Liang-Ge-San, a classic traditional Chinese medicine formula, protects against lipopolysaccharide-induced inflammation through cholinergic anti-inflammatory pathway

Abstract

Liang-Ge-San (LGS) is a classic formula in traditional Chinese medicine, which is widely used to treat acute lung injury (ALI), pharyngitis and amygdalitis in clinic. However, the underlying mechanisms remain poorly defined. In this study, we discovered that LGS exerted potent anti-inflammatory effects in lipopolysaccharide (LPS)-induced inflammation. We found that LGS significantly depressed the production of IL-6 and TNF-α in LPS-stimulated RAW 264.7 macrophage cells. The degradation and phosphorylation of IκBα and the nuclear translocation of NF-κB p65 were also inhibited. Moreover, LGS activated α7 nicotinic cholinergic receptor (α7nAchR). The blockage of α7nAchR by selective inhibitor methyllycaconitine (MLA) or α7nAchR siRNA attenuated the inhibitory effects of LGS on IκBα, NF-κB p65, IL-6 and TNF-α. Critically, LGS significantly inhibited inflammation in LPS-induced ALI rats through the activation of NF-κB signaling pathway. However, these protective effects could be counteracted by the treatment of MLA. Taken together, we first demonstrated anti-inflammatory effects of LGS both in vitro and in vivo through cholinergic anti-inflammatory pathway. The study provides a rationale for the clinical application of LGS as an anti-inflammatory agent and supports the critical role of cholinergic anti-inflammatory pathway in inflammation.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; Liang-Ge-San; NF-κB; inflammation; α7 nicotinic cholinergic receptor.

Figure 1. The cytotoxicity of LGS with or without LPS in RAW 264.7 macrophage cells

The viability was determined by MTT assay. A. Macrophages were incubated with LGS (25 ~ 400 μg/ml) alone for 24 h. B. Macrophages were treated with LGS at different concentrations in the presence of LPS (1 μg/ml) for 24 h. Data are represent as percentage of the cell viability as mean ± S.D. of three independent experiments.

Figure 2. LGS attenuates the production of IL-6 and TNF-α in LPS-stimulated RAW 264.7 cells

RAW 264.7 cells were cultured with indicated concentrations of LGS in the presence or absence of LPS (1 μg/ml) for 24 h. Quantitative analyses of IL-6 A. and TNF-α B. in supernatants of macrophages were measured by ELISA. ^##P < 0.01 versus control, *P < 0.05 and **P < 0.01 versus LPS treatment, one-way ANOVA, post hoc comparisons, Turkey, Cloumns, mean; error bar, S.D.

Figure 3. LGS inhibits the activation of NF-κB signaling in LPS-stimulated RAW 264.7 cells

A. LGS suppresses LPS-induced degradation and phosphorylation of IκBα. RAW 264.7 cells were stimulated with LPS (1 μg/ml) for 30 min and then treated with different concentrations of LGS for 24 h. The expression levels of IκBα and p-IκBα (Ser32) were examined by western blotting. B.-D. LGS blocks NF-κB p65 translocation from cytoplasm to nuclear. RAW 264.7 cells were stimulated with LPS (1 μg/ml) for 2 h, incubated with or without LGS (400 μg/ml) for 24 h and observed by confocal microscopy. (200 ×) (B). Expression of NF-κB p65 in nuclear was detected by western blotting (C) and NF-κB nuclear binding activity was measured by EMSA (D).

Figure 4. LGS suppresses inflammation and NF-κB pathway via α7nAchR activation

A. LGS increases the expression of α7nAchR. RAW 264.7 cells were pretreated with indicated concentrations of LGS for 24 h and then stimulated with LPS (1 μg/ml) for 2 h. The level of α7nAchR in total lysate was evaluated by western blotting. B.-C. LGS suppresses NF-κB pathway via α7nAchR. RAW 264.7 cells were transfected with α7nAchR siRNA (120 nM) for 24 h (B) or treated with methyllycaconitine (MLA, 20 μM) for 2 h (C) before cultured with 400 μg/ml LGS for 24 h and then stimulated with LPS (1 μg/ml) for 30 min or 2 h. The levels of α7nAchR (for 2 h), IκBα and p-IκBα (Ser32) (for 30 min) in total lysate and the expression of NF-κB in nuclear were detected by western blotting. D. LGS inhibits the production of IL-6 and TNF-α via α7nAchR. RAW 264.7 cells were transfected with α7nAchR siRNA (120 nM) for 24 h or pretreated with MLA (20 μM) for 2 h before culturing with LGS (400 μg/ml) in the presence or absence of LPS (1 μg/ml) for 24 h. IL-6 (left panel) and TNF-α (right panel) in the supernatants were determined by ELISA. ^##P < 0.01 versus control,*P < 0.05 and **P < 0.01 versus LGS and LPS treatment, one-way ANOVA, post hoc comparisons, Turkey, Cloumns, mean; error bar, S.D.

Figure 5. LGS attenuates LPS-induced acute lung injury (ALI) through cholinergic anti-inflammatory pathway

LGS with or without methyllycaconitine (MLA, a α7nAchR selective inhibitor) were treated with LPS-induced ALI rats. PNU282987 (PNU), a highly selective AChR α7 agonist, was used as a positive control. A. LGS decreases hyperaemia and swelling on the pulmonary surface in LPS-induced ALI rats which can be counteracted in the appearance of MLA. At 8 h after ALI, the rats were sacrificed and the representative lungs removed were photographed. B. MLA attenuates the influence of LGS on lung histopathological changes in LPS-induced ALI rats. Lungs were fixated. After paraffin embedding and sectioning at 5 μm thickness, lung tissues were stained by hematoxilin and eosin (H&E) staining. (200 ×) C. MLA counteracts the inhibitory effects of LGS on Acetyl cholinesterase (AChE) in LPS-induced ALI rats. At 8 h after ALI, the rats were sacrificed and the bronchoalveolar lavage fluid (BALF) was obtained. The activity of AChE in supernatants of BALF samples was measured using acetyl cholinesterase kit according to the manufacturer's protocol. *P<0.05 versus control, ^#P<0.05 and ^##P<0.01 versus LPS, one-way ANOVA, post hoc comparisons, Turkey, Cloumns, mean; error bar, S.D.

Figure 6. LGS inhibits edema and inflammation in LPS-induced ALI rats through cholinergic anti-inflammatory pathway

LGS with or without methyllycaconitine (MLA, a α7nAchR selective inhibitor) were treated with LPS-induced ALI rats. PNU282987 (PNU), a highly selective AChR α7 agonist, was used as a positive control. A. MLA attenuates the influence of LGS on lung ultrastructural changes in LPS-induced ALI rats. Ultrastructures of lung tissues were observed by transmission electron microscopy (15 000 ×, Blue arrow, normal lung type II alveolar cell; Red arrow, inflammatory cell in alveolar spaces; Yellow arrow, osmilphilic multilamellar body in alveolar spaces; Black arrow, lung type II alveolar cell in alveolar spaces). B. MLA reduces the decrease of the ratio of lung wet weight (W) and dry weight (D) induced by LGS in ALI rats. At 8 h after ALI, lungs were separated, and weighing to get the lung W/D ratio. BALF was obtained after the sacrifice of rats to calculate the total protein concentration (C), total cells count (D) and neutrophils count (E). F. MLA attenuates the regulatory activity of LGS on inflammatory factors. After rats were sacrificed, the BALF was obtained to measure the levels of MPO, MIP-1α, MIP-2, TNF-α and IL-6 by ELISA. **P<0.01 versus control, ^#P<0.05, and ^##P<0.01 versus LPS, one-way ANOVA, post hoc comparisons, Turkey, Cloumns, mean; error bar, S.D.

Figure 7. LGS inhibits the activation of NF-κB pathway in LPS-induced ALI rats through cholinergic anti-inflammatory pathway

A. MLA counteracts the inhibitory effects of LGS on the degradation and phosphorylation of IκBα in LPS-induced ALI rats. The lung tissues were removed and homogenated after the sacrifice of rats. The total protein was used to detect the expressions of IκBα and p-IκBα (Ser32) by western blotting. B.-C. MLA attenuates the inhibitory effects of LGS on the translocation of NF-κB p65 from cytoplasm to nuclear. The lung tissues were removed and homogenated after the sacrifice of rats. The nuclear protein was used to detect the expression of NF-κB p65 by western blotting (B). Lungs were fixated, embeded in paraffin and sectioned at 5 μm thickness. Lung sections were stained by immunohistochemistry (C).

Figure 8. The speculated anti-inflammatory network of LGS

┴ indicates an inhibitory effect. LGS exerts anti-inflammatory effects and suppresses the degradation and phosphorylation of IκBα and the translocation of NF-κB p65 from cytoplasm to nuclear as well as the release of inflammatory factors induced by LPS through cholinergic anti-inflammatory pathway in vitro and in vivo.