Estrogen is an important mediator of mast cell activation in ovarian endometriomas

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Endometriosis is an estrogen-dependent disease. Previous research has shown that abnormal enzymes associated with estrogen (E2) metabolism and an increased number of mast cells (MCs) in endometriomas are implicated in the pathogenesis of endometriosis. However, it remains unclear how MCs mediate the role of E2 in endometriosis. Accordingly, we investigated whether E2 was associated with the number of MCs, and the rate of degranulation, in local ovarian endometriomas, as well as the role of E2 on MCs during the pathogenesis of endometriosis. Using enzyme-linked immunosorbent assay and immunohistochemistry, we found that concentrations of E2, and the number and activity of MCs, were significantly higher in ovarian endometriomas than in controls, and that these parameters were correlated with the severity of endometriosis-associated dysmenorrhea. By measuring the release of hexosaminidase, we found that the rate of RBL2H3 cell degranulation increased after E2 treatment. Furthermore, activation of RBL2H3 cells by E2 was found to trigger the release of biologically active nerve growth factor, which promotes neurite outgrowth in PC12 cells and also sensitizes dorsal root ganglion cells via upregulation of Nav1.8 and transient receptor potential cation channel (subfamily V member 1) expression levels. When treated with E2, endometriotic cells could promote RBL2H3 cell recruitment by upregulating expression levels of stem cell factor, transforming growth factor-β and monocyte chemoattractant protein-1; these observations were not evident with control endometrial cells. Thus, elevated E2 concentrations may be a key factor for degranulation and recruitment of MCs in ovarian endometriomas, which play a key role in endometriosis-associated dysmenorrhea.

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  • Supplementary Figure 1 - The aberrant expression of estrogen-metabolizing enzymes in different endometrial tissues. (A) CYP19 (B) 18BHSD2 (C) Sulfate transferase. RQ: relative quantification. Data show mean ± SEM. For all experiments, n = 3. **P < 0.01, ***P < 0.001.

 

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    High concentrations of E2 in ovarian endometriotic lesions are positively correlated with endometriosis-related dysmenorrhea. (A) Correlation between concentrations of E2 in the serum and the degree of dysmenorrhea in patients with endometriosis. (B) Correlation between concentrations of E2 in endometriotic lesions and the degree of dysmenorrhea in patients with endometriosis. Each dot represents data from an individual patient. The Pearson coefficient of correlation and the significance of this correlation are shown in the lower right corner.

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    Immunohistochemistry and statistical analysis of MCs in different endometrial tissues following staining with tryptase (A) and c-kit (B). The graph represents the number of MCs counted in five fields (×20 objective, ×10 ocular) for each patient, with error bars representing s.e.m. Ec, ectopic endometrium; Eu, eutopic endometrium; Nm, normal endometrium. Scale bars = 500 μm. ***P < 0.001.

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    A high number of MCs and the rate of degranulation in ovarian endometriotic lesions were positively correlated with endometriosis-related dysmenorrhea but not the size of ovarian endometriotic cysts. (A) Correlation between the number of MCs and the degree of dysmenorrhea. (B) Correlation between the rate of MC degranulation and the degree of dysmenorrhea. (C) Correlation between the number of MCs and the size of ovarian endometriotic cysts. (D) Correlation between the rate of MC degranulation and the size of ovarian endometriotic cysts. Each dot represents data from an individual patient. The Pearson coefficient of correlation and the significance of this correlation are shown in the lower right corner.

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    Increased rate of RBL2H3 cell degranulation in high E2 concentrations. (A) Rates of degranulation after RBL2H3 cells were stimulated with various concentrations of E2 at 5, 10, 15, 30, 60 and 120 min; the most suitable E2 concentration of RBL2H3 cells degranulation was 500 pmol/L (indicated by the arrow). (B) Rates of degranulation after RBL2H3 cells were stimulated with 500 pmol/L E2 over 120 min; peak degranulation rate occurred at 15 min (indicated by the arrow). For all experiments, n = 3. ***P < 0.001.

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    Activation of RBL2H3 cells by E2 can trigger the release of biologically active NGF. (A) PC12 cell complete culture medium. (B) PC12 cells cultured for 24 h with 500 pmol/L E2. (C) Supernatants from RBL2H3 cells (1 × 106 cells/mL) cultured for 24 h without E2 stimulation. (D) Supernatants from RBL2H3 cells (1 × 106 cells/mL) cultured for 24 h with 500 pmol/L E2. (E) Replicate activated RBL2H3 cell supernatants with 100 ng/mL of anti-NGF antibody. (F) 2 ng/mL NGF. After 24 h, the number of PC12 cells with at least two neurites that were approximately 50 µm or longer was determined on a dark field inverted microscope. Data are shown as mean ± s.e.m. For all experiments, n = 5. Scale bars = 200 μm. ***P < 0.001.

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    Activation of RBL2H3 cells by E2 can promote the peripheral sensitization of DRG cells. (A) Nav1.8 (B) Trpv1. RQ, relative quantification. Data show mean ± s.e.m. For all experiments, n = 3. *P < 0.05, **P < 0.01.

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    Endometriotic cells with E2 treatment, and peritoneal fluid collected from patients with endometriosis, can promote RBL2H3 cell recruitment. (A) (Negative control) Endometrial cell complete culture medium. (B) (Standard control) Supernatants from normal endometrial cells cultured for 24 h without E2 stimulation. (C) Supernatants from normal endometrial cells cultured for 24 h with 10−7 mol/L E2. (D) Supernatants from ectopic endometrial cells cultured for 24 h with 10−7 mol/L E2. (E) Peritoneal fluid from patients without endometriosis. (F) Peritoneal fluid from patients with endometriosis. For all experiments, n = 3. Scale bars = 200 μm. **P < 0.01, ***P < 0.001.

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    Upregulation of the expression levels of SCF (A), TGFB (B) and MCP1 (C) in normal endometrial cells or ovarian endometriotic cells following E2 treatment. RQ, relative quantification. For all experiments, n = 3. *P < 0.05.

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    Schematic diagram of key findings. This study showed that high levels of E2 in ovarian ectopic lesions can promote MC recruitment by up-regulating the expression levels of SCF, TGFB, and MCP1 in ectopic endometrial cells. Additionally, high levels of E2 could directly trigger MC degranulation leading to the release of biologically- active NGF, which can promote nerve growth and the sensitization of nerve fibers by up-regulating the expression of Nav1.8 and Trpv1.

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