甾体衍生物02F04通过非核受体上调PAM212细胞胸腺基质淋巴细胞生成素的研究
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摘要
目的:探索02F04诱导TSLP表达是否通过核受体介导。方法:一系列浓度的6种核受体,包括视黄酸受体(RAR)、维甲酸X受体(RXR)、过氧化物酶体增生物激活受体(PPAR)、维生素D受体(VDR)、糖皮质激素受体(GR)和肝X受体(LXR)的激动剂和抑制剂,单用或者与10μmol/L 02F04联合刺激PAM212细胞24 h后,ELISA法测定培养上清中TSLP的蛋白水平,MTT法检测PAM212细胞的存活率。10μmol/L 02F04刺激PAM212细胞4、8、12或24 h,实时荧光定量PCR反应测定细胞中相应核受体靶向基因的表达。结果:VDR激动剂不能增加PAM212细胞中TSLP的表达。02F04不能诱导RAR、RXR和PPAR靶向基因的表达,且RARα、PPARγ和PPARδ的激动剂及抑制剂单用或与02F04合用后均不影响TSLP的产生; RARγ、RXR和GR激动剂可抑制细胞基本水平及02F04诱导水平的TSLP产生,而抑制剂未呈现出相反的结果。02F04可激活LXR并增加靶向基因ABCA1的表达,但LXR激动剂不能诱导TSLP产生,LXR抑制剂也不能抑制02F04诱导的TSLP表达。结论:02F04具有甾体结构,但诱导TSLP的产生不能通过核受体作用而介导。
Objective: To investigate that whether 02 F04-induced TSLP production was regulated via nuclear receptors.Methods: PAM212 cells were stimulated with agonists or antagonists of six nuclear receptors,including retinoic acid receptor( RAR),retinoid X receptor( RXR),peroxisome proliferator-activated receptor( PPAR),vitamin D receptor( VDR),glucocorticoids receptor( GR) and liver X receptor( LXR) at a series of concentrations for 24 h in the presence or absence of 10 μmol/L 02 F04. TSLP levels in culture supernatant and cell viability were determined by ELISA and MTT assay,respectively. Additionally,PAM212 cells were stimulated with 02 F04 at 10 μmol/L for 4,8,12 or 24 h,and mRNA expression of target gene of these nuclear receptors was determined by qRT-PCR. Results: TSLP production in PAM212 cells was not induced by VDR agonist. 02 F04 did not significantly change mRNA levels of target genes of RAR,RXR and PPAR. Both the agonists and antagonists of RARα,PPARγ and PPARδ had no effect on basal TSLP production and that induced by 02 F04. Though agonists of RARγ,RXR and GR inhibited basal TSLP production and that induced by 02 F04,antagonists of these nuclear receptors did not exhibit contrary effects on TSLP expression. Besides,02 F04 activated LXR to induce an increase of ABCA1 expression,but LXR agonist failed to induce TSLP production and LXR antagonist did not attenuate02 F04-induced TSLP production. Conclusion: 02 F04-induced TSLP production is not mediated by nuclear receptors although it has a steroidal structure.
Objective: To investigate that whether 02 F04-induced TSLP production was regulated via nuclear receptors.Methods: PAM212 cells were stimulated with agonists or antagonists of six nuclear receptors,including retinoic acid receptor( RAR),retinoid X receptor( RXR),peroxisome proliferator-activated receptor( PPAR),vitamin D receptor( VDR),glucocorticoids receptor( GR) and liver X receptor( LXR) at a series of concentrations for 24 h in the presence or absence of 10 μmol/L 02 F04. TSLP levels in culture supernatant and cell viability were determined by ELISA and MTT assay,respectively. Additionally,PAM212 cells were stimulated with 02 F04 at 10 μmol/L for 4,8,12 or 24 h,and mRNA expression of target gene of these nuclear receptors was determined by qRT-PCR. Results: TSLP production in PAM212 cells was not induced by VDR agonist. 02 F04 did not significantly change mRNA levels of target genes of RAR,RXR and PPAR. Both the agonists and antagonists of RARα,PPARγ and PPARδ had no effect on basal TSLP production and that induced by 02 F04. Though agonists of RARγ,RXR and GR inhibited basal TSLP production and that induced by 02 F04,antagonists of these nuclear receptors did not exhibit contrary effects on TSLP expression. Besides,02 F04 activated LXR to induce an increase of ABCA1 expression,but LXR agonist failed to induce TSLP production and LXR antagonist did not attenuate02 F04-induced TSLP production. Conclusion: 02 F04-induced TSLP production is not mediated by nuclear receptors although it has a steroidal structure.
引文
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[15] Weng Y,Mizuno N,Dong J,et al. Induction of thymic stromal lymphopoietin by a steroid alkaloid derivative in mouse keratinocytes[J]. Int Immunopharmacol,2017,55:28-37.
[16] Moon PD,Han NR,Ryu KJ,et al. A novel compound 2-(4-{2-[(phenylthio)acetyl]carbonohydrazonoyl}phenoxy)acetamide downregulates TSLP through blocking of caspase-1/NF-κB pathways[J]. Int Immunopharmacol,2016,38:420-425.
[17] Takai T. TSLP expression:cellular sources,triggers,and regulatory mechanisms[J]. Allergol Int,2012,61(1):3-17.
[18] Mizuno N,Abe K,Morishita Y,et al. Pentanoic acid induces thymic stromal lymphopoietin production through Gq/11 and Rhoassociated protein kinase signaling pathway in keratinocytes[J].Int Immunopharmacol,2017,50:216-223.
[2] Zhou B,Comeau MR,De Smedt T,et al. Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in mice[J]. Nat Immunol,2005,6(10):1047-1053.
[3] Sims JE,Williams DE,Morrissey PJ,et al. Molecular cloning and biological characterization of a novel murine lymphoid growth factor[J]. J Exp Med,2000,192(5):671-680.
[4] Liu YJ. Thymic stromal lymphopoietin and OX40 ligand Pathway in the initiation of dendritic cell-mediated allergic inflammation[J]. J Allergy Clin Immunol,2007,120(2):238-244.
[5] Ziegler SF,Roan F,Bell BD,et al. The biology of thymic stromal lymphopoietin(TSLP)[J]. Adv Pharmacol,2013,66:129-155.
[6] Ziegler SF. The role of thymic stromal lymphopoietin(TSLP)in allergic disorders[J]. Curr Opin Immunol,2010,22(6):795-799.
[7]孟平,陈壮桂,张天托,等. TSLP促进肌成纤维细胞向哮喘小鼠气道募集[J].中国免疫学杂志,2016,32(6):777-781,786.Meng P,Chen ZG,Zhang TT,et al. TSLP promotes recruitment of myofibroblasts into airway in asthmatic mice[J]. Chin J Immunol,2016,32(6):777-781,786.
[8] Satou N,Ishihara K,Hiratsuka M,et al. Induction of thymic stromal lymphopoietin production by xylene and exacerbation of picryl chloride-induced allergic inflammation in mice[J]. Int Arch Allergy Immunol,2012,157(2):194-201.
[9] Yamashita S,Segawa R,Satou N,et al. Induction of thymic stromal lymphopoietin production by nonanoic acid and exacerbation of allergic inflammation in mice[J]. Allergol Int,2013,62(4):463-471.
[10] Mihály J,Gericke J,Lucas R,et al. TSLP expression in the skin is mediated via RARγ-RXR pathways[J]. Immunobiology,2016,221(2):161-165.
[11] Lee HC,Headley MB,Iseki M,et al. Cutting edge:Inhibition of NF-κB-mediated TSLP expression by retinoid X receptor[J]. J Immunol,2008,181(8):5189-5193.
[12] Barroso E,Eyre E,Palomer X,et al. The peroxisome proliferatoractivated receptorβ/δ(PPARβ/δ)agonist GW501516 prevents TNF-α-induced NF-κB activation in human Ha CaT cells by reducing p65 acetylation through AMPK and SIRT1[J]. Biochem Pharmacol,2011,81(4):534-543.
[13] Li M,Hener P,Zhang Z,et al. Induction of thymic stromal lymphopoietin expression in keratinocytes is necessary for generating an atopic dermatitis upon application of the active vitamin D3analogue MC903 on mouse skin[J]. J Invest Dermatol,2009,129(2):498-502.
[14] Le TA,Takai T,Vu AT,et al. Glucocorticoids inhibit doublestranded RNA-induced thymic stromal lymphopoietin release from keratinocytes in atopic cytokine milieu more effectively than tacrolimus[J]. Int Arch Allergy Immunol,2010,153(1):27-34.
[15] Weng Y,Mizuno N,Dong J,et al. Induction of thymic stromal lymphopoietin by a steroid alkaloid derivative in mouse keratinocytes[J]. Int Immunopharmacol,2017,55:28-37.
[16] Moon PD,Han NR,Ryu KJ,et al. A novel compound 2-(4-{2-[(phenylthio)acetyl]carbonohydrazonoyl}phenoxy)acetamide downregulates TSLP through blocking of caspase-1/NF-κB pathways[J]. Int Immunopharmacol,2016,38:420-425.
[17] Takai T. TSLP expression:cellular sources,triggers,and regulatory mechanisms[J]. Allergol Int,2012,61(1):3-17.
[18] Mizuno N,Abe K,Morishita Y,et al. Pentanoic acid induces thymic stromal lymphopoietin production through Gq/11 and Rhoassociated protein kinase signaling pathway in keratinocytes[J].Int Immunopharmacol,2017,50:216-223.