| BackgroundEndometriosis,a benign condition in which the endometrial glands and stroma are present outside the uterine cavity. It is a common disease, which affects between 5 and 15% of`"normal women". It is a destructive disease, and benign only in its classification as having no abnormal meiotic activity. In many other respects, it is a malignant disease that causes considerable anatomical destruction and symptomatology, particularly pain. Endometriosis is thought to be a polygenically inherited disease with a complex, multifactorial etiology. In spite of surgical and medical treatments, recurrence as an important clinical problem remains frustrating. The mechanism of EMS is not well understood,but the most often cited and accepted theory is the"Implantation Hypotheses". The imbalance between phenomenon of retrograde menstruation and the relative low morbidity may be due to the characteristic features of eutopic endometrium. As more and more evidences suggest that eutopic endometrium of patients shares alterations with the ectopic tissue, we propose that the origin of disease is in uterus, and prospected the condition can be prevented or controlled by managing eutopic endometrium directly. Moreover, it is an estrogen-dependent disorder that tends to regress after estrogen deprivation. Our previous study suggested that traditional Chinese compounds"Jia Wei san-leng-wan"(SLW) reached good therapeutic effect on endometriosis. Accordingly, the study that aimed on the'estrogen dependent disease', and to explore the new target and mechanisms for SLW source therapy will provides basic research evidence for treatment of endometriosis.ObjectiveTo observe the effect of SLW on the level of E2 production in eutopic endometrium and the key enzyme and transcription factor participated in malignant positive feedback cycle in E2 production progress. To observe the effect of SLW on the development of rats' ectopic endometrium and the capability of production, adhesion of E2 and anti-apoptosis. Then to further approach on the main machanism of SLW in inhibiting the production of E2 at local focus of endometrium on anti- endometriosis.Methods1. At the cellular level, Eu and En cells were cultured. After Eu and En cells were intervened with different concentration of SLW respectively, the effect of SLW on proliferations of Eu and En cells were detected by WST-8, the expression of P450 arom, COX-2, SF-1, COUP-TF mRNA and protein and 17-beta-hydroxysteroid dehydrogenase 1 and 2 mRNA in En cells and Eu cells treated with SLW were detected by RT-PCR and western blot, the expression of SF-1and COUP-TF protein in En cells and Eu cells treated with SLW were detected by cellular immunofluorescence and the effect of SLW on the level of E2 and PGE2 in cell supernatant were detected by electrochemiluminescence immunoassay and radioimmunoassay respectively.2. Rats during estrus were selected for the surgery of autogeneic graft to establish endometriosis model of rats. Four weeks after model establishment, according to the volume of transplant generation xenograft, the rats were divided into SLW 1.02 mg/kg group,0.34 mg/kg group,0.11 mg/kg group,0.1mg/kg anastrozole group,model control group and normal control gruop. SLW were administered at a dose of 1.02 mg/kg,0.34 mg/kg,0.11 mg/kg by oral gavage every day for 28 days while 0.1mg/kg anastrozole of the same volume was administered by oral gavage as positive control. The volume of ectopic endometrium was examined and the microscopic sections were investigated with routine HE stain. The expression of P450 arom and COX-2 protein in ectopic endometrium were detected by immunohistochemistry and western blot. The effect of SLW on the level of E2 and PGE2 in ectopic endometrium were detected by electrochemiluminescence immunoassay and radioimmunoassay respectively. The expression of ICAM-1 protein and the effect on cell apoptosis in ectopic endometrium of rats were detected by immunohistochemistry and TUNEL method respectively.3. Firstly,The endometrial cells were divided into three groups:Eu control group,En control group,and 17μg/ml SLW group. The adherent capacity of endometrial cells was detected by crystal violet staining method. The activation of MMP-2,9 and their proenzyme was analyzed by using gelatinase zymography. The expression of TIMP-1,2 protein was observed by immunofluorescence method. The content of VEGF in culture supernatant of Eu cells was determined by ELISA. The early and advanced apoptosis rate of endometrial cells was detected by flow cytometry and TUNEL respectively. The best time-points of each testing index of SLW were selected to be the time-points for observation in later experiment. Then, 17μg/ml SLW and E2 at the final concentration (1.34pg/ml～6.36pg/ml) were mixed to combined intervene Eu cells for investigating whether the malignant biological behavior of Eu cells had recovered or not with the additional adverse treatment of E2. From the angle of E2 production, we approach the main machanism of the generation and development of SLW for anti-endometriosis. Meanwhile, we parallel designed aromatase inhibitor anastrozole at the concentration of 0.1μg/ml group and E2 at the concentration of 1.34pg/ml～6.36pg/ml group ,in order to prove that the malignant biological behavior of Eu cells was regulated by its local E2 production from positive and negative two aspects. Results1. The results of WST-8 method showed that the proliferation speed of Eu cells cultured in vitro is significantly higher than that of En cells at each time point (P<0.05), but there was not remarkable effect of SLW on the proliferation capability of Eu cells. The secretion level of E2 in Eu cells of 170μg/ml and 17μg/ml of SLW groups decreased obviously more than that of Eu cells without drug given at the time point 48 h, 72 h and 96h. There was significant difference (P<0.05). The level of E2 secretion in Eu cells of 1.7μg/ml SLW group decreased only at the time point 96 h. There was no significant difference between the level of E2 secretion in Eu cells of 1.7μg/ml SLW group and that of Eu cell control group at the other time points (P>0.05). The results of western blot and RT-PCR showed that compared with that of Eu control group, the expression of P450 arom, COX-2, SF-1 protein and mRNA ,HSD-1 mRNA decreased significantly at the time point 48 h after given 170μg/ml,17μg/ml and 1.7μg/ml dose of SLW,while the expression of COUP-TF protein and mRNA,HSD-2 mRNA increased significantly. There was significant difference (P<0.05).The results of cellular immunofluorescence also showed that compared with IOD of SF-1 positive cells of Eu control cells, that in 170μg/ml ,17μg/ml dose of SLW groups deceased significantly, but IOD of COUP-TF positive cells increased significantly. There was significant difference (P<0.05). The results were coincided with that of semi-quantitative analysis of western blot and RT-PCR. Meanwhile, the results of radioimmunoassay showed that the concentration of PGE2 in cell supernatant of 170μg/ml,17μg/ml and 1.7μg/ml dose of SLW groups was obviously lower than that of Eu cells without drug given group (P<0.05). The results summarized above indicated that through specifically down-regulating the expression of SF-1 and up-regulating the expression of COUP-TF, SLW could inhibit the expression of P450 arom in Eu cells, then decrease the activation of P450 arom on COX-2 to block positive feedback cycle of E2 production at local focus of endometrium in endometriosis. Meanwhile, the results of RT-PCR showed that compared with that of Eu cells without drug given group, the expression of HSD-1 mRNA decreased significantly while the expression of HSD-2 mRNA increased obviously in Eu cells with SLW treatment. There was significant difference (P<0.05).2. After the rats were auto-transplanted with endometrial for 28 days, it was observed that the grafts increased in size, much fluid accumulated to form the transparent saccule appearance, being covered by connective tissue or colic omentum and vascularized in it. There were endothelial cells, glandular organ and mesenchymal in grafts being observed with microscope. 40 rats appeared fluidify in 42 rats, the model achievement ratio was 95.24 percentage. According to the transplant volume, SLW 1.02 mg/kg group,0.34 mg/kg group and 0.11 mg/kg groups have the striking effect compared with model group(p<0.05). It was observed that the allotopic endometrial glandular epithelium became high- or low-column and located at the entocoele cavosurface of Ves., cell-cell junction tightly, oval-shap and large caryon located at basilar part and much kytoplasm was seen in model group. There was more endometrium interstitial substance and individual epithelial lamina emboled to form pseudo-glandular organ. Otherwise endometrial glandular epithelium became thin obviously, cells low-column or applanation appearance and loose after 1.02 mg/kg,0.34 mg/kg,0.11 mg/kg dose of SLW and 0.1mg/kg dose of anastrozole treated.The results of immunohistochemistry and western blot showed that compared with that of the model group, the expression of P450 arom protein in ectopic endometrium tissue of 1.02 mg/kg,0.34 mg/kg and 0.11 mg/kg dose of SLW groups decreased significantly(P<0.05). The expression of COX-2 protein in ectopic endometrium tissue of the model rats decreased significantly in high dose of SLW group(P<0.05). There was some decreasing tendency in 0.34 mg/kg,0.11 mg/kg dose of SLW groups, but there was no statistical significance(P>0.05). Meanwhile, the results of electrochemiluminescence immunoassay and radioimmunoassay method showed that the concentration of E2 and PGE2 in ectopic endometrium tissue of 1.02 mg/kg,0.34 mg/kg,0.11 mg/kg dose of SLW groups was obviously lower than that of Eu cells without drug given group. There was significant difference (P<0.05). The results of immunohistochemistry showed that ICAM-1 positive staining cells of heterotopic tissue in model control group were dense and of large quantity. The staining for HE in these cells was yellow or brown. The positive cells of 1.02 mg/kg,0.34 mg/kg and 0.11 mg/kg dose of SLW groups were of small quantity and light stained. Compared with that of the model control group, the positive degree and area score of the three groups had significant differences (P<0.05). The positive cells of 1.02 mg/kg and 0.34 mg/kg dose of SLW groups were of large quantity and deep stained. Compared with IOD of the positive cells of the model control group, that of the two groups had significant difference (P<0.05). The results of TUNEL method showed that by contrast with that of the model control group, the number of positive cells and the index of apoptosis of 1.02 mg/kg,0.34 mg/kg and 0.11 mg/kg dose of SLW groups increased significantly (P<0.05).3. 17μg/ml dose of SLW and Eu cells were co-incubated. The results of ELISA indicated that the content of VEGF in culture supernatant of Eu cell control group was higher than that of En control cell group significantly after 24 h (P<0.05). The results of crystal violet staining method, gelatinase zymography and cellular immunofluorescence method showed respectively that the percentage of adhesion and the activation of MMP-2,9 and its proenzyme of Eu cells were much higher than that of En control cells while the expression of TIMP-1,2 protein of Eu control cell group was significantly lower than that of En cell group after 48 h (P<0.05). The results of flow cytometry and TUNEL showed respectively that the percentage of early and advanced apoptosis of Eu control cell group was significantly lower than that of En control cell group after 72 h and the index of late apoptosis of Eu cell group was also significantly lower than that of En cell group after 96 h (P<0.05). Meanwhile, 17μg/ml dose of SLW group could decrease the content of VEGF secreted by Eu cells, cell adhesion percentage and the activation of MMP-2,9 and its proenzyme, upregulated the expression of TIMP-1,2 protein and increase early and advanced apoptotic cell number of Eu cells. Compared with that of Eu control cell group, there was significant difference(P<0.05). When 17μg/ml dose of SLW and 1.34pg/ml～6.36pg/ml dose of E2 were given simultaneously, the above malignant biological behavior of Eu cells recovered partly or completely, which had been already improved by SLW. No significant differences were found in the cell adhesion rate of Eu cells, the activation of MMP-2 and pro-MMP-9, the expression of TIMP-1,2 protein and the content of VEGF in culture supernatant of Eu control cells between combination of middle dose of SLW and E2 group and Eu control cell group (P>0.05). 1.34pg/ml～6.36pg/ml dose of E2 group could strengthen the capability of adhesion, invasion, promoting angiogenesis and anti-apoptosis, but the effect of positive drug anastrozole was just contrary to that of E2. Conclusion1. SLW has the inhibitory effect on the production of E2 in Eu cells, which is not dependant on the proliferation of Eu cells, but through inhibiting the expression of P450 arom in Eu cells specifically as the result of inhibiting the expression of SF-1 in Eu cells, promoting the expression of COUP-TF,the effect of P450 arom activation on COX-2 was inhibited and the level of PGE2 secretion of Eu cells decreased, thus, blocked positive feedback cycle of estrogen synthesis at local focus of eutopic endometrium of endometriosis. Additionally, SLW could inhibit the expression of HSD-1 mRNA in Eu cells in vitro and promote the expression of HSD-2 mRNA , which may be one of the machanisms in decreasing E2 production of Eu cells.2. SLW could inhibit the development of rats' endometriosis, reduce the volume of ectopic endometrium, reverse the histopathologic changes of ectopic endometrial tissue and decrease the content of E2 in ectopic endometrial tissue. It is the related machanism that SLW inhibiting P450 arom protein in ectopic endometrial tissue specifically and decreasing the activation effect of P450 arom on COX-2, then inhibiting the production of E2 and PGE2 to block the positive feedback cycle of estrogen production.3. SLW could inhibit the adhesive capability of endometrium in rats and promote apoptosis of ectopic endometrium cells. Its machanism is related to that SLW inhibiting the expression of ICAM-1 in ectopic endometrial tissue of rats and increasing the activity of endogenous endonuclease in ectopic endometrium cells.4. The capability of Eu cells in adhesion to the peritoneum, invasion to peritoneal basement membrane, promoting angiogenesis and anti-apoptosis of itself is stronger than that of En cells. The above characteristics of Eu cells are effected by the concentration of E2 in its local focus. SLW could inhibit adhesion and invasion of Eu cells, promote angiogenesis and Eu cell apoptosis which is related with SLW inhibiting specific adhesion of Eu cells to type-â… collagen, decreasing the activity of MMP-2,9, enhances the expression of TIMP-1,2, reducing VEGF secretion of Eu cells, promoting eversion of phosphatidylserine at Eu cell membrane and activating endogenous endonuclease in cells. Decreasing E2 concentration at local focus is the key mechansism for SLW to treat endometriosis, which is tightly related to the above-mentioned mechanisms... |
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