Effects And Mechanisms Of Genistein In The Rat Endometriosis Model

Objective:To investigate biological effects of Gen on treatment of Endometriosis and its mechanism we treated various doses of Gen on experimentally induced endometriosis in a rat model. After treatment Gen, we observed ectopic endometrium growth, measured apoptosis induced-area and expression of apoptosis related indicators-Bcl2and Bax; we also observed the expression of estrogen receptor alpha and beta; to evaluate invasion ability and vascular proliferation ability on ectopic endometrium, we examined the expression of VEGF, CD34, MMP-9, TIMP-1, COX-2; lastly, through monitoring Foxp3, checked the effect of immune tolerance.Methods:①Establishment of rat endometriosis model. The6-8weeks old female SD rats were selected and subcutaneous injected diethylstilbestrol to achieve the same estrous cycle, transplanted the autologous endometrial in the abdominal wall fascial layer, then injected17-beta-estradiol for3weeks.②Treatment method and dose selection. Control group was intragastric administratied7.5ml/(kg.d) of peanut oil; Treatment groups:Gen was suspended in7.5ml/(kg.d) of peanut oil and intragastric administrated:low-dose group, Gen50mg/kg/d; middle-dose group Gen150mg/kg/d; high-dose group Gen450mg/kg/d.③Medication persistence time and sampling. The medication time was6weeks. All animals were scarified24hours after last administration and recorded weight of wet tissue, some of specimen were cryopreserved in liquid nitrogen used for RT-PCR and the others fixed in10%neutral formalin for histological analysis.④We used TUNEL assay for detection apoptosis in ectopic endometrium after treatment, used RT-PCR and PCR technique for detection Bcl-2and Bax mRNA expression.⑤ELISA method was used for determination changes in serum E2, we also checked ER alpha, beta ER expression, both in mRNA level and protein level.⑥We performed SABC to detect tissue specimens of VEGF, CD34, MMP-9, TIMP-1, COX-2and Foxp3expression. Results:①Change of graft size:As Gen dose increased, the eutopic endometrium volume tends to smaller. Spatially in the high dose group (D group) it was significantly decreased(P<0.01), while the other Gen groups showed no significant difference compare with control group.②Induction of cell apoptosis:Tissue from high dose Gen group showed higher apoptotic index than the other groups, significant difference (P<0.01), middle dose group apoptotic index higher than low dosage group and control group, but no significant differences. Compare with control group, Bcl-2mRNA expression was significantly up-regulated and Bax mRNA was significantly down-regulated in high dose group (P<0.01).③With the increasing doses of Gen, serum E2although showed a downward trend, but has no statistical significance (P>0.05); compared with other groups high dose Gen significantly reduces ERbeta protein and ER beta mRNA expression (P<0.01), but no effect on ERalpha.④High dose Gen can significantly decrease the expression of VEGF, CD34and COX-2(P<0.01); Also decrease the expression of MMP-9while increasing TIMP-1expression (P<0.01), High dose Gen significantly down-regulated Foxp3expression (P<0.01).Conclusion:Gen can inhibit ectopic endometrium growth in our rat model and these inhibition effect expressed in a dose-dependent manner. Its mechanism as follows:①Induce apoptosis of ectopic endometrial cells through regulating apoptosis related gene expression and activity like Bcl-2and Bax;②Regulate expression of estrogen receptor subtypes:reflecting the beta ER antagonist effects and anti-estrogen effects;③Inhibit ectopic endometrium invasion and vascular proliferation activity through down-regulation of VEGF, CD34, COX-2and MMP-9/TIMP-1expression;④May decrease immune tolerance of ectopic endometrium or enhance immune surveillance and elimination by inhibiting Foxp3expression.