EGCG 对Ro52 介导的THP-1 细胞凋亡 及炎症因子的影响

Effect of EGCG on Ro52-induced apoptosis of THP-1 cells and inflammatory factors

摘要:

目的 探讨表没食子儿茶素没食子酸酯（EGCG）对SSA/Ro52（Ro52）介导的人单核- 巨噬 细胞株THP-1 凋亡及炎症因子分泌的影响。方法 构建稳定Ro52 过表达质粒（pCMV-Ro52），pCMVRo52 转染THP-1 细胞48 h，检测Ro52 mRNA 和蛋白表达水平的变化，采用膜联蛋白- 荧光素异硫氰酸酯 和碘化丙锭双染检测THP-1 细胞凋亡率，酶联免疫吸附法检测肿瘤坏死因子-α（TNF-α）、白介素-1β （IL-1β）、IL-13 水平。pCMV-Ro52 质粒转染48 h 后, 分别使用0、5、10、20 和50 mg/L EGCG 处理细 胞24 h，检测Ro52 表达的变化、THP-1 细胞的凋亡，以及TNF-α、IL-1β、IL-13 水平。结果 pCMVRo52 促进Ro52 表达（P <0.05），上调THP-1 细胞凋亡率及TNF-α、IL-1β、IL-13 水平（P <0.05）； 在Ro52 过表达的THP-1 细胞中，与0 mg/L EGCG 组相比，10、20 和50 mg/L EGCG 组Ro52 蛋白表达水 平、凋亡率及TNF-α、IL-1β、IL-13 水平降低（P <0.05）。结论 Ro52 过表达可促进人单核- 巨噬细胞 THP-1 凋亡及炎症因子TNF-α、IL-1β、IL-13 分泌；EGCG 可减弱Ro52 介导的THP-1 细胞凋亡，减 少炎症因子TNF-α、IL-1β、IL-13 的分泌。

Abstract:

Objective To explore the influence of epigallocatechin gallate (EGCG) on apoptosis of human monocyte macrophage cell line (THP-1) and expression of inflammatory cytokines through Ro52 overexpression. Methods Ro52 over-expression plasmid (PCMV-Ro52, 20 μg) was constructed and transfected into THP-1 cells for 48 h. Real-time fluorescent quantitative PCR was used to detect the mRNA level of Ro52 and the protein expression level of Ro52 was detected by Western blot. The apoptosis of THP-1 cells was detected by Annexin V-FITC and propidium iodide (PI) staining. The levels of TNF-α, IL-1β and IL-13 were measured by ELISA. After 48 h of pCMV-Ro52 plasmid transfection, the cells were treated with EGCG at different concentrations (0, 5, 10, 20 and 50 mg/L) for 24 h. The protein expression level of Ro52, the expression levels of TNF-α, IL-1β and IL-13 and the apoptosis rate of THP-1 cells were detected. Results pCMV-Ro52 (20 μg) significantly increased the mRNA and protein levels of Ro52 (P < 0.05) and up-regulated the apoptosis rate of THP-1 cells and the levels of TNF-α, IL-1β and IL-13 (P < 0.05). In the Ro52-overexpressed THP-1 cells, compared with the 0 mg/L group, the Ro52 protein levels, the cell apoptosis rates and the levels of inflammatory cytokines (TNF-α, IL-1β and IL-13) were significantly decreased in the 10 mg/L, 20 mg/L and 50 mg/L EGCG-treated groups (P < 0.05). Conclusions The over-expression of Ro52 could promote the apoptosis of THP-1 cells and the secretion of TNF-α, IL-1β and IL-13. EGCG can attenuate Ro52-induced apoptosis of THP-1 cells and secretion of inflammatory cytokines.