Study On The Expression Of Estrogen Effect Related Receptors In Adenomyosis Mice Model By Therapy Of Expelling Dampness And Dissipating Blood Stasis

Objective To investigate the mechanisms of adenomyosis(AM)by therapy of expelling dampness and dissipating blood stasis through observating the relative expression changes of ERα、ERβ、PR-A、PR-B、GPER、Akt、p-Akt、NF-κB m RNA and proteins in uterus tissues of AM mice before and after medication,and in different Course of disease which vary with estrus cycles.Methods We transplanted the pituitary gland of male mice into the uterine cavity of the female mice in order to construct the AM mice model at first.Then we divided these mice into seven groups randomly: high dose of Neiyikangfupian(expelling dampness and dissipating blood stasis)group(NYK group),medium dose of NYK group,low dose of NYK group;Danefukangjiangao group(DE group);Gestrinone group(GN group);3 months group and 6 months group.At the same time,we set the normal mice or mice with fake operation as the control groups.Each group had 24 mice.Breed the mice for 3 months.Then mice of 3 months model group and two control groups were killed in oestrus or anoestrus period respectively.Then we gave different drugs to each treatment group for another 3 months,and mice of 6 months group still fed by physiological saline for 3 months.Then we executed all the mice in the rest groups during in oestrus or anoestrus.By the technique of real-time PCR and western blot,testing ERα、ERβ、PR-A、PR-B、GPER、Akt、p-Akt、NF-κB m RNA and proteins expression of mice uterine tissues respectively.Results The results of the mechanisms of adenomyosis(AM):1.Normal group compared with the sham-operation group: the relative expression of ERα、ERβ 、 PR-A 、 PR-B 、 GPER 、 Akt 、 p-Akt 、 NF-κB m RNA and proteins had no statistical significance between the two groups(P>0.05).2.Model groups compared with the normal group: the relative expression of ERα、ERβ、PR-A、PR-B、GPER、Akt、p-Akt、NF-κB m RNA and proteins in 3 months model group and 6 months model group both had statistical significance(P<0.05).3.The 6 months model group compared with the 3 months model group:the relative expression of ERα 、 ERβ 、 GPER 、 Akt 、 p-Akt 、 NF-κB m RNA and proteins had statistical significance between the two groups(P<0.05).4.The gene and protein expression of PR-A、PR-B in the 6 group were significantly lower than those in the 3 group(P <0.05).5.The expression of ERα,ERβ,GPER,Akt,p-Akt and NF-κB was significantly higher than that of anoestrus in the 6 group and 3 group(P <0.05).6.The expression of PR gene and protein was significantly higher than that of oestrus in the 6 group and 3 group(P <0.05).The result of the function mechanisms of NYK:1.ERα At the m RNA and protein level,the expression of ERαin NYK dose group,DE group,and GN group were significantly decreased than 6 month group(P<0.05).The m RNA and protein expression of ERα in NYK high dose group were significantly decreased than DE group,and GN group(P<0.05).2.ERβ the expression of the m RNA and protein level of ERβin NYK dose group,DE group,and GN group were significantly decreased than 6 month group(P<0.05).And the m RNA and protein expression of ERα in NYK high dose group were significantly decreased than DE group,and GN group(P<0.05).3.PR-A At the m RNA and protein level,the expression of PR-A in NYK dose group,DE group,and GN group were obviously increased than 6 month group(P<0.05).The m RNA and protein expression of ERα in NYK high dose group were remarkable increased than DE group,and GN group(P<0.05).4.PR-B At the m RNA and protein level,the expression of PR-B in NYK dose group,DE group,and GN group were significantly increased than 6 month group(P<0.05).The m RNA and protein expression of ERα in NYK high dose group were significantly increased than DE group,and GN group(P<0.05).5.GPER At the m RNA and protein level,the expression of GPER in NYK dose group,DE group,and GN group were significantly decreased than 6 month group(P<0.05).The m RNA and protein expression of ERα in NYK high dose group were significantly decreased than DE group,and GN group(P<0.05).6.Akt At the m RNA and protein level,the expression of Akt in NYK dose group,DE group,and GN group were significantly decreased than 6 month group(P<0.05).The m RNA and protein expression of ERα in NYK high dose group were significantly decreased than DE group,and GN group(P<0.05).7.p-Akt At the m RNA and protein level,the expression of p-Akt in NYK dose group,DE group,and GN group were significantly decreased than 6 month group(P<0.05).The m RNA and protein expression of ERα in NYK high dose group were significantly decreased than DE group,and GN group(P<0.05).8.NF-κB At the m RNA and protein level,the expression of NF-κB in NYK dose group,DE group,and GN group were significantly decreased than 6 month group(P<0.05).The m RNA and protein expression of ERα in NYK high dose group were significantly decreased than DE group,and GN group(P<0.05).9.Comparison of all of the groups in oestrus or anoestrus :In this study,the expression of ERα,ERβ,GP,PR and Akt in the estrogen-related molecules was detected by western-blot.Both the eutopic endometrium and the ectopic endometrium were changed with different stages of the oestrus cycle,There was no statistically significant difference between PR-B in the DE group and GN group,and the other indicators were statistically significant in each group.The expression of progesterone receptor(PRA,PRB)was significantly higher than that of oestrus,while the others was significantly higher than that of anoestrus.Conclusions1.The occurrence of AM is related to the abnormal expression of estrogen and progesterone receptors and their pathways in endometrium.The results of this study show that overexpression of ERα and ERβ,especially the expression of ERα,increases estrogen and endometrial glands and interstitial Cell response,and GPER as an estrogen transmembrane receptor,which mediates the rapid genomic effect of estrogen can activate the Akt pathway,so that PI3 K / Akt / m TOR signaling pathway abnormalities,and activation of PI3 K / Akt pathway can Promote the activation of NF-κB,resulting in local estrogen levels are too high,promote cell proliferation,inhibit the normal role of apoptosis;and PR receptor subtype of low expression,especially PR-B protein deficiency,progesterone Activation and transcription weakened,progesterone can not fully play its normal role,and PR-A protein reduction so that it can not fully inhibit the transcriptional activation of ERα,so progesterone antagonism estrogen weakened,so that the excessive growth of endometrium and easy Uterine muscle and survive,leading to the occurrence of AM.2.The effect of estrogen on the proliferation of endometrial glands and stromal cells can be reduced by the therapy of expelling dampness and dissipating blood stasis through reducing the expression of estrogen and membrane receptors on the endometrium.Meanwhile,upregulates the expression of progesterone receptors to enhance the efficacy of progesterone antagonizes estrogen;so that the nuclear membrane is co-conditioned,and the two-way target controls the expression of estrogen-related receptors to improve the abnormal estrogenic effects.3.High dose of different anti-rehabilitation tablets in the regulation of estrogen-related factors is superior to gestrinone and Dan-E-Fu-Kang Decoction.The use of the method of the therapy of expelling dampness and dissipating blood stasis confirms the validity of the theory of collaterals and the theory of Xuanfu,which provides a new way of thinking for TCM treatment.It is worthy of further study and popularization.4.The expression of estrogen-related factors is regulated by hormones,with periodic changes in hormones,and estrogen effects in estrus than in intercourse,so this will be the best time choice for clinical treatment of AM to provide the basis.