Department of Cardiology, the First Hospital of Changsha, Changsha, 410008, China.
Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, 410008, China.
Curr Med Sci. 2020 Aug;40(4):646-653. doi: 10.1007/s11596-020-2229-6. Epub 2020 Aug 29.
In this study, we investigated the effects of nucleolin on lipopolysaccharide (LPS)-induced activation of MAPK and NF-KappaB (NF-κB) signaling pathways and secretion of TNF-α, IL-1β and HMGB1 in THP-1 monocytes. Immunofluorescence assay and Western blotting were used to identify the nucleolin expression in cell membrane, cytoplasm and nucleus of THP-1 monocytes. Inactivation of nucleolin was induced by neutralizing antibody against nucleolin. THP-1 monocytes were pretreated with anti-nucleolin antibody for 1 h prior to LPS challenge. The irrelevant IgG group was used as control. Secretion of inflammatory mediators (TNF-α, IL-1β and HMGB1) and activation of MAPK and NF-κB/I-κB signaling pathways were examined to assess the effects of nucleolin on LPS-mediated inflammatory response. Nucleolin existed in cell membrane, cytoplasm and nucleus of THP-1 monocytes. Pretreatment of anti-nucleolin antibody significantly inhibited the LPS-induced secretion of TNF-α, IL-1β and HMGB1. P38, JNK, ERK and NF-κB subunit p65 inhibitors could significantly inhibit the secretion of IL-1β, TNF-α and HMGB1 induced by LPS. Moreover, the phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65) was significantly increased after LPS challenge. In contrast, pretreatment of anti-nucleolin antibody could significantly inhibit the LPS-induced phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65). However, the irrelevant IgG, as a negative control, had no effect on LPS-induced secretion of TNF-α and IL-1β and phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65). We demonstrated that nucleolin mediated the LPS-induced activation of MAPK and NF-κB signaling pathways, and regulated the secretion of inflammatory mediators (TNF-α, IL-1β and HMGB1).
在这项研究中，我们研究了核仁素对脂多糖（LPS）诱导的 MAPK 和 NF-κB（NF-κB）信号通路的激活以及 THP-1 单核细胞中 TNF-α、IL-1β 和 HMGB1 的分泌的影响。免疫荧光法和 Western blot 用于鉴定 THP-1 单核细胞的细胞膜、细胞质和核仁素表达。用核仁素的中和抗体诱导核仁素失活。用抗核仁素抗体预处理 THP-1 单核细胞 1 h，然后用 LPS 刺激。无关 IgG 组作为对照。检测炎症介质（TNF-α、IL-1β 和 HMGB1）的分泌以及 MAPK 和 NF-κB/I-κB 信号通路的激活，以评估核仁素对 LPS 介导的炎症反应的影响。核仁素存在于 THP-1 单核细胞的细胞膜、细胞质和核仁素中。用抗核仁素抗体预处理可显著抑制 LPS 诱导的 TNF-α、IL-1β 和 HMGB1 的分泌。p38、JNK、ERK 和 NF-κB 亚基 p65 抑制剂可显著抑制 LPS 诱导的 IL-1β、TNF-α 和 HMGB1 的分泌。此外，LPS 刺激后 p38、JNK、ERK 和 p65 的磷酸化（或 p65 的核转位）明显增加。相反，用抗核仁素抗体预处理可显著抑制 LPS 诱导的 p38、JNK、ERK 和 p65 的磷酸化（或 p65 的核转位）。然而，作为阴性对照的无关 IgG 对 LPS 诱导的 TNF-α 和 IL-1β 的分泌以及 p38、JNK、ERK 和 p65（或 p65 的核转位）的磷酸化没有影响。我们证明核仁素介导了 LPS 诱导的 MAPK 和 NF-κB 信号通路的激活，并调节了炎症介质（TNF-α、IL-1β 和 HMGB1）的分泌。
