Bulb of Lilium longiflorum Thunb Extract Fermented with Lactobacillus acidophilus Reduces Inflammation in a Chronic Obstructive Pulmonary Disease Model

Abstract

Chronic obstructive pulmonary disease (COPD), one of the leading causes of death worldwide, is caused by repeated exposure to harmful matter, such as cigarette smoke. Although Lilium longiflorum Thunb (LLT) has anti-inflammatory effects, there is no report on the fermented LLT bulb extract regulating lung inflammation in COPD. Thus, we investigated the protective effect of LLT bulb extract fermented with Lactobacillus acidophilus 803 in COPD mouse models induced by cigarette smoke extract (CSE) and porcine pancreas elastase (PPE). Oral administration of the fermented product (LS803) suppressed the production of inflammatory mediators and the infiltration of immune cells involving neutrophils and macrophages, resulting in protective effects against lung damage. In addition, LS803 inhibited CSE- and LPS-induced IL-6 and IL-8 production in airway epithelial H292 cells as well as suppressed PMA-induced formation of neutrophil extracellular traps in HL-60 cells. In particular, LS803 significantly repressed the elevated IL-6 and MIP-2 production after CSE and LPS stimulation by suppressing the activity of the nuclear factor kappa-light-chain-enhancer of activated B (NFκB) in mouse peritoneal macrophages. Therefore, our results suggest that the fermented product LS803 is effective in preventing and alleviating lung inflammation.

Keywords: Lactobacillus acidophilus; Lilium longiflorum Thunb bulb; anti-inflammation; chronic obstructive pulmonary disease (COPD).

Fig. 1. Effect of LS803 on the BALF from the COPD model.

(A) Schematic of the CSE- and PPE-induced COPD model timeline. (B) The inflammatory cell infiltration was measured using Diff-Quik staining methods (scale bar: 50 μm). (C) Total cell, macrophage, and neutrophil numbers in the BALF. The number of immune cells was estimated using microscopy images. (D) The levels of cytokine and chemokines were measured using a Q-Plex Assay Kit (n = 5). The data were analyzed using one-way analysis of variance (ANOVA) and presented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001). LLT, bulbs of Lilium longiflorum Thunb (the raw material before fermentation); LS803, fermented material.

Fig. 2. Effect of LS803 on hematological parameters in the COPD model.

(A) The numbers of neutrophils, lymphocytes, monocytes, and eosinophils in the blood were estimated. (B) Immunoglobulin A (IgA) and G (IgG) levels in the serum were measured. Data were analyzed using one-way ANOVA and presented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001). LLT, bulbs of Lilium longiflorum Thunb (the raw material before fermentation); LS803, fermented material.

Fig. 3. Effect of LS803 on lung destruction and levels of cytokine and chemokines in lung tissues from COPD mice.

Lung tissues from each group were collected and used to assess lung destruction and inflammation. (A) Lung damage was confirmed using hematoxylin and eosin staining, followed by observation under a microscope (scale bar: 100 μm). (B) The mRNA expression levels of inflammation-related markers were measured using quantitative real-time polymerase chain reaction. The data were analyzed using one-way ANOVA. All values have been reported as mean ± SD. (*p < 0.05, **p < 0.01, and ***p < 0.001). LLT, bulbs of Lilium longiflorum Thunb (the raw material before fermentation); LS803, fermented material.

Fig. 4. Effect of LS803 on inflammation and NETosis.

(A) H292 cells were co-treated with 200 μg/ml LLT, 2% CSE and 100 ng/ml LPS for 24 h, after which the levels of human IL-6 and IL-8 in the culture supernatants were measured using ELISA. (B) HL-60 cells were co-treated with 200 μg/ml LLT and 100 nM PMA for 3 h, following which the cells were stained with CYTOX Green to assess NETosis. (C) Intraperitoneal macrophages were stimulated with CSE and LPS and the expression levels of IL-6 and MIP-2 and NFκB inhibition ability were evaluated. Data were analyzed using one-way ANOVA. All values have been reported as mean ± SD. LLT, bulbs of Lilium longiflorum Thunb (the raw material before fermentation); LS803, fermented material.