基于HMGB1/NF-κB信号通路评价参附注射液对脂多糖诱导RAW264.7细胞炎症的抑制作用

Inhibition of Shenfu Injection(参附注射液)on LPS-induced RAW264.7 Cells Inflammation Based on HMGB1/NF-κB Signal Pathway

目的探讨参附注射液对脂多糖(lipopolysaccharide, LPS)诱导的小鼠巨噬细胞(RAW 264.7细胞)中高迁移率族蛋白B1(high mobility group protein, HMGB1)/核转录因子-κB(Nuclear factor-κB, NF-κB)信号通路相关蛋白及通路下游炎症因子的干预作用。方法经LPS诱导RAW 264.7细胞建立炎性损伤模型,用3、6、12μL/mL剂量参附注射液干预细胞24 h。Western Blot法检测HMGB1、Toll样受体4(TLR4)、NF-κB抑制蛋白α(IκBα)、p65、p-p65和肿瘤坏死因子α(TNFα)的表达;细胞免疫荧光法观察p65的核转位情况;Elisa法检测细胞上清中炎症因子HMGB1、白细胞介素1β(IL-1β)和TNFα的分泌水平。结果与对照组比较,模型组RAW 264.7细胞中HMGB1、TLR4、p65、p-p65和TNFα蛋白表达均显著升高,IκBα的表达则显著降低;形态学观察到大量p65从胞浆向核内转移;细胞上清中HMGB1、IL-1β和TNFα的分泌水平显著升高。与模型组比较,参附注射液呈剂量依赖性抑制HMGB1、TLR4、p65、p-p65和TNFα的表达,上调IκBα的表达;有效抑制p65向核内移位;降低细胞上清中炎症因子HMGB1、IL-1β和TNFα分泌水平;上述作用均呈剂量依赖性。结论参附注射液可能通过抑制HMGB1/NF-κB信号通路的激活而减轻LPS诱导的RAW264.7细胞炎症反应。

Objective To investigate the effect of Shenfu Injection(参附注射液) on high mobility group protein B1(HMGB1)/nuclear factor κB(NF-κB) signal pathway related proteins and the downstream inflammatory factors in lipopolysaccharide(LPS)-induced macrophages(RAW264.7 cells). Methods RAW264.7 cells were induced by LPS to establish the inflammatory injury model. Different doses of SFI were used to treat the RAW264.7 cells for 24 hours respectively. Western blot was used to detect the expressions of HMGB1, toll-like Receptor 4(TLR4), inhibitor of NF-κBα(IκBα), p65, p-p65 and TNFα. Immunofluorescence was used to observe the nuclear translocation of p65. ELISA was used to detect the secretion of HMGB1, interleukin-1β(IL-1β) and tumor necrosis factor α(TNFα). Results Compared with those of the control group, the expressions of HMGB1, TLR4, p65, p-p65 and TNFα in RAW264.7 cells of the model group were significantly increased, while the expression of IκBα was significantly decreased. A large number of p65 was transferred from cytoplasm to nucleus. The levels of HMGB1, IL-1β and TNFα in the supernatant were significantly increased. Compared with the model group, Shenfu Injection can inhibit the expressions of HMGB1, TLR4, p65, p-p65 and TNF α, up-regulate the expression of IκBα, inhibit the migration of p65 to the nucleus effectively, and reduce the level of inflammatory factors HMGB1, IL-1β and TNFα in the supernatant in a dose-dependent manner. Conclusion Shenfu Injection may reduce the LPS-induced inflammatory of RAW264.7 cells by inhibiting the activation of HMGB1/NF-κB signal pathway.