Department of Surgical Sciences, University of Turin, 10126 Turin, Italy.
Department of Experimental Medicine, Sapienza University of Rome, 00185 Rome, Italy.
Molecules. 2017 Nov 17;22(11):1997. doi: 10.3390/molecules22111997.
We evaluated whether physiological and pre-eclamptic (PE) placentae, characterized by exacerbated inflammation, presented alterations in pro-inflammatory High Mobility Group Box 1 (HMGB1) and its Receptor of Advanced Glycation End products (RAGE) expression. Moreover, we investigated, in physiological placental tissue, the ability of Low Molecular Weight Heparin (LMWH) to modify HMGB1 structural conformation thus inhibiting RAGE binding and HMGB1/RAGE axis inflammatory activity. HMGB1, RAGE, IL-6 and TNFα (HMGB1/RAGE targets) mRNA expression were assessed by Real Time PCR. HMGB1, RAGE protein levels were assessed by western blot assay. Physiological term placental explants were treated by 0.5 U LMWH for 24 or 48 h. HMGB1 and RAGE expression and association were evaluated in LMWH explants by RAGE immunoprecipitation followed by HMGB1 immunoblot. HMGB1 spatial localization was evaluated by immuofluorescent staining (IF). HMGB1 expression was increased in PE relative to physiological placentae while RAGE was unvaried. 24 h LMWH treatment significantly up-regulated HMGB1 expression but inhibited HMGB1/RAGE complex formation in physiological explants. RAGE expression decreased in treated relative to untreated explants at 48 h. IF showed HMGB1 localization in both cytoplasm and nucleus of mesenchymal and endothelial cells but not in the trophoblast. IL-6 and TNFα gene expression were significantly increased at 24 h relative to controls, while they were significantly down-regulated in 48 h vs. 24 h LMWH explants. Our data depicted a new molecular mechanism through which LMWH exerts its anti-inflammatory effect on PE placentae, underlying the importance of HMGB1/RAGE axis in PE inflammatory response.
我们评估了生理性和先兆子痫（PE）胎盘是否存在促炎因子高迁移率族蛋白 1（HMGB1）及其晚期糖基化终产物受体（RAGE）表达改变。此外，我们研究了低分子肝素（LMWH）在生理性胎盘组织中改变 HMGB1 结构构象从而抑制 RAGE 结合和 HMGB1/RAGE 轴炎症活性的能力。通过实时 PCR 评估 HMGB1、RAGE、IL-6 和 TNFα（HMGB1/RAGE 靶标）的 mRNA 表达。通过 Western blot 分析评估 HMGB1、RAGE 蛋白水平。用 0.5 U LMWH 处理生理足月胎盘外植体 24 或 48 h。通过 RAGE 免疫沉淀后 HMGB1 免疫印迹评估 LMWH 外植体中 HMGB1 和 RAGE 的表达和关联。通过免疫荧光染色（IF）评估 HMGB1 的空间定位。PE 胎盘组织中 HMGB1 的表达高于生理性胎盘组织，而 RAGE 表达无差异。24 h LMWH 处理显著上调了生理外植体中 HMGB1 的表达，但抑制了 HMGB1/RAGE 复合物的形成。与未处理的外植体相比，处理后的外植体中 RAGE 表达在 48 h 时降低。IF 显示 HMGB1 定位于间质和内皮细胞的细胞质和细胞核中，但不在滋养层中。与对照组相比，24 h 时 IL-6 和 TNFα 基因表达显著增加，而与 24 h LMWH 外植体相比，48 h 时它们显著下调。我们的数据描绘了 LMWH 对 PE 胎盘发挥抗炎作用的新分子机制，表明 HMGB1/RAGE 轴在 PE 炎症反应中的重要性。
