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

Study of a steroid alkaloid derivative 02F04 on upregulation of thymic stromal lymphopoietin in PAM212 cells via non-nuclear receptors

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10 μmol/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。10 μmol/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 02F04-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 02F04.TSLP levels in culture supernatant and cell viability were determined by ELISA and MTT assay,respectively.Additionally,PAM212 cells were stimulated with 02F04 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.02F04 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 02F04.Though agonists of RARγ,RXR and GR inhibited basal TSLP production and that induced by 02F04,antagonists of these nuclear receptors did not exhibit contrary effects on TSLP expression.Besides,02F04 activated LXR to induce an increase of ABCA1 expression,but LXR agonist failed to induce TSLP production and LXR antagonist did not attenuate 02F04-induced TSLP production. Conclusion: 02F04-induced TSLP production is not mediated by nuclear receptors although it has a steroidal structure.