Jinhong decoction protects sepsis-associated acute lung injury by reducing intestinal bacterial translocation and improving gut microbial homeostasis

Abstract

Background: Currently no specific treatments are available for sepsis and the associated syndromes including acute lung injury (ALI). Jinhong Decoction (JHD) is a traditional Chinese prescription, and it has been applied clinically as an efficient and safe treatment for sepsis, but the underlying mechanism remains unknown. The aim of the study was to explore the potential mechanisms of JHD ameliorating sepsis and concurrent ALI. Methods: The cecum ligation puncture (CLP)- induced murine sepsis model was established for determining the efficacy of JHD protecting CLP and ALI. The role of gut microbiota involved in the efficacy of JHD was evaluated by 16S rRNA sequencing and fecal microbiota transplantation (FMT). Translocation of intestinal Escherichia coli (E. coli) to lungs after CLP was verified by qPCR and in vivo-imaging. Intestinal permeability was analyzed by detecting FITC-dextran leakness. Junction proteins were evaluated by Western blotting and immunofluorescence. Results: JHD treatment remarkably increased survival rate of septic mice and alleviated sepsis-associated lung inflammation and injury. FMT suggested that the protective role for JHD was mediated through the regulation of gut microbiota. We further revealed that JHD administration partially restored the diversity and configuration of microbiome that was distorted by CLP operation. Of interest, the intestinal bacteria, E. coli particularly, was found to translocate into the lungs upon CLP via disrupting the intestinal mucosal barrier, leading to the inflammatory response and tissue damage in lungs. JHD impeded the migration and hence lung accumulation of intestinal E. coli, and thereby prevented severe ALI associated with sepsis. This effect is causatively related with the ability of JHD to restore intestinal barrier by up-regulating tight junctions. Conclusion: Our study unveils a mechanism whereby the migration of gut bacteria leads to sepsis-associated ALI, and we demonstrate the potential of JHD as an effective strategy to block this bacterial migration for treating sepsis and the associated immunopathology in the distal organs.

Keywords: JHD; acute lung injury; bacterial translocation; gut microbiome; sepsis.

FIGURE 1

JHD attenuated symptoms of CLP-induced sepsis in mice. (A) Flow chart of model establishment and JHD administration; Sepsis-characterized symptoms including (B) body temperature, (C) body weight, (D) food intake, and (E) water intake; Serum level of (F) ALT and (G) AST; (H) Bacterial load in circulation; (I) Survival rates. (mean ± SD, n = 5; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).

FIGURE 2

JHD alleviates sepsis-induced lung injury. (A) H&E staining of lung tissues and the correlated pathological scores (magnification: ×400, scale bar = 200 μm); (B) Total cells infiltrated and (C) total protein leaked in BALF; (D) MPO activity in lung tissue; (E) Bacterial loads in lung tissues; (F) qPCR assay of Il6, Il1β, Il17a and Tnf levels in lung tissues; (G) ELISA assay of IL-6, IL-1β, IL-17A and TNF-α levels in lung homogenate; Flow cytometry analysis of (H) CD11b⁺Ly6G⁺ neutrophils and (I) CD3ε⁺TCRγδ⁺ γδ T cells in BALF (%). (mean ± SD, n = 5–6;^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).

FIGURE 3

JHD modulates the composition of intestinal microbiome in sepsis mice. (A) Principal Component Analysis (PCA) and Principal Co-ordinates Analysis (PCoA) of gut microbiome. Orange, red and blue represents the sham (S), CLP (C) and CLP + JHD (J) groups, respectively; (B) Linear discriminant analysis Effect Size (LefSE) analysis. Deep blue, red and green represents the sham (S), CLP (C) and CLP + JHD (J) groups, respectively; (C) The composition of gut microbiome at phylum level, and (D) the representative bacteria with significant changes between the indicated groups (Bacteroidota, Firmicutes and Proteobacteria). (E) The composition of gut microbiome at genus level, and (F) the representative bacteria with marked changes (Bacteroides, Citrobacter, Escherichia-Shigella, Enterococcus); (G) The composition of gut microbiome at species level, and (H) the representative bacteria with significant change (Bacteroides, Citrobacter, Escherichia coli and Enterococcus faecalis). (mean ± SD, n = 5; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).

FIGURE 4

JHD confers the protective effect on sepsis-associated ALI via FMT. (A) Experimental flow chart; (B) Body weight, food consumption and water intake; and (C) Survival rates of mice with different FMT; (D) Bacterial load in lungs; (E) Total cell counts, and (F) protein leakage in BALF; (G) MPO activity in lungs. (H) H&E staining of lung sections and correlated pathological scores (magnification: ×400, scale bar = 200 μm); (I) ELISA assays of the levels of inflammatory cytokines in lung homogenates; (J) CD3ε⁺TCRγδ⁺ γδT (%) and IL-17A⁺ γδT (%) in BALF were analyzed by flow cytometry. (mean ± SD, n = 6, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).

FIGURE 5

Sepsis causes impaired intestinal barrier and gut-lung translocation of E. coli to induce lung pathology. Intestinal permeability was determined by measuring (A) fluorescence intensity of FITC-dextran and (B) serum FITC-dextran concentration. (C) H&E staining of colon tissues and correlated pathological scores (magnification: ×400, scale bar = 200 μm); (D) Immunoblotting of ZO-1, Occludin and Muc-1; (E) immunofluorescent staining of ZO-1 and Muc-1; (F) Relative abundance of (E) coli in intestinal contents and lung tissues; (G) DiL-labeled E. coli was given rectally to mice (10⁶ CFU per mouse), and 2 h later, the mice were subjected to CLP; (H) Frozen section of lung tissues was detected for DiL-labeled E. coli; (I) H&E staining of lungs and correlated pathological scores (magnification: ×400, scale bar = 200 μm) (mean ± SD, n = 5 or 6; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).

FIGURE 6

JHD improves intestinal mucosal barrier and prevents gut bacterial translocation for inducting lung inflammation. (A) Relative abundance of E. coli in intestinal contents and lung tissues; (B)Intestinal barrier permeability measured by in vivo imaging of FITC-dextran; (C) H&E staining of colon tissues and correlated histological analysis (magnification: ×400, scale bar = 200 μm); ZO-1 and Muc-1 in colon tissues were determined by (D) Western blotting and (E) immunofluorescence; (F) Experimental flow chart; (G) DiL-labeled E. coli in frozen lung sections; (H) H&E staining of lung and correlated pathological scores (magnification: ×400, scale bar = 200 μm) (mean ± SD, n = 6;^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).