肝X受体激动剂上调人肾小球内皮细胞血栓调节蛋白表达的机制和作用

Mechanism and role of LXR agonist upregulating thrombo-modulin expression in human glomerular endothelial cells

摘要:

目的  探讨肝X受体（LXR）激动剂T0901317上调人肾小球内皮细胞（HRGEC）血栓调节蛋白（TM）表达的机制和作用。方法  Western blot检测25 mmol高糖和2μmol T0901317刺激后的HRGEC上IκBα、磷酸化IκBα、核转录因子-κB（NF-κB）p65、磷酸化NF-κB p65表达；免疫共沉淀法检测LXR与P300之间有无结合；重组腺病毒AdTMshRNA转染HRGEC，观察其对高糖下HRGEC分泌炎症介质的影响。结果 T0901317能显著降低高糖刺激后的IκBα和NF-κB p65磷酸化（P <0.05），LXR-α沉默后NF-κB活性增强；2μmol T0901317刺激HRGEC 24 h后以Co-IP检测LXR与P300的表达上调；T0901317明显抑制高糖刺激下的HRGEC培养上清中TNF-α、IL-1β浓度（P <0.05），AdTMshRNA转染HRGEC后，有或无T0901317刺激，HRGEC培养上清中TNF-α、IL-1β浓度与高糖组比较，差异无统计学意义（P >0.05）。结论  LXR可能通过与P300发生相互作用阻断了NF-κB与P300之间的竞争性结合而提高TM的表达并抑制炎症介质的分泌。

Abstract:

Objective To explore the effect of liver X receptor (LXR) agonist T0901317 on thrombomodulin (TM) expression in human glomerular endothelial cells (HUGECs) and its mechanism. Methods Western blot was used to detect the expressions of IκBα, p-IκBα, nuclear transcription factor-κB (NF-κβ) p65 and p-NF-κB p65 in HUGECs stimulated by 25 mmol high glucose and 2 μmol T0901317. The interaction between LXR and the transcriptional coactivator p300 in HUGECs was detected using co-immunoprecipitation (Co-IP) assay. The concentrations of IL-1β and TNF-α in the supernatant of HUGECs transfected with or without AdTMshRNA were determined using commercially available ELISA kits. Results T0901317 (2 μmol) significantly reduced the phosphorylation of IκBα and NF-κB p65 in the HUGECs stimulated by high glucose (P < 0.05). The activity of NF-κB was increased by LXR-α silencing despite the presence of T0901317. Co-IP revealed up-regulation of LXP and p300 in the HUGECs 24 h after stimulation by 2 μmol T0901317. T0901317 inhibited the secretion of high glucose-induced inflammatory cytokines such as TNF-α and IL-1β in the HUGECs (P < 0.05). The levels of TNF-α and IL-1β were increased by TM silencing with AdTMshRNA despite the presence of T0901317, but the differences were not significant (P > 0.05). Conclusions LXR agonist increases TM expression and inhibits secretion of inflammatory mediators probably by competitively blocking the interaction between NF-κB and p300 through interaction with p300.