| Objective:(1)To investigate the estrognic effects of environmental endocrine disruptors(EEDs) and to test the antagonistic effects of traditional Chinese medicine on the estrogenic animal model induced by EEDs.(2) To investigate the adverse effects of EEDs on the gene regulative network related to pituitary-gonadal axis in rats using the functionally genomic analysis.Furthermore,the mechanisms of estrogen antagonistic action of traditional Chinese medicine on the estrogenic rats were also explored.Methods:(1) The estrogenic animal models were induced by the administration of the representative EEDs such as 4-nonylphenol(NP) alone,or by the co-administration of NP and bisphenol A(BPA) in the female SD rat at the age of three-week-old.For testing the preventive action of traditional Chinese medicine on the estrogenic rat induced by the abovementioned EEDs,traditional Chinese medicine was also i.g.administered to the rat when the animal model was producing.After 15-day course of treatment with or without traditional Chinese medicine,the uterine wet weight,uterus organ coefficient,height of endometrium epithelium,endometrial glands,myometrium thickness,protein level of proliferating cell nuclear antigen (PCNA) in the endometrium and myometrium and the estrogen receptor(ER) expression in the rat uterine were measured and analyzed by one-way ANOVA.(2) The gene expression profiles in pituitary—ovarian axis of rats which were fed with NP+BPA or co-treatment with traditional Chinese medicine respectively were detected by Illumina Beadchip(ratref-12).The expression of genes which could be influenced significantly by EEDs and reversely affected by traditional Chinese medicine was analyzed.Moreover,the screened genes which are closely related to sexual development were further verified by real-time polymerase chain reaction (real-time PCR)and western blotting.Significance was analyzed by one-way ANOVA. (3)All the screened and verified genes were categorized into three groups according to their functions,i.e.1) Genes related to GnRHR and GnRHR receptor-mediated signal transduction pathway;2) Genes related to ER and ER-mediated signal transduction pathway and 3) Enzymes involved in the estrogen metabolism in ovary.Results:(1) The uterine wet weight,uterus organ coefficient,the height of endometrium epithelium,myometrium thickness,endometrial glands significantly increased in the NP or NP+BPA groups as compared with those of control group.In addition,PCNA and ER expression in endometrium and myometrium were also significantly up-regulated in the EEDs groups.However,all the increased or up-regulated indicators abovementioned were notably decreased in the animals which were co-treated with traditional Chinese medicine(p＜0.05).(2) The data of gene expression profiles screened and analyzed by Illumina Beadchip(ratref-12) demonstrated that the mRNA levels of LHβand FSHβ,which involve in the functional group of GnRHR and GnRHR receptor-mediated signal transduction pathway significantly up-regulated in the EEDs exposure animals.With traditional Chinese medicine treatment,the up-regulated mRNA expressions of LHβand FSHβby EEDs were significantly inhibited.Moreover,the mRNA expression profile of genes related to the GnRHR receptor-medicated signal pathway,including phospholipase family(PLCd1,PLC 11,PLCb2,PLCg2),diacylglycerol(DG)family (DGat2,DGkq),lipoxygenase family(Alox15,Alox5,Alox3,Alox12)and leukotriene family(ltb4r2,ltc4s,ltb4dh) also showed the similar patterns as those of LHβand FSHβ,which significantly increased in the EEDs exposure groups and reduced in the traditional Chinese medicine treatment groups.Among them, lipoxygenase Alox15 was the differential gene displayed by the ratref-12 analysis.In the group of genes related to ER and ER-receptor mediated signal pathway,the mRNA expression of ERαand ERβwas significantly increased in rats exposure to the EEDs for 8 and 15 days and decreased in the rat with traditional Chinese medicine treatment.Furthermore,the increases in ER coactivator Sral,cross-talker EGF, EFGR,IGFβ2,IGFr2 mRNA expression induced by EEDs decreased with 15 days-treatment with traditional Chinese medicine.Among them,EGFR was the differential gene,which showed the similar trend as that of ER.Our data also showed that among the enzymes participate in the estrogen metabolism,the gene expression of stAR and CYP19al was significantly increased in the EEDs exposure groups as compared to the control group.However,traditional Chinese medicine treatment significantly down-regulated stAR and CYP11A1 expression when compared to the animals with the EEDs exposure alone.(3) The expression levels,including mRNA and protein of ERα,ERβ,EGFR,stAR,CYP19al and Cyp11A1 were further validated by real-time PCR and western blot.The results indicated that the mRNA levels of ERα,ERβ,EGFR,stAR,CYP19al on ovary were significantly raised(p＜0.05) after exposed to NP+BPA.The protein expressions of ERα,ERβ,stAR, CYP19al,Cyp11A1 were significantly increased(p＜0.05) as well.After treatment with traditional Chinese medicine the upregulated-mRNA levels of ERα,ERβ,EGFR, stAR and Cyp11al were significantly down-regulated(p＜0.05) and the protein expression of ERα,ERβ,stAR,Cyp11A1 significantly reduced(p＜0.05).Conclusions:(1) The estrogenic effects of EEDs were tested and validated by uterotrophie assays in immature female SD rats.Our data demonstrated that treatment with the traditional Chinese medicine could inhibit the estrogenic effects induced by EEDs,which was consistent with our previous studies.(2) With the analysis of gene expression profiles,EEDs could up-regulate the mRNA levels of genes which promote pituitary-gonadal axis function and therefore elicited estrogenic activity.(3) Traditional Chinese medicine could significantly inhibit the adversely estrogenic effect of EEDs by intervening the gene expression related to pituitary-gonadal axis function.
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