| Objective:(1)To replicate the anmimal model of precocious puberty induced by environment endocrine disruptors (EEDs) in cynomolgus monkey,which will more accurately imitate human disease progress and drug treatment function.(2) To verify the antagonistic effect of Traditional Chinese medicine on the estrogenic anmimal model induced by EEDs.(3)To investigate the mechanism of the antagonistic action of Traditional Chinese medicine on gene and protein level.The research will provide more scientific and appropriate theorial support for the Traditional Chinese medicine.Methods:(1) Preliminary experiment:The animal models were induced by the co-administration of the representative EEDs such as4-nonylphenol (4-NP) and bisphenol A (BPA) in female precocious cynomolgus monkey. These monkeys were divided into two age groups, each one including the high dose group, the low dose group and the control group. We observed EEDs estrogenic effect by analyzing the maturation value of vaginal keratinocytes, uterine volume, uterine wet weight, organ coefficient, endometrium thickness, myometrium thickness, endometrial epithelial cell height, the uterine glandular epithelium height and so on. And by then, we can be sure about the best age, the best exposure dose and the optimal exposure time to establish the precocious puberty models.(2)The prepubertal female cynomolgus monkeys at the best age were randomly divided into the exposed group, the treatment group and the control group. The exposed group was fed with4-NP and BPA, the treatment group was fed with4-NP, BPA and Traditional Chinese medicine, and the control group was fed with corn oil. We can observe the EEDs estrogenic effect and the antagonistic effect of Chinese medicine by analyzing the indicators mentioned above.(3) In order to investigate the mechanism,we tested some factors by the real-time polymerase chain reaction (RT-PCR) and Western blotting, respectively on the gene and protein expression level. We validated the change of the estrogen receptor a (ERa), estrogen receptor β (ERβ), epidermal growth factor receptor (EGFR), insulin-like growth factor1receptor (IGF-1R), the cholesterol side-chain cleavage enzyme (cytochrome P450, family11, subfamily A, polypeptide1, CYP11A1), aromatase(cytochrome P450, family19, subfamily A, polypeptide1CYP19A1). Significance was analyzed by one-way ANOVA.Results:(1)Replicating the cynomolgus monkey model of precocious puberty induced by EEDs, we should ensure that20-month old female cynomolgus monkeys as the best experimental animals, low dose BPA and4-NP as the best exposure dose, and4weeks as the optimal exposure time.(2)Serum estradiol, the maturation value of vaginal keratinocytes, the uterine weight, uterus organ coefficient,endometrial thickness, myometrium thickness myometrium thickness,height of endometrium epithelium and endometrial glands were significantly increased in the exposed group compared to the control group(p<0.05). These markers were sharply reduced (p<0.05) in the treated group than the exposed group.(3)The mRNA expression levels of ERa, ERp, EGFR, IGF-1R, CYP11A1and CYP19A1were validated by RT-PCR.The results indicated that the mRNA levels of these relevant markers were significantly raised (p<0.05) after exposed to4-NP and BPA. Howerer, after treatment with Traditional Chinese medicine the up-regulated mRNA levels were significantly down-regulated (p<0.05).(4)The potein expressions of ERa, ERβ, EGFR and CYP19A1were significantly increased (p<0.05) after exposed to4-NP and BPA.After treatment,the protein levels of ERa, ERP, EGFR and CYP19A1were reduced (p<0.05) as well.Conclusions:(1) We successful replicated the cynomolgus monkey model of precocious puberty induced by EEDs.(2)The findings on this model demonstrated that Traditional Chinese medicine can antagonize the estrogenic effect of EEDs.(3) Traditional Chinese medicine could significantly inhibit the adverse effect of EEDs, on multiple levels and targets. |
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