孕酮对绵羊输卵管上皮细胞β-防御素2表达的影响
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摘要
研究孕酮(P4)对绵羊输卵管上皮细胞β-防御素2(SBD2)基因表达的影响,探讨P4调节SBD2表达的潜在机制。建立绵羊输卵管上皮细胞体外培养体系,用不同浓度(10~(-6)、10~(-7)、10~(-8)、10~(-9)、10~(-10)mol/L)P4分别处理绵羊输卵管上皮细胞0、2、6、12、24、48 h,筛选P4诱导绵羊输卵管上皮细胞SBD2 mRNA表达的最佳条件。然后使用10~(-6)mol/L孕激素核受体拮抗剂RU486,50μmol/L蛋白激酶C(PKC)信号通路阻断剂H7预处理细胞1 h,再使用P4诱导SBD2 mRNA表达的最佳条件处理细胞,同时设只添加阻断剂(RU486和H7)处理组、只添加孕酮(P4)处理组和空白对照组,通过RT-qPCR技术检测SBD2 mRNA的表达变化。10~(-9)mol/L P4诱导绵羊输卵管上皮细胞6 h和24 h时SBD2 mRNA表达显著高于对照组(P<0.01),且与P4组相比,添加RU486和H7后显著抑制了P4诱导的SBD2 mRNA的表达水平(P<0.01)。P4以浓度和时间依赖性方式显著增加SBD2 mRNA表达,并且诱导可能通过孕酮核受体(PR)介导的基因组途径以及PKC信号通路来提高绵羊输卵管上皮的先天免疫防御能力。
To investigate the effect of progesterone(P4) on β-defensin-2(SBD2) gene expression in ovine oviduct epithelial cells and to explore the underlying mechanism of P4 in regulating SBD2 expression,the ovine oviduct epithelial cells in vitro culture system were established and treated with different concentrations(10~(-6),10~(-7),10~(-8),10~(-9),10~(-10)mol/L)P4 for the indicated time(0,2,6,12,24,48 h) in order to screen the optimal conditions for P4-induced SBD2 mRNA expression in ovine oviduct epithelial cells.Then ovine oviduct epithelial cells were pre-incubated with 10~(-6)mol/L progesterone nuclear receptor antagonist RU486 and 50 μmol/L PKC signal pathway inhibitors H7 for 1 h and subsequently treated with P4 to induce the optimal expression of SBD2 mRNA,and compared to cells that were only pre-treated with RU486 and H7,P4 and control.The expression of SBD2 mRNA was detected by RT-qPCR.The expression of SBD2 mRNA was increased significantly in ovine oviduct epithelial cells induced by 10~(-9)mol/L P4 at 6 h and 24 h compared to untreated control(P<0.01).In addition,the addition of RU486 and H7 significantly inhibited the expression of SBD2 mRNA induced by P4 compare to P4 group(P<0.01).P4 significantly increases the expression of SBD2 mRNA in a concentration and time-dependent manner,and induces the innate immune defenses of ovine oviduct epithelial cells through the progesterone nuclear receptor(PR)-mediated genomic pathway and PKC signaling pathway.
To investigate the effect of progesterone(P4) on β-defensin-2(SBD2) gene expression in ovine oviduct epithelial cells and to explore the underlying mechanism of P4 in regulating SBD2 expression,the ovine oviduct epithelial cells in vitro culture system were established and treated with different concentrations(10~(-6),10~(-7),10~(-8),10~(-9),10~(-10)mol/L)P4 for the indicated time(0,2,6,12,24,48 h) in order to screen the optimal conditions for P4-induced SBD2 mRNA expression in ovine oviduct epithelial cells.Then ovine oviduct epithelial cells were pre-incubated with 10~(-6)mol/L progesterone nuclear receptor antagonist RU486 and 50 μmol/L PKC signal pathway inhibitors H7 for 1 h and subsequently treated with P4 to induce the optimal expression of SBD2 mRNA,and compared to cells that were only pre-treated with RU486 and H7,P4 and control.The expression of SBD2 mRNA was detected by RT-qPCR.The expression of SBD2 mRNA was increased significantly in ovine oviduct epithelial cells induced by 10~(-9)mol/L P4 at 6 h and 24 h compared to untreated control(P<0.01).In addition,the addition of RU486 and H7 significantly inhibited the expression of SBD2 mRNA induced by P4 compare to P4 group(P<0.01).P4 significantly increases the expression of SBD2 mRNA in a concentration and time-dependent manner,and induces the innate immune defenses of ovine oviduct epithelial cells through the progesterone nuclear receptor(PR)-mediated genomic pathway and PKC signaling pathway.
引文
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[3] Soehnlein O.Direct and alternative antimicrobial mechanisms of neutrophil-derived granule proteins [J].J Mol Med,2009,87(12):1157-1164.
[4] Cohen C,Mugo N,Astete S,et al.Detection of Mycoplasma genitalium in women with laparoscopically diagnosed acute salpingitis [J].Sex Transm Infect,2005,81(6):463-466.
[5] Huttner K M,Lambeth M R,Burkin H R,et al.Localization and genomic organization of sheep antimicrobial peptide genes [J].Gene,1998,206(1):85-91.
[6] Teilmann S C,Clement C A,Thorup J,et al.Expression and localization of the progesterone receptor in mouse and human reproductive organs [J].J Endocrinol,2006,191(3):525-535.
[7] Wira C R,Rodriguez-Garcia M,Patel M V.The role of sex hormones in immune protection of the female reproductive tract [J].Nat Rev Immunol,2015,15(4):217-230.
[8] Han J H,Kim M S,Lee M Y,et al.Modulation of human beta-defensin-2 expression by 17beta-estradiol and progesterone in vaginal epithelial cells [J].Cytokine,2010,49(2):209-214.
[9] Ibarra Velarde F,Alcala Canto Y.Downregulation of the goat β-defensin-2 gene by IL-4 in caprine intestinal epithelial cells infected with Eimeria spp[J].Parasitol Res,2007,101(3):613-618.
[10] Kuwahara K,Yoshimura Y,Isobe N.Effect of steroid hormones on the innate immune response induced by Staphylococcus aureus in the goat mammary gland.[J].Reprod Domest Anim,2017,52(4):579-584.
[11] Castro Leyva V,Zaga Clavellina V,Espejel-Nuez A,et al.Decidualization mediated by steroid hormones modulates the innate immunity in response to group B streptococcal infection in vitro[J].Gynecol Obstet Invest,2017,82(6):592-600.
[12] Liu Z,Wong J,Tsai S Y,et al.Sequential recruitment of steroid receptor coactivator-1 (SRC-1) and p300 enhances progesterone receptor-dependent initiation and reinitiation of transcription from chromatin [J].Proc Natl Acad Sci USA,2001,98(22):12426-12431.
[13] Lyons T J,Moussatche P.Non-genomic progesterone signalling and its non-canonical receptor [J].Biochem Soc Trans,2012,40(1):200-204.
[14] Jacobsen B M,Horwitz K B.Progesterone receptors,their isoforms and progesterone regulated transcription [J].Mol Cell Endocrinol,2012,357(1-2):18-29.
[15] Nutu M,Weijdegard B,Thomas P,et al.Distribution and hormonal regulation of membrane progesterone receptors beta and gamma in ciliated epithelial cells of mouse and human fallopian tubes [J].Reprod Biol Endocrinol,2009,7:89.
[16] Tubbs C,Thomas P.Functional characteristics of membrane progestin receptor alpha (mPRalpha) subtypes:a review with new data showing mPRalpha expression in seatrout sperm and its association with sperm motility [J].Steroids,2008,73(9-10):935-941.
[17] Monkkonen K S,Aflatoonian R,Lee K F,et al.Hormonal regulation of Galphai2 and mPRalpha in immortalized human oviductal cell line OE-E6/E7[ J].Mol Hum Reprod,2007,13(12):845-851.
[18] Monkkonen K S,Aflatoonian R,Lee K F,et al.Localization and variable expression of G alpha(i2) in human endometrium and Fallopian tubes [J].Hum Reprod,2007,22(5):1224-1230.
[19] Fujinoki M.Progesterone-enhanced sperm hyperactivation through IP3-PKC and PKA signals [J].Reprod Med Biol,2013,12(1):27-33.
[20] Breton A,Descoteaux A.Protein kinase C-alpha participates in FcgR-mediated phagocytosis in macrophages [J].Biochem Biophys Res Commun,2000,276(2):472-476.