| Objective To nvestigate the mechanisms of adenomyosis by therapy of expelling dampness and dissipating blood stasis through observating the relative expression changes of ER alpha, ER beta, GPER, PR-A. PR-B mRNA 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 firstly transplanted the pituitary gland of male mice into the uterine cavity of the female mice in order to construct the AM mice model. Then we divided these mice into seven groups randomly:high dose of Neiyikangfupian (expelling dampness and dissipating blood stasis) group (NYK gropu), 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, set the mice without operation or with fake operation as the control groups. Each group had 12 mice. Breed the mice for 3 months. Then mice of 3 months model group and two control groups were killed in estrum and interval period respectively. Then gave different drugs for another 3 months in each treatment group, 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 estrus and diestrus.By the technique of real-time PCR and western blot, testing the ER alpha, ER beta, GPER, PR-A, PR-B mRNA and proteins expression of in mice uterine tissues respectively.Results1. Normal group compared with the sham-operation group:the relative expression of ER alpha, ER beta, GPER, PR-A, PR-B mRNA 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 ER alpha, ER beta, GPER, PR-A, PR-B mRNA 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 ER alpha, ER beta, GPER, PR-A, PR-B mRNA and proteins had no statistical significance between the two groups (P>0.05).4.Comparison of the treatment groups and the 6 months model group:(1) ER alpha The relative expression of ER alpha mRNA and protein in NYK high dose group, DE group, and GN group were significantly decreased in estrus and diestrus (P<0.05). NYK low dose group decreased significantly only in diestrus (P<0.05).(2) ER beta At the mRNA level, NYK high dose group, DE group, GN group were both significantly decreased in estrus and diestrus (P<0.05);and NYK dose group decreased significantly only in diestrus (P<0.05). At the protein level, all of the treatment groups were decreased significantly (P<0.05).(3) GPER The relative expression of GPER mRNA and protein in NYK high dose group, DE group, and GN group were both significantly decreased in estrus and diestrus cy (P<0.05). There were no significant difference in the other treatment groups (P>0.05).(4) PR-A At the mRNA level. NYK high dose group, NYK medium dose group, DE group, and GN group were both significantly decreased in estrus and diestrus (P<0.05); and NYK low dose group only showed significantly increased in estrus (P<0.05). At the protein level, NYK high dose group, DE group, and GN group were both significantly decreased in estrus and diestrus (P<0.05). And NYK dose group was significantly up-regulated only in diestrus(P<0.05).(5) PR-B In estrus, the relative expression of PR-B mRNA and proteins were significantly up-regulated only in NYK high dose group and DE group (P<0.05). In diestrus, there was significantly increased only in the NYK high dose group and DE group, and just at the mRNA level (P<0.05).5. Comparison of all of the groups in estrus and diestrus:There was statistically significant between the estrus and diestrus cycle in normal group and the sham operation group about the relative expression of ER alpha, ER beta, GPER, PR-A and PR-B mRNA and protein (P<0.05). But in 3 months model group and 6 months model group, there was no statistically significant difference(P>0.05), except the relative expression of PR-A mRNA(P<0.05). In the treatment groups, the relative expression of ER alpha. ER beta, GPER, PR-A mRNA and protein did not show the statistically significant between the estrus and diestrus cycle (P>0.05). Also did the expression of PR-B mRNA(P>0.05). But the PR-B protein in NYK tablet of high dose groups, DE group and Decoction group showed the statistically significant difference between the estrus and diestrus cycle (P<0.05).Conclusions1. The occurrence of AM is related to the amount change of local estrogen and progesterone receptors expression in uterine. This experimental data show that the up-regulation of estrogen receptor and down-regulation of progesterone receptors may lead the occurrence of AM. Among the abnormal expression of estrogen receptors, the new estrogen receptor GPER, togther with the nuclear receptors ER alpha and ER beta, play a synergistic role. But the nuclear receptors are in advantage, especially ER alpha. In addition, the distribution pattern changes of estrogen and progesterone receptor subtype and the periodic loss may also be the biological basis of the pathogenesis of AM.2. The therapy of expelling dampness and dissipating blood stasis may down-regulate the expression of ER alpha, ER beta, GPER to reduce the proliferation response of endometrial glands and stromal cells, and up-regulate the expression of PR-A and PR-B in order to block estrogen effect.3. The different treatment methods all play a role in relating the estrogen effects. The high dose of Nei-Yi-Kang-Fu Tablet have the same regulating power with Dan-E-Fu-Kang Decoction and Gestrinone Capsules in regulating the expression of ER alpha, ER beta, GPER, PR-A; but better than Gestrinone Capsules in regulating the expression of PR-B. Chinese traditional medicine can avoid the side effects, and the therapy of expelling dampness and dissipating blood stasis may provides a new method for TCM as an new treatment of AM, and Nei-Yi-Kang-Fu Tablet is a traditional Chinese medicine compound which is worthy of further research and promotion.4. The Prescription which have the effect of expelling dampness and dissipating blood stasis may recover the periodic expression of estrogen effect related receptors, but receptor types and their regulatory mechanisms still needs further experimental studies to confirm. |
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