Liang-Ge-San Attenuates Inflammation Through Cholinergic Anti-inflammatory Pathway In LPS-stimulated RAW 264.7Macrophage Cells

BackgroundRecent research indicated that central nervous system through vagus nerve could observably regulate peripHeral inflammatory responses and this is termed as cholinergic anti-inflammatory pathway. The physiological mechanism potential originates from activating the vagus nerve to release acctylcholine (Ach), which binds to the α7 cholinergic receptor on immune cells and then suppresses the production of inflammatory cytokine, including interleukin-6 (IL-6) and tumor necrosis factor (TNF-a), high-mobility group box 1 proteins and matrix metalloproteinase 9.α7 subtype expresses on macrophages and plays an important role in cholinergic anti-inflammatory pathway. Studies in animal-models under the experimental conditions of sepsis, shock have demonstrated that nicotine could inhibit the production of pro-inflammatory cytokines througha7 nicotinic cholinergic receptor (a7nAchR).Lianggesan (LGS) is a classic Chinese medicine formuladescribed in "Taiping huiminheji Jufang". This decoction contains seven herbs, including Forsythiasuspense, Rheum palmatum, Scutellaria baicalensis, Gardenia jasminoides, Glycyrrhiza uralensis, Mentha haplocalyx and Natrii Sulfas. In traditional Chinese medicine, LGS has been widely used for centuries to clear heat and fire. Nowadays, it has curative effect on respiratory inflammations, such as pharyngitis, amygdalitis, pneumonia andacute lung injury (ALI) in clinic.In this study, we first found that LGS exerted anti-inflammatory effects through activating cholinergic anti-inflammatory pathway which lead to the inhibition of NF-κB pathway. Our research provided scientific evidence for the therapeutic application of LGS.ObjectThis object of study is to investigate anti-inflammatory effects of LGS and its mechanisms in LPS-stimulated RAW 264.7macrophage cells to support clinical application.Methods1. Cell viability assay was performed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-dipHenyltetrazolium bromide (MTT) assay.2. Anti-inflammatory effects of LGSin LPS-stimulated RAW 264.7 macrophage cells were measured from the release of interleukin-6 (IL-6) and tumor necrosis factor (TNF-a) using Enzyme-linked immunosorbent assay (ELISA).3. The expressions of phospho-IκBα (Ser32), IκBα and NF-κB p65 in NF-κB signaling pathway and the level ofa7nAchRwere analyzed by western blotting. The nuclear translocation of NF-κB p65 was further conformed by confocal microscopy and electrophoretic mobility shift assay.4. After the inhibition of a7nAchR by using special siRNA to knock-down α7nAchR or a selective inhibitor Methyllycaconitine (MLA) to abrogate a7nAchR, the expressions of IκBα, pHospHo-IκBα (Ser32) and NF-κB p65 were analyzed by western blotting and the release of IL-6 andTNF-a was detected by ELISA.Results1. Cytotoxicity of LGS in RAW 264.7 macrophage cellsMTT assay was used to investigate the cytotoxicity of LGS in RAW 264.7 cells. Results showed that LGS (25～400 μg/ml) had no obvious effects on cell viability after 24 h treatment. Furthermore, we evaluated the cell viability of LGS with LPS (1 μg/ml). It was found that both of LGS and LPStreatments didn’t show significant cytotoxicity in RAW 264.7 cells. Therefore, LGS at the concentrations of 25～400 μg/ml was selected in the subsequent experiments.2. LGS inhibits the release of IL-6, TNF-a in RAW 264.7 macrophage cellsELISAassay showed that resting RAW 264.7 cells released little IL-6and TNF-α. After stimulated by LPS, IL-6 and TNF-awere significantly released from RAW 264.7 cells which were inhibited in the presence of LGS. Collectively, these results suggest that LGS can inhibit inflammatory effects in LPS-stimulated RAW 264.7 macrophage cells.3. LGS attenuates NF-κB activation in LPS-stimulated RAW 264.7 macrophage cellsOur results showed that LPS treatment induced the degradation and phosphorylation of IκBα in RAW 264.7 cells, while LGS treatment prevented this phenomenon. In addition, we found that NF-κB p65 mainly existed in the cytoplasm of RAW 264.7 cells, which was transferred into nuclear after LPS stimulation. Notably, this migration was blocked in the presence of LGS (400 μg/ml). Finally, we confirmed the nuclear translocation of NF-κB p65 by western blotting and EMSA, revealing that LGS markedly suppressed endogenous NF-κB p65 nuclear translocation in LPS-stimulatedRAW 264.7 cells. Taken together, these findings demonstrate that LGS attenuates inflammation by NF-κB signaling pathway.4. LGS suppresses the inflammation and NF-κBpathway via the cholinergic anti-inflammatory pathwayWestern blotting showed that LGS increased the expression level ofa7nAchR in RAW 264.7 cells by western blotting, suggesting that LGS could activate the cholinergic anti-inflammatory pathway. We further explored the role of a7nAchRin anti-inflammatory effects of LGS. Special a7nAchRsiRNA or MLAwas used to abrogate this protein. The results showed that the decrease of a7nAchRcounteracted the inhibitory effects of LGS on the degradation and phosphorylationof IκBα and the nuclear translocation of NF-κBin LPS-stimulated RAW 264.7 cells.Moreover, the inhibition ofa7nAchR could also decrease the suppressive effects of LGS on the levels of IL-6 and TNF-α. These data demonstrate that LGS inhibits inflammation and NF-κB pathway which is associated with the activation of cholinergic anti-inflammatory pathway.ConclusionLGS exerts anti-inflammatory effects through the activation of cholinergic anti-inflammatory pathway in LPS-stimulated RAW 264.7 macrophage cells. The study may provide a rationale for the clinical application of LGS for respiratory inflammations.