The Activation of Akt/p38MAPK Signaling and M1/M2 Polarization of Lipopolysaccharide Induced RAW264.7 Cells Regulated by Tibetan Eighteen Flavor Dangshen Pills
CHEN Zi-lu1, WU Jin-jie2, ZHI Wei2, HAN Qi1*
1. Department of Pharmacy, Zhejiang Hospital,Hangzhou 310013, China; 2. School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China
Abstract：OBJECTIVE To explore the effect and mechanism of Tibetan Eighteen flavor Dangshen Pills (TEP) in regulating lipopolysaccharide (LPS) induced inflammatory response in macrophages RAW264.7. METHODS Alarmarblue assay was used to evaluate the effect of TEP on the viability of RAW264.7. The reactive oxygen species (ROS) level in RAW264.7 induced by LPS was quantified by fluorescence microplate. Griess assay was used to detect the secretion of nitric oxide (NO), and the surface markers CCR7 and CD206 of M1/M2 phenotype were detected by flow cytometer. RT-qPCR method was used to detect gene expression of IL-1β, IL-6, CCL2, iNOS, ARG-1 and TNF-α. Western blot was used to detect the expression of iNOS, pAkt, p38MAPK and p65NF-κB signal pathway related protein. RESULTS The viability of RAW264.7 cells induced by two dose groups was significantly improved compared with the model group. Meanwhile, the intracellular ROS level induced by LPS IN RAW264.7 cells was inhibited via the addition of TEP. The secretion of NO in LPS group increased significantly, reaching the highest expression after 24 hours, but that in two dose groups decreased significantly. The surface marker CCR7 of M1 pro-inflammatory phenotype was increased in RAW264.7 induced by LPS, however the addition of TEP decreased the expression of CCR7, and increased the expression of surface marker CD206 of anti-inflammatory M2 phenotype. The gene expression of IL-1β, IL-6, CCL2, iNOS, ARG and TNF-α in two dose groups was significantly reduced. The protein expression levels of iNOS, pAkt and p38MAPK in two dose groups were significantly reduced, however the expression levels of p65NF-κB were significantly increased. CONCLUSION TEP can suppress the ROS expression level, reduce cellular NO secretion and inflammatory gene expression of RAW264.7 cells, and inhibit M1 pro-inflammatory phenotype and promote M2 anti-inflammatory phenotype of macrophages. The mechanism may be related to the inhibition of iNOS, pAkt and p38MAPK signal pathway protein expression.
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2022, Vol. 57 
