四黄清灵液通过MAPKs/NF-κB信号通路保护脂多糖诱导的RAW264.7细胞炎症反应

Anti-inflammation action of SHQYL by regulating MAPKs/NF-κB signaling pathways in RAW264.7 macrophage cells

目的 研究中药复方四黄清灵液水提物对脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7炎症的保护作用及其作用机制。方法 把处于对数生长期的RAW264.7细胞随机分为Control、LPS组、SHQLY浓度组,采用CCK-8试剂检测SHQLY提取物对RAW264.7细胞的活性影响,酶联免疫法检测培养基上清中TNF-α和IL-6的含量,后续选取10μg和20μg两个浓度,利用DCFH-DA荧光探针法测定活性氧,Western-blot法检测P-ERK/ERK、P-p38/p38、P-JNK/JNK、P-Iκβα/Iκβα和P-NF-κB/NF-κB等蛋白表达情况,激光共聚焦显微镜观察经免疫荧光染色NF-κB入核情况。结果 SHQLY可以呈剂量依赖的抑制LPS诱导RAW264.7细胞炎症反应,可以抑制IL-6和TNF-α炎症因子的释放,抑制ROS的产生,Western-blot结果显示SHQLY通过抑制丝裂原活化蛋白激酶(MAPKs)和核因子(NF-κB)信号通路中磷酸化蛋白P-ERK、P-p38、P-JNK、P-IKβ-α和P-p65等蛋白磷酸化,同时抑制NF-κB转移入核。结论 SHQLY抑制LPS诱导的RAW264.7细胞炎症,可能的抗炎机制是其抑制MAPKs/NF-κB信号通路蛋白磷酸化相关。

Objective Four Yellow Qing Ling Water(SHQLY),a traditional Chinese medicine formula has been used for dry eye;However,anti-inflammatory activities of SHQYL are poorly understood.We elucidated the protective effect of SHQLY on the LPS-induced inflammation in RAW264.7 macrophage cells and to explore its potential mechanism.Methods RAW264.7 cells have been stimulated with LPS and treated with SHQLY.The cell viability of the LPS-treated RAW264.7 cells was examined by CCK-8 method.The modulation of SHQLY on anti-inflammation was detected by Elisa by measuring the release of inflammatory cytokines(TNF-αand IL-6),DCFH-DA fluorescence probe method was used to detect reactive oxygen species(ROS).Further,the content P-ERK/ERK,P-p38/p38,P-JNK/JNK,P-Iκβα/Iκβαand P-NF-κB/NF-κB in MAPKs/NF-κB signaling pathway were detected by western-blot method.The nuclear translocation of NF-κB was detected by immunofluorescence using laser confocal scanning.Results Anti-inflammation assay demonstrated that SHQLY inhibited the production of the pro-inflammatory cytokines IL-6 and TNF-αin LPS-stimulated RAW264.7 macrophages.SHQLY decreased ROS accumulation.SHQLY down-regulated the protein levels of phosphorylated proteins such as P-ERK,P-p38,P-JNK,P-IKβαand P-p65 in MAPKs/NF-κB pathway and inhibited NF-κB translocation into nucleus.Conclusion SHQLY protects LPS-induced inflammation in RAW264.7 cells,which may be related to its inhibition of phosphorylation of MAPKs/NF-κB pathway.