Herbal formula BaWeiBaiDuSan alleviates polymicrobial sepsis-induced liver injury via increasing the gut microbiota Lactobacillus johnsonii and regulating macrophage anti-inflammatory activity in mice

Abstract

Sepsis-induced liver injury (SILI) is an important cause of septicemia deaths. BaWeiBaiDuSan (BWBDS) was extracted from a formula of Panax ginseng C. A. Meyer, Lilium brownie F. E. Brown ex Miellez var. viridulum Baker, Polygonatum sibiricum Delar. ex Redoute, Lonicera japonica Thunb., Hippophae rhamnoides Linn., Amygdalus Communis Vas, Platycodon grandiflorus (Jacq.) A. DC., and Cortex Phelloderdri. Herein, we investigated whether the BWBDS treatment could reverse SILI by the mechanism of modulating gut microbiota. BWBDS protected mice against SILI, which was associated with promoting macrophage anti-inflammatory activity and enhancing intestinal integrity. BWBDS selectively promoted the growth of Lactobacillus johnsonii (L. johnsonii) in cecal ligation and puncture treated mice. Fecal microbiota transplantation treatment indicated that gut bacteria correlated with sepsis and was required for BWBDS anti-sepsis effects. Notably, L. johnsonii significantly reduced SILI by promoting macrophage anti-inflammatory activity, increasing interleukin-10⁺ M2 macrophage production and enhancing intestinal integrity. Furthermore, heat inactivation L. johnsonii (HI-L. johnsonii) treatment promoted macrophage anti-inflammatory activity and alleviated SILI. Our findings revealed BWBDS and gut microbiota L. johnsonii as novel prebiotic and probiotic that may be used to treat SILI. The potential underlying mechanism was at least in part, via L. johnsonii-dependent immune regulation and interleukin-10⁺ M2 macrophage production.

Keywords: 16S PacBio SMRT sequencing; BaWeiBaiDuSan; Interleukin-10; Lactobacillus johnsonii; Macrophages; Network pharmacology; Sepsis-induced liver injury.

Graphical abstract

Figure 1

BWBDS protects mice against sepsis-induced liver injury. (A) Survival rate (n = 9–11). Serum levels of (B) IL-6, (C) IL-1β, (D) TNF-α and (E) IL-10 (n = 5). (F) mRNA levels of Il-6, Tnf-α, Il-1β, Ccl2, Ccl3, Ccl7, Cxcl1, and Cxcl10 in liver (n = 5). (G) H&E staining and (H) histological scores of livers to visualize the histomorphological features and quantitation analysis (n = 5). (I) The proportions of F4/80⁺CD11b⁺CD45⁺ macrophages in the liver (n = 5). (J, K) Serum transaminase levels ALT and AST (n = 5). Scale bar =100 μm. Data are expressed as mean ± SEM. ^####P < 0.0001 vs. Sham; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. CLP; ns, not significant.

Figure 2

BWBDS decreases the M1 macrophages population and increases the M2 macrophages population after CLP. (A) PMs, spleen and pPBMC were analyzed by flow cytometry for (F4/80⁺CD11b⁺CD45⁺) macrophages cells in mice in the sham group, CLP vehicle group and BWBDS + CLP group (n = 6). (B–F) M1 macrophages, M2 macrophages, expression of TNF-α, INOS among M1 macrophages and IL-10 among M2 macrophages in the PMs, spleen and pPBMC (n = 6). Each symbol represents an individual animal. Data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. CLP.

Figure 3

BWBDS-treated mice have unique intestinal microbiological composition. (A) Observed features in different groups (n = 8). (B) Shannon index in different groups (n = 8). (C) The level of Firmicutes in the feces (n = 8). (D) The Firmicutes/Bacteroidetes ratio in the feces (n = 8). (E) The level of Bacteroidetes in the feces (n = 8). (F) The level of Proteobacteria in the feces (n = 8). (G) Beta diversity analysis in different groups mice (n = 8). (H) Relative abundance of top 15 genera in different treatment groups (n = 8). (I) LEfSe analysis for differential abundant taxa detected between BWBDS + CLP group and CLP control group (n = 8). (J, K) The relative mRNA levels of Zo-1 and occludin in the colon (n = 5). Data are expressed as the mean ± SEM. ^####P < 0.0001 vs. Sham; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. CLP; ns, not significant.

Figure 4

Gut microbiota from BWBDS-treated mice can independently alleviate sepsis-induced liver damage. (A) Survival rate (n = 5). (B) Serum levels of IL-6, IL-1β, TNF-α and IL-10 in the feces recipient mice (n = 5). (C, D) H&E staining and histological scores of livers in different feces recipient mice (n = 5). (E) The proportions of F4/80⁺CD11b⁺CD45⁺ macrophages in the liver (n = 5). (F, G) Serum transaminase levels ALT and AST (n = 5). (H, I) The relative mRNA levels of Zo-1 and occludin in the colon (n = 5). (J) Beta diversity analysis (abund_jaccard) (n = 5). (K) Relative abundance of top 15 genera in different feces recipient mice (n = 5). (L) LEfSe analysis for differential abundant taxa detected between BWBDS feces recipient mice and CLP feces recipient mice (n = 5). Scale bar = 100 μm. Data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. CLP feces + CLP; ns, not significant.

Figure 5

L. johnsonii improves sepsis-induced liver injury. (A) The survival rates (n = 8). (B–E) Serum levels of IL-6, IL-1β, TNF-α, and IL-10 (n = 5). (F) mRNA levels of Il-6, Tnf-α, Il-1β, Ccl2, Ccl3, Ccl7, Cxcl1, Cxcl10 in liver (n = 5). (G, H) H&E staining and histological scores of livers (n = 5). (I) The proportions of F4/80⁺CD11b⁺CD45⁺ macrophages in the liver (n = 5). (J, K) Serum transaminase levels ALT and AST (n = 5). (L, M) The relative mRNA levels of Zo-1 and occludin in the colon were measured (n = 5). Scale bar = 100 μm. Data are expressed as the mean ± SEM. ^##P < 0.01, ^###P < 0.001 vs. Sham; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. CLP; ns, not significant.

Figure 6

L. johnsonii decreases the M1 macrophages population and increases the M2 macrophages population. (A) PMs, spleen and pPBMC macrophages were analyzed by flow cytometry for (F4/80⁺CD11b⁺CD45⁺) macrophages cells in different group mice (n = 5). (B–F) M1 macrophages, M2 macrophages, and expression of TNF-α, INOS among M1 macrophages and IL-10 among M2 macrophages in the PMs, spleen and pPBMC. Each symbol represents an individual animal (n = 5). Data are expressed as the mean ± SEM. ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. CLP.

Figure 7

L. johnsonii improves sepsis-induced liver injury dependent on the participation of macrophages. (A) PMs, spleen and pPBMC were analyzed by flow cytometry for (F4/80⁺CD11b⁺CD45⁺) cells in mice (n = 5). (B) mRNA levels of Il-6, Tnf-α, Il-1β, Ccl2, Ccl3, Ccl7, Cxcl1, Cxcl10 in liver (n = 5). (C, D) H&E staining and histological score of livers (n = 5). (E) The proportions of F4/80⁺CD11b⁺CD45⁺ macrophages in liver (n = 5). (F, G) Serum transaminase levels of ALT and AST (n = 5). (H, I) The relative mRNA levels of Zo-1 and occludin in the colon (n = 5). Scale bar = 100 μm. Data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001; ns, not significant.

Figure 8

L. johnsonii regulates M2 macrophages to alleviate sepsis-induced liver injury. (A) The proportions of IL-10⁺ M2 macrophages were analyzed by flow cytometry in PMs, spleen and pPBMC in group mice (n = 5). (B) The relative serum protein levels of IL-10 (n = 5). (C) Colonic mRNA levels of Il-10 (n = 5). (D) mRNA levels of Il-6, Tnf-α, Il-1β, Ccl2, Ccl3, Ccl7, Cxcl1, Cxcl10 in liver (n = 5). (E) The proportions of F4/80⁺CD11b⁺CD45⁺ macrophages in the liver (n = 5). (F, G) HE staining and the quantitation analysis of livers (n = 5). (H, I) Serum transaminase levels of ALT and AST (n = 5). (J, K) The relative mRNA levels of Zo-1 and occludin in the colon (n = 5). Scale bar = 100 μm. Data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001; ns, not significant.

Figure 9

HL-L. johnsonii can improve sepsis-induced liver injury. (A) The survival rates (n = 8). (B–E) Serum levels of IL-6, IL-1β, TNF-α, and IL-10 (n = 5). (F, G) Serum transaminase levels ALT and AST (n = 5). (H, I) The relative mRNA levels of Zo-1 and occludin in the colon were measured (n = 5). (J, K) The proportion of total macrophages and IL-10⁺ M2 macrophages in liver were analyzed by flow cytometry in liver (n = 5). Data are expressed as the mean ± SEM. ^###P < 0.001 vs. Sham; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. CLP; ns, not significant.