The aim of the present study was to evaluate the effects of baicalein on human endometrial stromal cells in vitro. Ectopic endometrium samples were obtained from 6 female patients with ovarian endometriosis who underwent laparoscopic surgical procedures from July to September 2015. After culturing the cells, immunocytochemistry was performed to verify the purity and homogeneity of the endometrial stromal cells, and a Cell Counting Kit-8 assay was used to evaluate cell viability. In addition, cell cycle progression was analyzed using flow cytometry, and the effects of baicalein on the expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), proliferating cell nuclear antigen (PCNA) and cyclin D1 in endometrial stromal cells were evaluated using western blot analysis. The related signaling pathways were also investigated by incubating cells with inhibitors of signaling pathways, prior to adding 40 µM baicalein for 48 h, followed by analysis of cell viability using a Cell Counting Kit-8 assay. The results indicated that treatment with baicalein significantly induced a dose-dependent decrease (P<0.05) in the viability of human endometrial stromal cells, which was abolished by inhibition of the nuclear factor (NF)-κB signaling pathway. However, baicalein treatment did not induce a time-dependent decrease in viability, as cell viabilities between the 24, 48 and 72 h treatment groups did not differ significantly. The number of cells in the G0/G1 phase significantly increased following treatment with baicalein (P<0.05), while the number of cells in the S and G2/M phases significantly decreased (P<0.05). Baicalein-treated cells also exhibited significantly reduced expression of Bcl-2, PCNA and cyclin D1 compared with control cells (P<0.05). These results suggested that baicalein may suppress the viability of human endometrial stromal cells through the NF-κB signaling pathway in vitro, and may induce apoptosis and promote cell cycle arrest at the G0/G1 phase. Thus, baicalein may provide a novel treatment option for endometriosis.
Despite its prevalence and the severity of symptoms, little is known about the pathogenesis and etiology of adenomyosis. In previous studies, the protein expression level of the polarity protein Scribble in the eutopic endometrium of patients with adenomyosis was found to be significantly decreased; however, little is known about its regulatory mechanism. In consideration of the important role of supraphysiologic estrogen production in the endometrium in the development of adenomyosis, we analyzed the effect and mechanism of estrogen on the expression of Scribble in vivo and in vitro. Firstly, we found Scribble was down-regulated in eutopic endometrium and negatively related with aromatase P450 in Tamoxifen-induced adenomyosis. Then, we established a 3D culture of primary endometrial epithelial cells and used found that estrogen could disrupt apical-basal polarity of endometrial glandular epithelial cells. Based on the following experiments and GEO datasets screening, we found estrogen regulates the expression level of Scribble by HECW1 through ubiquitination of Scribble protein. At last, we verified the expression of Scribble, HECW1 and aromatase P450 in eutopic endometrium of human and mouse specimens and found the expression of HECW1 and aromatase P450 was significantly increased, while the expression of Scribble was significantly downregulated. Furthermore, a positive correlation was found between HECW1 and aromatase P450, while a negative correlation was found between HECW1 and Scribble in human clinical tissue specimens. Therefore, our research may provide a new understanding of the pathogenesis of adenomyosis.
The aim of the present study was to evaluate the effects of the selective cyclooxygenase (COX)-2 inhibitor celecoxib on the development of uterine adenomyosis in mice. ICR neonatal mice were first exposed to tamoxifen to establish a mouse model of adenomyosis. Following 60 days of celecoxib treatment, pathological formation of adenomyosis lesions and the depth of myometrial infiltration were evaluated using hematoxylin and eosin staining. To examine thermal pain modulation in mice, a hotplate test was conducted every 15 days from postnatal day 30 onwards. Immunohistochemistry was performed to assess the expression of aromatase P450, N-cadherin, E-cadherin, COX-2 and cluster of differentiation 31, whereas the levels of estrogen were analyzed in uterine tissue homogenates using ELISA. Masson trichrome staining was performed to assess the extent of fibrosis in the uterus. Celecoxib treatment significantly inhibited the depth of infiltration into the myometrium, resulting in significantly reduced disease severity. Treatment with high doses of celecoxib significantly prolonged thermal response latency. Following celecoxib treatment, the expression of E-cadherin was significantly increased whereas the expression of N-cadherin was significantly decreased. Concomitantly, the extent of fibrosis was also reduced following celecoxib treatment. Uterine tissue homogenates isolated from mice treated with both high and low doses of celecoxib exhibited lower concentrations of estrogen and decreased expression of aromatase P450. These observations suggest that celecoxib reduces adenomyosis severity by suppressing estrogen production in the uterus, reversing epithelial-mesenchymal transition and relieving fibrosis. Taken together, the results of the present study support the potential use of celecoxib, a selective COX-2 inhibitor, for the treatment of adenomyosis.
Differentiation of endometrial stromal cells (ESCs) into secretory decidualized cells (dESCs) is essential for embryo implantation. Adenomyosis is a common benign gynecological disease that causes infertility. However, whether adenomyosis affects decidualization of human endometrial stromal cells is elusive. Primary eutopic ESCs were obtained from patients with adenomyosis (n = 9) and women with non-endometrial diseases (n = 12). We determined the capacity of decidualization of human ESCs by qRT-PCR, Edu proliferation assay, cytokine array and ELISA assay. We found that the expression of decidualization markers (IGFBP1 and PRL) in ESCs of adenomyosis was reduced, concomitant with increased cell proliferation. Differential secretion of cytokines in dESCs, including CXCL1/2/3, IL-6, IL-8, MCP-1, VEGF-A, MIP-3α, OPN, SDF-1α, HGF and MMP-9, were observed between adenomyosis and non-adenomyosis. Moreover, the expression of decidualization regulators (HOXA10 at both mRNA and protein levels, FOXO1, KLF5, CEBPB and HAND2 at mRNA levels) in the eutopic endometrium of adenomyosis were lower than that of non-adenomyosis. We propose that ESCs from adenomyosis have defected ability to full decidualization, which may lead to a non-receptive endometrium.
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