Study On The Regulation Of Total Flavonoids Of Astragalus On NF-κB Signaling Pathway In RAW264.7 Cells

Astragalus membranaceus is a traditional Chinese medicine which has a long history in China. Total flavonoids of Astragalus (TFA) is a kind of flavonoids isolated from Astragalus membranaceus as the main active ingredient, which has a wide range of biological activities, including anti-inflammatory, anti-cancer, anti-tumor and antioxidant. Previous research in our lab demonstrated that TFA had significant anti-inflammatory and immune enhanced effect in vitro and in vivo, and confirmed that it was associated with MAPK signaling pathway. However, whether it is associated with NF-κB signaling pathway has not been studied and reported, its regulatory mechanism needs to be further explored. In this study, RAW264.7 cells were used as experimental subjects, the effects of TFA on transcription levels of inflammatory factors in normal or LPS-stimulated RAW264.7 cells were studied. By detecting the expressions of the key protein molecules in the NF-κB signaling pathway, the anti-inflammatory and immune regulation mechanisms of TFA were further discussed, which provided scientific basis for the development and clinical application of the total flavonoids of Astragalus.Firstly, the MTT method was used to determine the safe dose of the total flavonoids of Astragalus. The experimental results showed that there was no significant cytotoxic effect of 0-150μg/mL TFA, so we proposed TFA 10 μg/mL,25 ug/mL,100 μg/mL as the TFA low, medium and high dose groups. RT-PCR method was used to detect the effects of TFA on the transcription levels of IL-1β, IL-6, TNF-a and IL-10 in RAW264.7 cells under two kinds of conditions. Western-blot method was used to determine the effects of TFA on the expressions of key protein IKKα/β, p-IKKα/β, IκBα, p-IκBα, C-NF-κB p65 and N-NF-κB p65 in the NF-κB signaling pathway, to explore the anti-inflammatory and immune regulation mechanisms of total flavonoids of Astragalus.Electrophoresis results showed that the target genes were amplified single electrophoretic bands in 535 bp,384 bp,383 bp and 367 bp, which was consistent with the primer design. TFA could dose-dependently upregulate IL-1(3, IL-6, TNF-a and IL-10 mRNA transcription levels in normal RAW264.7cells (P<0.05 or P<0.01). TFA could dose-dependently inhibit of IL-1β, IL-6, TNF-a mRNA overexpression, but increase IL-10 mRNA transcription levels in LPS-stimulated RAW264.7 cells (P <0.05 or P<0.01). The results showed that TFA could regulate the expressions of cytokine mRNA in the gene transcription levels, and regulate the immune response of the organism.The results of Western blot showed that TFA could inhibit IKKα/β, IκBα, and C-NF-κBp65 protein expressions, upregulate p-IKKa/p, p-IκBα and N-NF-icBp65 protein expressions in normal RAW264.7cells in a dose-dependent relationship (P <0.05 or P<0.01). TFA could upregulate IKKa/p, IκBα, C-NF-κBp65 proteins expressions, decrease p-IKKα/β, p-IκBα, N-NF-icBp65 proteins expressions in LPS-stimulated RAW264.7 cells in a dose-dependent relationship (P<0.05 or P <0.01). The results showed that the total flavonoids of Astragalus could regulate the phosphorylation degradation of IKKα/β, IκBα, and NF-κBp65 from the cytoplasm to nuclear translocation through the regulation of NF-κB pathway and play anti-inflammatory and immune effects.The results demonstrated that TFA could regulate the critical factor IKKa/p, p-IKKa/p, IκBα, p-IκBα, C-NF-κBp65 and N-NF-κBp65 protein expressions of NF-κB signaling pathway, activate or inhibit the NF-κB signaling pathway, regulate cytokine mRNA expressions, and play its anti-inflammatory and immune regulatory effects.