Protective effect of Haoqin Qingdan decoction on pulmonary and intestinal injury in mice with influenza viral pneumonia

Abstract

Background: Haoqin Qingdan decoction (HQQD), composed of eleven herbs, is a traditional Chinese formula widely recognized for its efficacy in treating pulmonary inflammation induced by viral infections. Despite its extensive use, the potential pulmonary and intestinal protective effects of HQQD on influenza viral pneumonia (IVP) and the underlying molecular mechanisms remain unclear.

Materials and methods: Ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) was employed to identify the major chemical constituents of the prescription. Subsequently, network analysis was conducted to predict the potential therapeutic targets of HQQD in IVP. The mechanisms by which HQQD mitigates lung and intestinal damage were further elucidated by assessing NP protein expression, inflammatory factors, TLR7/MyD88/NF-κB signaling pathway mRNAs and proteins, and through intestinal flora analysis.

Results: The protective effects of HQQD on pulmonary and intestinal injuries induced by IVP were thoroughly investigated using comprehensive network analysis, signaling pathway validation, and gut microflora analysis. UHPLC-MS analysis identified the primary chemical constituents. Validation experiments demonstrated a significant reduction in NP protein expression in the lungs. HQQD notably alleviated immune damage in the lungs and intestines of mice by inhibiting NP protein expression and the release of inflammatory factors such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ); downregulating the expression levels of TLR7, MyD88, and phospho-NF-κB p65 (p-p65); lowering serum LPS levels; and reducing the relative abundance of Proteobacteria.

Conclusion: HQQD exerts therapeutic effects against influenza viral pneumonia through antiviral and anti-inflammatory mechanisms and by remodeling the intestinal flora. This study provides initial insights into the "gut-lung" axis mechanism of HQQD in combating respiratory influenza virus infection.

Keywords: Haoqin Qingdan decoction; influenza viral pneumonia; intestinal flora; network analysis; traditional Chinese medicine.

FIGURE 1

Evaluation of the efficacy of HQQD Decoction. (A) Schematic diagram of the drug efficacy experiment. (B) Body weight trend chart following virus infection. (C) Lung indices in the validation experiment. (D) HE staining scores of lung tissue in mice. (E) HE staining images of lung and intestine tissues in mice (scale bar: 200 and 50 μm). (F) Western blot analysis showing the inhibitory effect of HQQD against influenza virus (n = 4). Data are presented as means ± standard errors of the means.

FIGURE 2

Results of Network Analysis. (A) Venn diagram illustrating 364 common targets between HQQD and IVP. (B) PPI network diagram depicting protein interactions, where targets are represented by nodes and protein-protein interactions by edges. The size and color of nodes are governed by degree centrality, with larger nodes and red underpainting indicating higher degree centrality. (C) Top 20 signaling pathways identified through KEGG enrichment analysis, with larger points indicating greater gene enrichment. (D) Sankey diagram of “formula-components-targets-pathways”. The lines represent the properties of targets and pathways.

FIGURE 3

Effect of the HQQD on serum and lung cytokine levels. (A), (B), (C) ELISA analysis of serum inflammatory factor levels (IFN-γ, TNF-α, and LPS) in mice (n = 6). (D), (E), (F) ELISA analysis of lung inflammatory factor levels (TNF-α, IL-6, and IL-1β) in mice (n = 6). Data are presented as means ± standard errors of the means.

FIGURE 4

Treatment Mechanism of HQQD for IVP. (A), (B) Expression levels and quantitative analysis of TLR7 and MyD88 mRNA in lung homogenates (n = 6). (C), (D), (E) Immunohistochemical staining and AOD calculation of NF-κB p65 and p-p65 in lung tissue of model mice (scale bar: 50 μm; n = 4). Data are presented as means ± standard errors of the means.

FIGURE 5

Intestinal Flora Diversity Analysis on Day Six. (A) Venn diagram showing different strains identified through OTU analysis. (B) Relative abundance and composition of microbial communities at the phylum level. (C) Relative abundance and composition of microbial communities at the family level. (D) Relative abundance and composition of microbial communities at the genus level. (E) Alpha diversity analysis represented by the Chao1 index. (F) PCA analysis showing differences between samples at the OTU level.

FIGURE 6

LEfSe Analysis of Intestinal Flora on Day Six. (A) LEfSe analysis at the phylum and family levels in the VC and control groups. (B) LEfSe analysis at the phylum and family levels in the VC and HD groups.