Interleukin-25 induced by human chorionic gonadotropin promotes the proliferation of decidual stromal cells by activation of JNK and AKT signal pathways

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Interleukin-25 induced by human chorionic gonadotropin promotes the proliferation of decidual stromal cells by activation of JNK and AKT signal pathways Ying Wang, B.Sc., Yuan Zhang, B.Sc., Ming-Qing Li, M.D., Ph.D., Deng-Xuan Fan, M.S., Xiao-Hui Wang, B.Sc., Da-Jin Li, M.D., Ph.D., Li-Ping Jin, M.D., Ph.D. Fertility and Sterility Volume 102, Issue 1, Pages 257-263 (July 2014) DOI: 10.1016/j.fertnstert.2014.03.025 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Interleukin-25/IL-17RB is coexpressed by DSCs in human first-trimester pregnancy. (A) The expression of IL-25 and its receptor IL-17RB in human DSCs (n = 6) of normal pregnancy was analyzed by flow cytometry. (B) After culture for 24, 48, 72, and 96 hours, the secretion of IL-25 in the supernatant of DSCs (2 × 105 cell per well) (n = 6) was analyzed by ELISA. Data are mean ± SD. **P<.01 compared with the 24-hour group. Fertility and Sterility 2014 102, 257-263DOI: (10.1016/j.fertnstert.2014.03.025) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Human chorionic gonadotropin (HCG) up-regulates the expression of IL-25 and IL-17RB. (A–D) We treated DSCs (n = 6) with hCG (0.316–2.5 KU/L) for 24 hours in vitro, with the medium as control. Then the expression of IL-25 and IL-17RB was analyzed by flow cytometry. FSC = forward scatter. Data are mean ± SD. *P<.05, **P<.01 compared with control. Fertility and Sterility 2014 102, 257-263DOI: (10.1016/j.fertnstert.2014.03.025) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Interleukin-25 stimulates proliferation of DSCs by activating JNK and AKT signals. Primary DSCs were incubated with rhIL-25 (0–100 ng/mL) (A, B) or anti-IL-25 neutralizing antibody (anti-IL-25) (0–1 μg/mL) (C, D) for 24 or 48 hours, and then the proliferation of DSCs was analyzed by BrdU proliferation assay. (E) The cell number count assay was performed to analyze numbers of the seeding cells and final cells of DSCs in 12 microplates after treatment with rhIL-25 and/or anti-IL-25. (G) After stimulation with rhIL-25 for 5, 15, 30, or 60 minutes, the phosphorylation level of AKT, ERK1/2, JNK, and NF-κB in DSCs (n = 6) was detected by Western blot assay. (F) DSCs (n = 6) were treated with rhIL-25 (1 ng/mL), rhIL-25 plus different signal inhibitors (mitogen-activated protein kinase/ERK1/2 inhibitor, U0126, 10 μM; JNK inhibitor, SP600125, 10 μM; NF-κB inhibitor, BAY, 2.5 μM; AKT inhibitor, LY294002, 10 μM) for 24 hours. In addition, vehicle was added as negative control. Then the ability of DSCs to proliferate was detected by BrdU cell proliferation assay. IL-25 = rhIL-25; anti-IL-25 = treatment with anti-human IL-25 neutralizing antibody; SP = SP600125; LY = LY294002. Data are mean ± SD. *P<.05, **P<.01 compared with vehicle control. Fertility and Sterility 2014 102, 257-263DOI: (10.1016/j.fertnstert.2014.03.025) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 4 The expression of IL-25/IL-17RB decreased in DSCs from abortion. (A–C) Western blot assay was used to evaluate the expression of IL-25 and IL-17RB in DSCs from normal pregnant women (n = 6) and women with abortion (n = 6). Normal DSC = DSCs from normal pregnancy; abortion DSC = DSCs from abortion. Data are mean ± SD.*P<.05 compared with the normal DSC group. Fertility and Sterility 2014 102, 257-263DOI: (10.1016/j.fertnstert.2014.03.025) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions