Xuanfei Baidu Decoction protects against macrophages induced inflammation and pulmonary fibrosis via inhibiting IL-6/STAT3 signaling pathway

Abstract

Ethnopharmacological relevance: Xuanfei Baidu Decoction (XFBD), one of the "three medicines and three prescriptions" for the clinically effective treatment of COVID-19 in China, plays an important role in the treatment of mild and/or common patients with dampness-toxin obstructing lung syndrome.

Aim of the study: The present work aims to elucidate the protective effects and the possible mechanism of XFBD against the acute inflammation and pulmonary fibrosis.

Methods: We use TGF-β1 induced fibroblast activation model and LPS/IL-4 induced macrophage inflammation model as in vitro cell models. The mice model of lung fibrosis was induced by BLM via endotracheal drip, and then XFBD (4.6 g/kg, 9.2 g/kg) were administered orally respectively. The efficacy and molecular mechanisms in the presence or absence of XFBD were investigated.

Results: The results proved that XFBD can effectively inhibit fibroblast collagen deposition, down-regulate the level of α-SMA and inhibit the migration of fibroblasts. IL-4 induced macrophage polarization was also inhibited and the secretions of the inflammatory factors including IL6, iNOS were down-regulated. In vivo experiments, the results proved that XFBD improved the weight loss and survival rate of the mice. The XFBD high-dose administration group had a significant effect in inhibiting collagen deposition and the expression of α-SMA in the lungs of mice. XFBD can reduce bleomycin-induced pulmonary fibrosis by inhibiting IL-6/STAT3 activation and related macrophage infiltration.

Conclusions: Xuanfei Baidu Decoction protects against macrophages induced inflammation and pulmonary fibrosis via inhibiting IL-6/STAT3 signaling pathway.

Keywords: COVID-19; IL-6/STAT3; Macrophage polarization; Pulmonary fibrosis; Xuanfei baidu decoction.

Graphical abstract

Fig. 1

Network Pharmacology analysis on the potential mechanisms of XFBD against Idiopathic Pulmonary fibrosis (IPF). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (A) and gene ontology (GO) analysis of biological processes (B) were investigated using the DAVID website. PPI interactions and hub genes of XFBD targets related to inflammatory response were analyzed by Cytoscape software (the larger the node, the higher the degree) (C–E).

Fig. 2

Identification of active ingredients in Xuanfei Baidu Decoction. The typical chromatograms of standard compounds of 210 nm (A), 254 nm (B) and sample of 210 nm (C), 254 nm (D). (1) ephedrine, (2) amygdalin, (3) sinapine, (4) hastatoside, (5) verbenalin, (6) polydatin, (7) liquiritin, (8) acteoside, (9) naringin and (10) glycyrrhizic acid.

Fig. 3

XFBD inhibits the activation and migration of fibroblasts. (A, B) The inhibitory effect of XFBD on TGF-β1-induced fibroblast activation (magnification × 200). (C, D) The effect of XFBD on TGF-β1-induced α-SMA expression was evaluated by immunofluorescence analysis. (E, F) Inhibition of XFBD on the migration of activated fibroblasts. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, vs. control group, *p < 0.05, **p < 0.01, ***p < 0.001, vs. Model group. Scale bar = 100 μm.

Fig. 4

XFBD inhibits the inflammatory response of macrophages (A, B) XFBD inhibits IL-4 induced polarization of M2 macrophages in vitro. (C) Flowcytometry result of the proportion of CD206 positive cells (D) The inhibitory effect of XFBD on LPS-induced IL-6 expression. (E) The inhibitory effect of XFBD on LPS-induced iNOS expression. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, vs. control group, *p < 0.05, **p < 0.01, ***p < 0.001, vs. Model group. Scale bar = 100 μm.

Fig. 5

XFBD has protective effect on mice treated with bleomycin. (A) Timeline of XFBD administration. (B) Body weight changes of mice in each group. (C) Survival rates of mice after different treatment. (D) Scanning of pulmonary fibrosis after BLM treatment at 10th day. (E) μCT score of pulmonary fibrosis. *p < 0.05, **p < 0.01, ***p < 0.001, vs. Model group. Scale bar = 100 μm.

Fig. 6

XFBD inhibits pathological changes of pulmonary fibrosis. (A) The histopathological changes of lung tissues were examined using H&E staining (magnification × 200) and (B) Alveolar structural changes of pulmonary vascular thickness and the value changes (magnification × 400). (C) Masson staining to assess the deposition of collagen in the lungs (Magnification 200 × ). The detect (D) and quantify (H) of α-SMA by Immunohistochemical staining. (E) The morphological damage score for the lung tissues. (F) Improvement of XFBD on vascular remodeling in pulmonary fibrosis mice. (G) The collagen deposition. (I) The level of Arg-1 in lung tissue. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, vs. control group, *p < 0.05, **p < 0.01, ***p < 0.001, vs. Model group. Scale bar = 100 μm.

Fig. 7

XFBD inhibits pulmonary fibrosis by down-regulating M2 polarization and IL-6/STAT3 pathway. (A, B, E) XFBD inhibits fibrosis-induced macrophage infiltration and M2 polarization in mouse lungs in vivo. (C, D, F) Representative microphotographs of immunohistochemical analysis for expression of IL-6 and STAT3 in lung tissue sections showing reduced immunopositivity upon XFBD treatment as compared to BLM alone treated lung sections (Magnification: 20 × ). (G, H) Quantitative analyses of mRNA of the IL-6 and STAT3 genes in mice. (I) Quantitative analyses of cytokine content of IL-6 by ELISA in mice serum and lung tissue. (J) Western blot result of the expression of total STAT3 in polarized macrophages group and XFBD group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, vs. control group, *p < 0.05, **p < 0.01, ***p < 0.001, vs. Model group. Scale bar = 100 μm.