The body weight and the wet weight of testis were determined, and the weight coefficient of testis was calculated. Radioimmunoassay was used to determine serum testosterone level. Ultrastructural-level histopathological changes of the testis were examined by transmission electron microscopy. The mRNA and protein expression of follicle stimulating hormone receptor (FSHR), androgen binding protein (ABP), inhibin beta-B (INHBB) and vimentin (VIM) were analyzed by real-time PCR and Western blot, respectively. Factorial design analysis of variance was used to compare differences between groups; interaction diagrams were used to determine the interaction between DEHP and CYP.</p><p><b>RESULTS</b>Compared with those of the control group, the testis weights and testis coefficients of the DEHP, CYP, and combined exposure groups significantly decreased by 39.3-59.2%and 19.7-58.6%, respectively, and all exposure groups showed significant reductions in serum level of testosterone, ranging from 49.1% to 62.7% (P < 0.05 or P < 0.01). And all the exposure groups showed different levels of ultrastructural damages in the testes. Compared with that in the control group, the mRNA expression of FSHR, ABP, INHBB, and VIMin the DEHP group was down-regulated by 1.72, 2.64, 2.83 and 1.79 times, and their protein levels were significantly reduced by 65.2%, 53.7%, 70.1%, and 51.9% (P < 0.05 or P < 0.01). Significant decreases in mRNA expression of ABP (down 1.72 times) and INHBB (down 2.06 times) were observed in the CYP group, and their protein levels decreased by 38.3% and 49.7%, respectively (P < 0.05). The combined exposure to both DEHP and CYP resulted in big decreases in the mRNA levels of FSHR (down 1.62 times), ABP (down 2.00 times), INHBB (down 2.35 times), and VIM (down 1.54 times) and protein levels of FSHR (down 52.1%), INHBB (down 53.9%), and VIM (down 58.8%) (P < 0.05). Factorial design analysis of variance showed that the combination of two substances had an antagonistic effect on the expression of ABP and INHBB (P < 0.05).</p><p><b>CONCLUSION</b>DEHP and CYP, alone or combined, can lead to gonadal dysgenesis in prepubertal male rats. Both of them can disrupt functional mRNA and protein expression in Sertoli cells to certain levels. The combination of DEHP and CYP shows antagonistic effects, and DEHP has a stronger reproductive toxicity than CYP.</p>
Subject(s)
Animals , Male , Rats , Diethylhexyl Phthalate , Toxicity , Gonadal Dysgenesis , Pyrethrins , Toxicity , Rats, Sprague-Dawley , Sertoli Cells , Metabolism , Testis , Cell BiologyABSTRACT
BACKGROUND: In association with advanced startover of hypothalamus-pituitary-sexual gland axis, the growth of skeleton is accelerated and epiphyseal fusion occurred earlier in puberty precocity children. In clinic,Chinese drug for nourishing yin and reducing fire retards bone age and the advanced fusion in the disease. At the age of sexual development, Chinese drug for benefiting kidney to supplement essence is replaced to promote sexual development and accelerate skeleton growth. The patients with puberty precocity are the same to that in normal puberty in sexual development and bone growing, just being advanced in time. But, it is still unknown the mechanism of Chinese drugs on bone growth and effects on somatostatin (SS) in bone growth.OBJECTIVE: To explore the regulation of kidney-nournishing Chinese drug on periventricular nucleus (PeVN) SS.DESIGN: Completely randomized group division and control experiment were designed.SETTING: Department of Endocrinology, Children's Hospital Affiliated to Shanghai Jiaotong University MATERIALS: The experiment was performed in Institute of Pediatrics of Children's Hospital Affiliated to Shanghai Fudan University from October 2001 to April 2002, in which, 24 SD white rats were employed, of purebred, female and clean grade. 4 groups were randomized, named the control A, the group with drugs of nourishing yin and reducing fire (experiment group No. 1), the control B and group with drugs of nourishing yin and reducing fire + benefiting kidney to supplement essence (experiment group No.2),6 rats in each group.METHODS: ① Control A:physiological saline was applied for gastricperfusion, 5 mL each time, once per day, totally for 1 month. Experiment No. 1 group: Chinese mixture for nourishing yin and reducing fire was applied for gastric perfusion, composed of shengdihuang (Radix Rehmanniae),zhiguiban (Chinemys reevesii.), huangbai (Cortex Phellodendri) and zhimu (Rhizoma Anemarrhenae) etc, 5 mL each time, once per day, totally for 1 month. Control B: physiological saline wasapplied for gastric perfusion,5 mL each time, once per day, totally for 2 months. Experiment No.2 group:Firstly, Chinese mixture for nourishing yin and reducing fire was applied for gastric perfusion, 5 mL each time, once per day, totally for 1 month.And then Chinese mixture for benefiting kidney to supplement essence was applied for breeding, composed of shudihuang (Radix Rehmanniae Preparata), guibanjiao (Colla Carapacis Testudinis), xianlingpi, lujiaojiao (Colla Cornus Cervi) etc., 5 mL each time, once per day, totally for 1 month.② In situ hybridization staining procedure with floating and routine immunohistochemical technique. Medical imaging analysis software was used to assay PeVN SS mRNA, SS immunoreactive neuron counts and absorption (A value) of positive cell area that reflects the gene expression and protein expression. Number of positive neurons of unit area refers to the positive neuron counts in each square millimeter.③ Singer-factoranalysis of variance was used for the comparison of measurement data. F test was adopted for difference comparison among groups.MAIN OUTCOME MEASURES: Effects of Chinese drugs for nourishing yin and reducing fire and benefiting kidney to supplement essence on PeVN SS gene mRNA and protein expressionRESULTS: 24 rats were all in result analysis. ① PeVN SS mRNA positive neuron counts, positive neuron counts of unit area and A value of positive cell area: Those in experiment No.1 group was higher remarkably than the those of control A (P ＜ 0.05); and those in experiment No.2 group were lower remarkably than those of experiment No. 1 group (P ＜ 0.05). ② PeVN SS positive neuron counts, positive neuron counts of unit area and A value of positive cell area: those in experiment No. 1 group: those in experiment No.1 group were higher remarkably than those of control A (P ＜ 0.05) and those in experiment No.2 group were lower remarkably than those of experiment No. 1 group (P ＜ 0.05).CONCLUSION: By controlling hypothalamus SS gene expression and protein expression, Chinese drugs for nourishing yin and reducing fire and benefiting kidney to supplement essence regulate the synthesis and releasing of pituitary growth hormone.
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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.
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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).
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Objective:To compare therapeutic effects of Chinese medicine prescriptions for nourishing Yin to purge pathogenic fire,and prescriptions for relieving the depressed liver in treatment of sexual precocity of girls.Methods:The girls with sexual precocity were divided with random,double blind and control methods into a treatment group treated with the prescription for nourishing Yin to purge pathogenic fire,Zaoshu No.Ⅱ,and a control group treated with the prescription for relieving the depressed liver, Xiaoyao Pill.The medicines were given with double blind and double simulated methods.The mammary nucleus index,mammary Tanner phasing,cumulative scores of TCM syndromes,B type ultrasonography(volume of the uterus,ovary,the biggest follicle diameter),change of bone age were followed up and compared before and after treatment in the two groups.Follow-up was completed in 132 cases.Results:The treatment group in the mammary nucleus index,mammary Tanner phasing,decrease of cumulative scores of TCM syndromes after treatment was better than the control group(P0.05).Conclusion:Chinese medicine for nourishing Yin to purge pathogenic fire,Zaoshu No.Ⅱ,in therapeutic effect for treatment of sexual precocity of girls is better than the Chinese medicine for relieving the depressed liver,Xiaoyao Pill.
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Objective To explore the antagonistic effect of Ziyin Xiehuo Recipe(ZYXHR,Recipe for nourishing kidney yin and purging ministerial fire) on estrogen-like activity of environmental endocrine disruptors,nonylphenol(NP) and mixture of NP and bisphenol A(BPA). Methods Totally 30 three-week-old female SD rats were randomly divided into control group(fed with corn oil),exposed group A (feeding NP100 mg / kg ),exposed group B (feeding NP 50 mg/kg+ BPA 200 mg/kg ),treatment group A (NP 100 mg/kg + ZYXHR),treatment group B (NP 50 mg/kg+ BPA 200 mg/kg + ZYXHR). After 15 days of treatment,the uterine weight gain and PCNA protein expression were detected. Results As compared with those of the control group,Uterine wet weight,uterus organ coefficient,thickness of endometrium and smooth muscle,and the height of endometrial glands and glandular epithelium significantly increased in exposed group A and B (P
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The function of hypothalamic-pituitary-ovarian axis and the size of uterus and ovary were determined in 57 girls with idiopathic true precocious puberty at various stages of pubertal development. The results showed: 1) The extent of precocious puberty correlated closely with the course of the disease, the degree of bone growth accelaration, the size of uterus and ovary, and the hyperfunctional state of the hypothalamic-pituitary-ovarian axis, however irrelevant to the age at onset of the disease; 2) The hypothalamic-pituitary-ovarian axis in girls with idiopathic precocious puberty started beforehand and was in a state of hyperfunction. There were characteristic changes in their reactive curve in response to LHRH-stimulating test; 3) In comparison with normal pubertal development, the development of internal genitalia in the girls with idiopathic precocious puberty was more immature and the size was relatively smaller.