Pathogenesis Of Precocious Puberty Induced By Environmental Endocrine Disruptors And Therapeutic Effect Of Traditional Chinese Medicine

Objective: To explore the role of environmental endocrine disruptors(EEDs) in the onset of precocious puberty(PP) and the therapeutic effect of traditional Chinese medicine.Methods: (1) The blood samples were collected from 73 cases of precocious puberty patients and 38 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 the blood serum samples were measured by using reversed-phase high performance liquid chromatography(HPLC). The volume of uterus and ovary, bone density, and content of estradiol(E2) in the serum were determined at the same time. Contents of EEDs in the blood serums of precocious puberty cases and indices of the target organs were analyzed by correlation and regression methods. (2) The vehicle (peanut oil), ethinyl estradiol(EE, 0.3mg/kg positive control) , 4-NP(50mg/kg,100mg/kg,200mg/kg), bisphenol(BPA,200mg/kg,400mg/kg,600mg/kg),DEHP(100mg/kg,250mg/kg,500mg/ kg) and a mixture of 4-NP 50mg/kg and BPA 200mg/kg were given orally(by gavage ) q.d. to immature female Sprague-Dawley rats on the 21^sd, 22^nd,23^rd postnatal days. The rats were sacrificed 24 hours after the last administration. Uterine wet weight, uterine/body weight ratio, height of the luminal epithelium, thickness of the myometrium and protein level of proliferating cell nuclear antigen(PCNA) of rat uterine were analysed by one-way ANOVA. (3) The therapeutic project of traditional Chinese medicine was established to treat PP children and the experimental animals. Developing parameters of reproductive organs and bone were observed. Using methods of real— time fluorescence quantitative PCR and immunohistochemistry, we detected the expression level of four receptors on target organs(uterine, breast, bone), including estrogen receptor alpha(ERα) estrogen receptor beta(ERβ) epidermal growth factor receptor(EGFR) and insulin like factor-1 receptor( IGF-1R). The data were analysed by one-way ANOVA and T test.Results: (1) In the normal control group, p,p'-DDE was detected in all theblood samples(14.93 - 40.39ng/ml), but 4-NP and DEHP were only detected in some of the samples(ND - 6.77 ng/ml,ND - 17.61 ng/ml). Levels of 4-NP p,p'-DDE and DEHP in the serum of the precocious puberty group were notably higher than that of the control group (P < 0.01) .In the precocious puberty group, positive correlations were found between 4-NP and volume of uterus, volume of ovary and bone density ((r=0.394, 0.286,0.237; P < 0.01); p,p'-DDE and volume of uterus also had a positive correlation((r=0.306;P < 0.01). The influence intensity of 4-NP was higher than that of p,p'-DDE. (2) In animal experiments, uterine wet weight and uterine/body weight ratio were significantly higher in the 4-NP 200mg/kg and 100mg/kg, BPA 600mg/kg and 400mg/kg, and mixture group(4-NP 50mg/kg and BPA 200mg/kg) (P < 0.05) . Height of luminal epithelium, thickness of myometrium,as well as protein level of PCNA of rat uterine were significantly increased in the three groups of 4-NP, the three groups of BPA, and the mixture group (P < 0.05) , and a dose-response relationship was observed. Levels of aboved parameters in the mixture group were significantly higher than that in the single dose groups (P < 0.05) . (3) Volume of uterus, content of estradiol(E2) in the serum and bone density, bone mineral content, content of osteocalcin(OST) in the serum were decreased in patients after treating with traditional Chinese medicine (P < 0.05) . Uterine wet weight, uterine/body weight ratio, height of the luminal epithelium, thickness of the myometrium, protein level of PCNA of rat uterine and bone density of rats were notably decreased in animals treated with Chinese traditional medicine (P < 0.05) . After the therapy, mRNA and protein levels of ER α and ER β were significantly down regulated in uterine, breast and bone(P < 0.05). The mRNA and protein level of EGFR were significantly regulated down-ward in uterine and breast (P < 0.05) . The mRNA and protein level of IGF-1R were also significantly regulated down-ward in bones (P<0.05) .Conclusions: (1) Normal children and children with precocious puberty are all contaminated by EEDs, and the later are exposured to higher EEDs. There is a close relationship between EEDs and the onset of precocious puberty, and EEDs are important factors inducing the disease. Different kinds of EEDs have different influence intensity on target organs. (2) The estrogenic effects of EEDs and their dose-response relationship were validated by uterotrophic assay in immature SD rats. Different kinds of EEDs below no effect levels, if combined, could also evoke additive effects, exerting estrogenic activitiy. (3) Traditional Chinese medicine couldregulate and decrease the expression of ERα ,ERβ,EGFR and IGF-1R on the target organs. Through this mechanism, treatment with traditional Chinese medicine, the medicine could inhibit the estrogenic effects of EEDs and slow down the accelerated development of reproductive organs and skeleton in PP patients.