摘要
背景
子宫内膜异位症(以下简称内异症)是生育年龄妇女的常见良性疾病。它可浸润病灶周围的正常组织,治疗后易于复发,具有一定的“恶性”行为。近年来发病率有逐年升高趋势,成为妇科疾病中的热点问题。尽管EMS的发病机制仍未阐清,但在经血逆流“种植学说”的基础上,越来越多的资料表明,EMS患者的在位内膜存在与异位内膜相似而与正常内膜迥异的遗传和功能特性。因此我们推测,在位内膜是EMS病变的源头,而直接干预在位内膜可达到防治EMS的效果。
内异症是一种雌激素依赖性疾病,雌激素的合成代谢在内异症疾病的发生、发展中占有重要的地位。既往研究提示,加味三棱丸有效成分(以下简称SLW)具有良好的抗子宫内膜异位症作用。因此,以“EMS是雌激素依赖性疾病”为切入点,探讨SLW源头治疗内异症的作用靶点及机制,可为其早日成为新的内异症治疗药物提供基础研究依据。
目的
观察SLW对内异症在位内膜雌二醇生成水平的影响,及其对参与雌二醇生成过程中恶性正反馈循环的关键酶及转录因子的影响;观察SLW对大鼠异位子宫内膜的发展、雌二醇生成、粘附及抗凋亡能力的影响,并进一步探讨SLW抑制内异症内膜细胞中雌二醇的生成在其抗子宫内膜异位症中的关键作用机制。
方法
1.培养内异症和非内异位症在位内膜(Eu and En)细胞,分为非内异症内膜细胞组、内异症内膜细胞组、SLW 170μg/ml组、17μg/ml组、1.7μg/ml组及0.1mg/kg阿那曲唑组。采用WST-8法检测SLW对Eu和En细胞增殖情况的影响,RT-PCR法和western blot检测En细胞及SLW处理后的Eu细胞中细胞色素P450芳香化酶(P450 arom)、环氧合酶-2(COX-2),类固醇类生成因子-1(SF-1)、小鸡卵清蛋白上游启动子转录因子(COUP-TF)mRNA及其蛋白的表达,17-β-羟甾类脱氢酶1,2(17-β-HSD 1,2)mRNA的表达情况;细胞免疫荧光法观察En细胞及SLW处理后的Eu细胞中SF-1和COUP-TF蛋白的表达,电化学发光免疫法和放射免疫法分别检测SLW对Eu细胞培养液上清中雌二醇(E2)和前列腺素E2(PGE2)水平的影响。
2.选择动情期大鼠行子宫内膜自体移植术,建立大鼠子宫内膜异位症疾病模型,取移植物积液高度≥2mm,病理学检查有子宫内膜组织生长的成功模型动物,分为SLW 1.02 mg/kg组、0.34 mg/kg组和0.11 mg/kg组、0.1mg/kg阿那曲唑组、模型对照组,同时设立正常对照组。用药前经统计学检验,除正常对照组外,各组异位内膜体积间无显著性差异。灌胃给药,每天1次,连续28天。检测异位内膜体积,常规HE染色,免疫组织化学法和western blot检测异位组织中P450 arom和COX-2蛋白的表达;电化学发光免疫法和放射免疫法分别观察SLW对异位内膜组织中E2和PGE2水平的影响;免疫组织化学法和TUNEL法分别检测SLW对大鼠异位子宫内膜中细胞间粘附分子(ICAM-1)蛋白表达及细胞凋亡的影响。
3.首先将内膜细胞分为非内异症内膜细胞对照组、内异症内膜细胞对照组和SLW 17μg/ml组,采用结晶紫染色法检测内膜细胞的粘附能力,明胶酶谱法分析内膜细胞分泌基质金属蛋白酶2,9(MMP-2,9)及其酶原的活性,细胞免疫荧光法观察组织型金属蛋白酶抑制剂1,2(TIMP-1,2)蛋白的表达,ELISA法检测内膜细胞培养上清中血管内皮生长因子(VEGF)的含量,流式细胞术和TUNEL法分别检测内膜细胞早期和晚期凋亡率。同时选取SLW的各测定指标最佳作用时间点,作为后续实验的检测时间点。随后,将SLW 17μg/ml与终浓度为1.34pg/ml～6.36pg/ml的雌二醇进行联合干预,并同时设计0.1μg/ml阿那曲唑组和终浓度为1.34pg/ml～6.36pg/ml的雌二醇组作为正反对照,在各检测时间点上按照上述方法对各项指标进行检测。
结果
1. WST-8法结果提示,体外培养的Eu细胞在各时相点的增殖速度,明显高于En细胞(P<0.05),但SLW对Eu细胞的增殖能力没有显著的影响;SLW 170μg/ml和17μg/ml组在作用48h、72h和96h时,分泌雌二醇的水平较Eu细胞对照组明显降低,差异显著(P<0.05)。而SLW 1.7μg/ml组仅在96h时显著降低内异症在位内膜细胞分泌雌二醇水平,其余各时间点与Eu细胞对照组比较,差异无显著性(P>0.05)。western blot和RT-PCR结果显示,给予SLW 170μg/ml、17μg/ml和1.7μg/ml 48h后的Eu细胞,P450 arom、COX-2、SF-1蛋白和mRNA表达较Eu细胞对照组明显降低,而COUP-TF蛋白和mRNA表达显著升高(P<0.05);细胞免疫荧光结果也显示,SLW 170μg/ml、17μg/ml组SF-1阳性细胞累积光密度值与Eu细胞对照组比较显著降低,而COUP-TF阳性细胞累积光密度值显著升高(P<0.05),与western blot和RT-PCR半定量分析结果一致。同时,放射免疫法结果显示,SLW 170μg/ml、17μg/ml和1.7μg/ml组细胞上清中PGE2的浓度,明显低于Eu细胞对照组(P<0.05)。RT-PCR结果同时显示,经SLW处理后, Eu细胞中HSD-1 mRNA表达显著减少,而HSD-2 mRNA表达明显升高,与Eu细胞对照组比较差异显著(P<0.05)。
2.造模28天后,腹壁移植物体积增大,出现液体积聚,呈隆起透明的小囊状,被结缔组织或大网膜覆盖并有血管形成。以抑制物积液高度≥2mm,病理学检查有子宫内膜组织生长为模型成功标准,42只大鼠中40只符合上述标准,造模成功率为95.24%。SLW 1.02 mg/kg组、0.34 mg/kg组和0.11 mg/kg组的移植物体积与模型对照组相比明显减小,差异显著(P<0.05)。HE染色光镜下观察模型对照组异位内膜腺上皮细胞呈高柱状或矮柱状,排列在囊泡内腔面,细胞间连接较紧密,细胞核大,呈椭圆形,位于基底部,胞浆丰富,内膜间质丰富,其中有个别部位上皮层内陷形成假腺体;SLW 1.02 mg/kg组、0.34 mg/kg组和0.11 mg/kg组和0.1mg/kg阿那曲唑组的内膜腺上皮层明显变薄,细胞呈矮柱状甚至扁平状,松散排列。
免疫组织化学和western blot结果显示,SLW 1.02 mg/kg组、0.34 mg/kg组和0.11 mg/kg组异位内膜组织中P450 arom蛋白的表达值与模型对照组比较,显著降低(P<0.05);SLW 1.02 mg/kg组可以显著降低模型大鼠异位内膜组织中COX-2蛋白的表达(P<0.05),SLW 0.34 mg/kg组和0.11 mg/kg组虽有一定的降低趋势,但无统计学差异(P>0.05)。同时,电化学发光免疫法和放射免疫法结果也显示,SLW 1.02 mg/kg组、0.34 mg/kg组和0.11 mg/kg组异位内膜组织中E2和PGE2的浓度,也明显低于模型对照组(P<0.05)。
免疫组织化学结果显示,模型对照组异位组织ICAM-1阳性染色细胞数量多而密集,染色呈淡黄色或棕黄色;SLW 1.02 mg/kg组、0.34 mg/kg组和0.11 mg/kg剂量组阳性细胞数量少,浅染,阳性程度和面积评分与模型对照组比较有显著性差异(P<0.05)。SLW 1.02 mg/kg组、0.34 mg/kg组的阳性细胞累积光密度值也显著低于模型对照组(P<0.05)。TUNEL法结果显示,SLW 1.02 mg/kg组、0.34 mg/kg组和0.11 mg/kg组内阳性细胞数显著增加,细胞凋亡指数增加,与模型对照组比较,差异显著(P<0.05)。
3.将SLW 17μg/ml与Eu细胞共同孵育24h后,ELISA法检测结果显示,Eu细胞培养液上清中VEGF的含量显著高于En细胞组(P<0.05);结晶紫染色法、明胶酶谱法和细胞免疫荧光结果分别显示,48h后Eu细胞的粘附百分数,MMP-2,9及其酶原的活性显著高于En细胞,而TIMP-1,2蛋白的表达明显低于En细胞组(P<0.05);流式细胞术和TUNEL法结果分别显示,72h后的Eu细胞早期凋亡百分数显著低于En细胞组,96h后Eu细胞晚期凋亡指数也显著低于En细胞组(P<0.05);同时,SLW 17μg/ml可同时降低Eu细胞分泌的VEGF含量、细胞粘附百分数、MMP-2,9及其酶原的活性,上调TIMP-1,2蛋白的表达,增加Eu细胞早期和晚期凋亡数目,与Eu细胞对照组比较,差异显著(P<0.05)。当SLW 17μg/ml与雌二醇1.34pg/ml～6.36pg/ml联合给予时,Eu细胞被SLW改善的恶性生物学行为被反向添加的雌二醇部分或完全恢复。其中,Eu细胞的粘附百分率、MMP-2和pro-MMP-9活力、TIMP-1,2蛋白的表达以及Eu细胞培养液上清中VEGF的含量与与Eu细胞对照组比较,已无显著性差异(P>0.05)。单加雌二醇可进一步加强Eu细胞的粘附、侵袭、促血管生成及抗凋亡能力,而特异性的芳香化酶抑制剂阿那曲唑0.01μg/ml的作用恰与雌二醇相反。
结论
1. SLW体外可抑制内异症在位内膜细胞雌二醇的生成,这种抑制作用非依赖于抑制Eu细胞的增殖,而与抑制Eu细胞SF-1表达,促进COUP-TF表达,特异性地抑制Eu细胞中P450 arom的表达,进而降低P450 arom对COX-2的激活作用,并降低Eu细胞分泌PGE2的水平,从而阻断内异症内膜局部雌激素合成的正反馈机制密切有关。此外,SLW体外抑制Eu细胞HSD-1 mRNA表达、促进HSD-2 mRNA表达的作用可能也是其降低Eu细胞雌二醇生成机制之一。
2. SLW可抑制大鼠子宫内膜异位症的发展,缩小异位内膜体积,改善异位内膜组织病理学变化,降低异位内膜组织中雌二醇含量,其机制与SLW特异性地抑制异位内膜组织中P450 arom蛋白,降低P450 arom对COX-2的激活作用,进而抑制E2和PGE2的生成,阻断雌激素生成的正反馈环有关。
3. SLW可抑制大鼠异位子宫内膜细胞的粘附能力,促进异位内膜细胞凋亡,其机制与SLW抑制大鼠异位内膜组织ICAM-1表达,激活异位内膜细胞中内源性核酸内切酶有关。
4. Eu细胞在体外与En细胞比较,粘附腹膜、侵袭腹膜基底膜、促血管生成及抗自身凋亡的能力更强,Eu细胞的上述特质受其局部雌二醇浓度的影响。SLW可抑制Eu细胞粘附、侵袭、促血管生成,促进Eu细胞凋亡,其机制与SLW抑制Eu细胞与Ⅰ型胶原的特异性粘附,降低MMP-2,9活力,提高TIMP-1,2表达,降低Eu细胞VEGF的分泌,促进其胞膜内层磷脂酰丝氨酸外翻,激活细胞中内源性核酸内切酶有关。降低Eu细胞局部雌二醇合成是SLW治疗内异症的关键,它与上述作用机制密切相关。
Background
Endometriosis,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.
Objective
To 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.
Methods
1. 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.
Results
1. 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.
Conclusion
1. 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-Ⅰcollagen, 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
引文
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