The Animal Modeling Of Gonadal Dysgenesis Induced By Environmental Endocrine Disruptors And The Research On The Mechanism Of Antagonistic Effect Of Traditional Chinese Medicine Therapy

Objective:(1)To build the animal model of gonadal dysgenesis induced by environmental endocrine disruptors (EEDs) in male Sprague-Dawley rats.(2) To verify the antagonistic effect of Traditional Chinese medicine on the animal model induced by EEDs.(3)To investigate the mechanism of the antagonistic action of Traditional Chinese medicine on gene and protein level by molecular biology experimental methods.The research will provide more scientific and appropriate theoretical support for the Traditional Chinese medicine.Methods:(1) Preliminary experiment:The animal models were induced by the oral administration of the representative EEDs such as di-2-ethylhexylphthalate (DEHP) and Cypermethrin (CYP) in male Sprague-Dawley rats.30healthy male SD rats were randomly divided into5groups (N=6/group), including the high dose group (DEHP750mg/kg or CYP100mg/kg),the medium dose group (DEHP500mg/kg or CYP80mg/kg) and the control group(feeding corn oil). We observed EEDs anti-androgen effect by analyzing testicular descent,preputial separation.anogenital distance and genitalia development situation,testicular wet weigh.organ coefficient,serum level of testosterone,histopathological changes and so on. As a result, we can be sure about he best exposure dose and the optimal exposure time to establish the gonadal dysgenesis models.(2)The prepubertal male Sprague-Dawley rats (21days old) were randomly divided into the control group(feeding corn oil), the DEHP exposed group, the CYP exposed group and the DEHP+CYP combined exposed group(N=6/group).All the rats were administered with cypermethrin (80mg/kg) or DEHP(500mg/kg) by gavage daily. The testicular descent,preputial separation,anogenital distance,genitalia development situation.the wet weight of the testis, testis coefficient, the serum testosterone level,testis histopathological parameterize and ultrastructural lesions and the activity of lactate dehy drogenase(LDH), acid phosphatase(ACP),alkaline phosphatase(ALP) and succinic dehydrogenase(SDH) in testis homogenate were the criteria used to evaluate the anti-androgen effect and reproductive toxicity of EEDs.Statistical analysis were done using the2x2variance of factorial design.(3) Seventy male Sprague-Dawley rats (21days old) were randomly divided into seven groups (N=10/group).They were control group(feeding corn oil), exposed group A(feeding DEHP500mg/kg),exposed group B (feeding CYP80mg/kg),exposed group C(500mg/kg DEHP+CYP80mg/kg), respectively. The treatment groups were fed with Chinese medicine at the same time.We can observe the EEDs anti-androgen effect and the antagonistic effect of Chinese medicine by analyzing the indicators mentioned above.(4) In order to investigate the mechanism,we tested some factors by the real-time polymerase chain reaction (FQ-PCR) and Western Blot, respectively on the gene and protein expression level. We validated the change of the steroidogenic acute regulatory protein(StAR),cytochrome P450,family11,subfamily A,polypeptide1(CYP11A1),17a-hydroxylase/17,20-Iyase (CYP17A1),3β-hydroxysteroid dehydrogenase(3β-HSD),17β-hydroxysteroid dehydrogenase(17β-HSD),androgen receptor(AR) i.Data were analyzed by one-way ANOVA and the Fisher’s least significant difference (LSD) method to determine treatment differences.Results:(1)Replicating the Sprague-Dawley rats model of gonadal dysgenesis induced by EEDs, we should ensure that21days old male SD rats as the best experimental animals, DEHP (500mg/kg) and CYP (80mg/kg) as the best exposure dose, and4weeks as the optimal exposure time.(2) Study showed significant decline in the wet weight of testes and testis coefficient,and reduction in the serum testosterone level in chemical-treated animals. The activity of LDH, ACP, ALP was significantly increased, while SDH activity was markedly diminished compared to the control group(17<0.05or0.01). These markers were drastically increased (P<0.05or0.01) in the treated group than the exposed group.(3)The mRNA levels of StAR,CYP11A1,CYP17A1,3β-HSD,17β-HSD and AR in testes were verified by FQ-PCR.The results indicated that the mRNA levels of these relevant markers were significantly down-regulated (p<0.05or0.01) in EEDs-treated rat.However, after treatment with Traditional Chinese medicine the down-regulated mRNA levels were significantly up-regulated (p<0.05or0.01).(4) Correspondingly, protein expressions of testicular CYP11A1、CYP17A1and AR were significantly decreased (p<0.05or 0.01) in EEDs-treated rat. After treatment,the protein levels of CYP11A1,CYP17A1and AR were up-regulated (p<0.05or0.01) as well.Conclusions:(1) We successful replicated the male SD rats model of gonadal dysgenesis induced by EEDs.(2) DEHP and CYP had obviously separate and combined reproductive toxicity to prepubertal male rats.DEHP significantly disrupted T synthesis.The decreased T synthesis might be associated with EEDs-induced impairment in spermatogenesis in rat.while CYP interfered with the combination of androgen and the receptor via reducing the number of androgen receptors (3) The findings on this model demonstrated that Traditional Chinese medicine can antagonize the anti-androgen effect of EEDs.(4) Traditional Chinese medicine can significantly antagonize the reproductive toxicity of EEDs, on multiple levels and targets.