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.

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.

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.

Objective. To evaluate the effect of GuiXiong Xiaoyi Wan (GXXYW) on the development of endometriosis in a rat model. Methods. Sprague-Dawley rats with surgically induced endometriosis were randomly treated with low-dose GXXYW, high-dose GXXYW, or vehicle (negative control) for 28 days. Immunohistochemistry was used to assess cell proliferation in the lesions. The terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP biotin nick end labelling (TUNEL) method was performed to analyse the apoptosis induced by GuiXiong Xiaoyi Wan. The percentages of CD3+ lymphocytes, CD4+ lymphocytes, and CD8+ lymphocytes in the spleens of the rats were evaluated using flow cytometric analysis. Results. Treatment with GXXYW significantly decreased the lesion size, inhibited cell proliferation, and induced apoptosis in endometriotic tissue. The spleens of GXXYW-treated rats also demonstrated a significant increase in the percentage of CD4+ lymphocytes and a significant decrease in the percentage of CD8+ lymphocytes. Conclusions. These results suggest that, in a rat model, GXXYW may be effective in the suppression of the growth of endometriosis, possibly through the inhibition of cell proliferation, the induction of apoptosis of endometriotic cells, and the regulation of the immune system.

Interleukin (IL)-6 regulates several cellular processes such as proliferation, differentiation and immune regulation. The pathogenesis of cervical cancer is closely related to abnormal secretion and regulation of cytokines. IL-6 in the pathogenesis of cervical cancer was investigated in the present study. In our work, we collected cervical cancer tissues and adjacent normal tissues first and then established mouse model of cervical cancer for following assays. Immunohistochemistry and Western blot analysis were performed to detect IL-6, JAK3 and STAT6 and qRT-PCR determined VEGF, MMP-2, MMP-9 and others in adjacent tissues and cancer tissues. MTT was used to detect the capability of proliferation, invasion and migration. Chromatin immunoprecipitation was employed to verify the binding of STAT6 to IL-6. The effect of STAT6 inhibitors, AS1517499 on JAK3/STAT6 axis was also detected through in vivo experiments. Except IL-6, the related proteins were upregulated in cervical cancer tissues. IL-6 (25–100 ng/mL) accelerated the growth of Hela cells. IL-6 improved cell migratory and invasive ability when recombinant IL-6 promoted phosphory-lation and JAK3/STAT6. The activity of IL-6 in Hela was mediated by JAK3/STAT6 signaling pathway as AS1517499 inhibited IL-6-induced JAK3/STAT6 phosphorylation and STAT6 bound to the IL-6 promoter. STAT6 inhibitors significantly suppressed tumor growth and reduced p-STAT3 level. IL-6 contributes to cervical cancer through JAK3/STAT6 signaling which could be a cornerstone of subsequent anti-cervical cancer treatment.

STUDY QUESTION Does Scribble (SCRIB) contribute to aberrant decidualization of endometrial stromal cells (ESC) in adenomyosis? SUMMARY ANSWER SCRIB knockdown impairs decidualization of ESC by decreasing Fork-head box O1A (FOXO1) expression through the protein kinase B (AKT) and atypical protein kinase C (aPKC) activated pathways. WHAT IS KNOWN ALREADY Stromal SCRIB is required for primary decidual zone formation and pregnancy success in mice. In our previous studies, decidualization was dampened in ESC isolated from adenomyosis patients, yet the underlying molecular mechanisms remain elusive. STUDY DESIGN, SIZE, DURATION Eutopic endometrium tissue samples from diffuse adenomyosis and non-adenomyosis patients in proliferative, early-secretory and mid-secretory phase (n = 10 per phase for each group) were explored. In parallel, in vitro decidualization studies were carried out in ESC isolated from non-adenomyosis women (n = 8). PARTICIPANTS/MATERIALS, SETTING, METHODS The endometrial SCRIB expression was analyzed using immunohistochemistry staining and western blot. Quantitative RT-PCR (qRT-PCR), western blot and immunofluorescence staining were used to explore the expression of SCRIB in ESC during in vitro decidualization. siRNA-mediated SCRIB knockdown followed by decidual markers expression analysis, flow cytometry for cell cycle analysis and phalloidin staining for morphological analysis were performed to examine the function of SCRIB in ESC decidualization. RNA-sequencing was performed to examine the SCRIB-mediated transcriptional changes in decidualized ESC (DSC). Rescue experiments using an AKT inhibitor MK2206 and aPKC inhibitor NSC37044 were used to investigate the signaling pathways through which could mediate SCRIB-regulated FOXO1 protein expression and ESC decidualization. MAIN RESULTS AND THE ROLE OF CHANCE We found that the expression of SCRIB in the mid-secretory phase eutopic endometrial stroma of adenomyosis patients was significantly lower than that of non-adenomyosis. SCRIB knockdown reduced the expression of decidual markers, abrogated the epithelioid-like morphological changes, inhibited the mesenchymal-to-epithelial transitions process and promoted the cell cycle progression of ESC during in vitro decidualization. SCRIB knockdown-induced decidualization defects were attributed to a decrease in expression of transcription factor FOXO1, known to regulate decidualization. Furthermore, we found that SCRIB knockdown induced the aberrant activation of AKT and aPKC, which led to FOXO1 phosphorylation and degradation. Rescue assay confirmed that restoring the expression of FOXO1 effectively reversed the decidualization defects and cell cycle progression caused by SCRIB knockdown. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION In this study, it was demonstrated that SCRIB knockdown mediated the activation of AKT and aPKC, contributing to FOXO1 degradation and aberrant decidualization, however, the molecular link between AKT and aPKC signaling was not determined, and still requires further exploration. WIDER IMPLICATIONS OF THE FINDINGS Our findings support the hypothesis that adenomyosis interferes with embryo implantation due to insufficient endometrial receptivity. Abnormal decidualization of the endometrial stroma may clarify the possible association between adenomyosis and infertility. Our findings may be clinically useful for counseling and treatment of infertile adenomyosis patients. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the National Natural Science Foundation of China (82001523 and 82171639). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER N/A.

Purpose To test the expression profile of transient receptor potential channels (TRPs) in adenomyosis patients and evaluate the effects of primidone on tamoxifen-induced adenomyosis mice. Methods Eutopic endometrium from adenomyosis patients (n = 20) was collected and subjected to mRNA analysis of TRP channels. TRPA1, TRPV1 and TRPM3 in adenomyosis patients (n = 50) and tamoxifen-induced adenomyosis mice (n = 6) were examined by immunohistochemistry. From 10 weeks after birth, primidone (2 mg/kg/d) and atosiban (1 mg/kg/d) were given separately to adenomyotic mice by intraperitoneal injection for 3 weeks. The hotplate test was conducted once a week beginning at 10 weeks, and then uterine samples were harvested for HE staining and RNA-seq at 13 weeks. Results The mRNA expression of 15 TRPs was significantly increased in the proliferative phase of the adenomyotic endometrium. TRPV1, TRPM3 or TRPA1 staining levels were positively correlated with dysmenorrhea severity, menses amount and uterine size. In tamoxifen-induced adenomyosis mice, primidone had a significant effect on both the depth of myometrial infiltration and analgesia. Forty-seven DEGs were identified after primidone treatment, and bioinformatics analysis predicted that they were enriched in the cell cycle and cell division. Conclusion The expression profile of TRP channels varies significantly in adenomyosis patients, and primidone may provide a potential therapeutic method for adenomyosis management.