Shu-Feng-Jie-Biao Formula Ameliorates Influenza A Virus-Induced Acute Lung Injury by Inhibiting NF-κB and ERK MAPK Signaling Pathways

Abstract

Purpose: Shu-Feng-Jie-Biao formula (SFJBF) has been used to treat acute respiratory infections for a dozen years. This study aimed to explore its mechanisms and effects for the treatment of influenza.

Methods: Network pharmacology was used to explore the underlying mechanism of SFJBF against influenza. The protective effects of SFJBF in vivo were evaluated by lung indexes, body weight loss and pathological changes in lungs. The anti-inflammatory effects in vivo were evaluated by flow cytometry and ELISA. RAW264.7 cells stimulated with imiquimod (R837) were used to determine the anti-inflammatory effects of SFJBF. Neutrophils isolated from bone marrow were activated by phorbol 12-myristate 13-acetate (PMA) to validate the effects of the active components of SFJBF.

Results: SFJBF protected body weight loss, decreased lung indexes, reduced total protein content in lungs and mitigated pathological changes in mice. SFJBF inhibited the expression of chemokines (Cxcl2 and Ccl2) and cytokines (Il1b and IL-6) accompanied by the decreased infiltration of neutrophils in lungs. SFJBF inhibited the expression of iNOS and MPO in lungs. The synergistic role of OSV and SFJBF was exhibited by suppressing virus-induced cytokine expression and reducing the infiltration of inflammatory monocytes in lungs. SFJBF inhibited the phosphorylation of ERK1/2 and NF-κBp65, thereby reducing the secretion of MIP-2, TNF-α, MCP-1 and CCL5 in vitro The active components of SFJBF, including baicalin and wogonin, reduced the production of reactive oxygen species (ROS), MIP-2, MCP-1, and IL-6 in vitro.

Conclusion: SFJBF ameliorated virus-induced lung injury by suppressing overactivated immune responses via NF-κB and ERK MAPK signaling pathways, thereby protecting mice from influenza virus infection. SFJBF could be considered a potent therapeutic agent for treating influenza.

Keywords: ERK MAPK signaling pathway; NF-κB signaling pathway; TCM; acute lung injury; anti-inflammatory; influenza virus; traditional Chinese medicine.

Figure 1

Potential targets of SFJBF against influenza virus infection. (A) The venn diagram of targets between active compounds-related targets and influenza-related targets. The overlapping shape represented 61 influenza-related targets regulated by SFJBF. (B) The protein–protein interactions of 61 targets. The darker and larger circles represent the more important targets regulated by SFJBF.

Figure 2

Top 10 GO terms regulated by SFJBF.

Figure 3

Top 20 signaling pathways regulated by SFJBF.

Figure 4

Components-target network. Purple nodes represented the influenza-related targets, circles represented active ingredients and red nodes represented eight herbs including Schizonepeta tenuifolia (Jingjie, JJ), Saposhnikovia divaricata (Fangfeng, FF), Bupleurum chinensis (Chaihu), Radix Angelicae Dahuricae (Baizhi, BZ), Ligusticum chuanxiong (Chuanxiong, CX), Sojae Semen Praeparatum (Dandouchi, DDC), Glycyrrhiza uralensis (Gancao, GC) and Notopterygium incisum Ting ex (Qianghuo, QH).

Figure 5

UPLC-MS/MS analysis of SFJBF. (A) Positive ion mode; (B) Negative ion mode. (C) The 11 representative compounds in positive or negative ion modes of SFJBF and their chemical structures.

Figure 6

Base peak intensity chromatograms of 71 compounds with a score of 1 in positive ion mode (A) and negative ion mode (B) using UHPLC-QE-MS analysis.

Figure 7

The protective effect of SFJBF(L), SFJBF(M), SFJBF(L), OSV, or the combination OSV and SFJBF(M) on PR8-infected mice. (A) The scheme of exploring the protective mechanisms of SFJBF against influenza in vivo. (B) Weight changes of mice in different groups at 6 dpi (n=6). (C) Lung indexes of mice in different groups at 6 dpi (n=6). (D) Total protein content in lungs in different groups at 6 dpi (n=6). (E and F) Histopathological score of lung tissues and the statistic histopathological changes of lung tissues at 6 dpi (n=3). scale bar = 250 µm. Data were shown as mean ± SD and analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests according to homogeneity of variance test. *, p < 0.05; **, p < 0.01 or ***, p < 0.001. vs PR8 group. #, p < 0.05 or ##, p < 0.01 vs PR8 + OSV group.

Figure 8

SFJBF(H), SFJBF(M), OSV, or the combination of OSV and SFJBF(M) decreased the excessive innate immune cells recruitment in lung tissues of flu mice. (A) Gating strategy. (B–D) Typical diagrams of neutrophils, inflammatory monocytes and T cells in each group. (E-G) The proportion of neutrophils, inflammatory monocytes and T cells (n=6). The values were presented as mean ± SD and analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests according to homogeneity of variance test. **, p < 0.01 or ***, p < 0.001. vs PR8 group. #, p < 0.05 vs PR8 + OSV group.

Figure 9

SFJBF, OSV, or the combination of OSV and SFJBF(M) inhibited the overexpression of cytokines and chemokines in lung tissues of flu mice at 6 dpi. (A–G) The mRNA expression of Cxcl2, Ccl2, Il1b, Ccl4, Ccl3, Tnf and Cxcl10 (n=6). (H) The expression of IL-6 in lung homogenates by ELISA assays (n=6). The data were shown as mean ± SD and analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests according to homogeneity of variance test. *, p < 0.05; **, p < 0.01 or ***, p < 0.001. vs PR8 group. #, p < 0.05; ##, p < 0.01 or ###, p < 0.001 vs PR8 + OSV group.

Figure 10

SFJBF, OSV, or the combination of OSV and SFJBF(M) inhibited the overexpression of mediators in lung tissues of flu mice at 6 dpi. (A–D) The expression of iNOS, MPO, SOD and ICAM-1 in lung homogenates by ELISA assays (n=5). The values were presented as mean ± SD and analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests according to homogeneity of variance test. *, p < 0.05; **, p < 0.01 or ***, p < 0.001. vs PR8 group.

Figure 11

SFJBF-containing sera inhibited the expression of mediators in RAW264.7 cells. (A–D) The expression of MIP-2, MCP-1, CCL5 and TNF-α in RAW264.7 cells stimulated with R837 (n=3). (E) The expression of p-p65, p65, p-ERK1/2, ERK1/2 and β-Actin in RAW264.7 cells stimulated with R837 (n=3). (F and G) The relative expression of p-p65 and p-ERK1/2 analyzed by Image J. The values were analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests according to homogeneity of variance test. *, p < 0.05; ***, p < 0.001. vs ctrl group.

Figure 12

The active components of SFJBF including wogonin and baicalin inhibited neutrophil activation induced by PMA. (A–D) The production of ROS, MIP-2, MCP-1 and IL-6 with the stimulation of PMA in different groups (n=3). The values were analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests according to homogeneity of variance test. *, p < 0.05; **, p < 0.01 or ***, p < 0.001. vs PMA group.