Loranthus tanakae Franch. & Sav. Suppresses Inflammatory Response in Cigarette Smoke Condensate Exposed Bronchial Epithelial Cells and Mice

Abstract

Loranthus tanakae Franch. & Sav. found in China, Japan, and Korea is traditionally used for managing arthritis and respiratory diseases. In this study, we analyzed the components of L. tanakae 70% ethanol extract (LTE) and investigated the therapeutic effects of LTE on pulmonary inflammation using cells exposed to cigarette smoke condensate (CSC) and lipopolysaccharide (LPS) in vitro and in vivo in mice and performed a network analysis between components and genes based on a public database. We detected quercitrin, afzelin, rhamnetin 3-rhamnoside, and rhamnocitrin 3-rhamnoside in LTE, which induced a significant reduction in inflammatory mediators including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and inflammatory cells in CSC exposed H292 cells and in mice, accompanied by a reduction in inflammatory cell infiltration into lung tissue. In addition, LTE increased translocation into the nuclei of nuclear factor erythroid-2-related factor 2 (Nrf2). By contrast, the activation of nuclear factor (NF)-κB, induced by CSC exposure, decreased after LTE application. These results were consistent with the network pharmacological analysis. In conclusion, LTE effectively attenuated pulmonary inflammation caused by CSC+LPS exposure, which was closely involved in the enhancement of Nrf2 expression and suppression of NF-κB activation. Therefore, LTE may be a potential treatment option for pulmonary inflammatory diseases including chronic obstructive pulmonary disease (COPD).

Keywords: Loranthus tanakae Franch. & Sav.; NF-κB; Nrf2; chronic obstructive pulmonary disease; cigarette smoke condensate.

Figure 1

Analysis of ingredients of LTE. (a) HPLC chromatogram of LTE and active molecules at 254 nm, (b) verification of four main peaks comparing λ_max, m/z for [M−H]⁻; quercitrin (purple, λ_max = 255.8, m/z = 447.1), afzelin (olive green, λ_max = 262.9, m/z = 431.2), rhamnetin 3-rhamnoside (pink, λ_max = 253.4, m/z = 461.2), and rhamnocitrin (orange, λ_max = 264.1, m/z = 445.2), and (c) overlay of four SIR peaks in LTE; quercitrin (purples; 14.265 min, m/z = 447.15), afzelin (olive green, 17.037 min, m/z = 431.18), rhamnetin 5-rhamnoside (pink, 25.229 min, m/z = 461.15), and rhamnocitrin (orange, 29.953 min, m/z = 445.18).

Figure 2

Network of four molecules (pink oval) and 70 potential target genes (cyan rectangles); this network consisted of 74 nodes and 160 edges.

Figure 3

Topology analysis of PPI related COPD disease: the higher degree genes are expressed as large circles and in orange color.

Figure 4

Network pharmacological analysis of LTE. (a) Top 10 KEGG pathways related to COPD with p-value, (b) group of COPD-related KEGG pathways; signal transduction and immune system were potential COPD-related pathways. (c) Network consisting of potential target genes (cyan rectangle) and KEGG pathways (orange triangles) with 61 nodes and 155 edges.

Figure 5

Effects of LTE on the production of inflammatory cytokines in CSC stimulated H292 cells. (a) cell viability, (b) IL-1β level, and (c) IL-6 level. This experiment was performed in triplicate. Data shown as the mean ± SD. ^##, vs. Control, p < 0.01, *, **, vs. CSC stimulated cells, p < 0.05 and <0.01, respectively.

Figure 6

Effects of LTE on Nrf2 and NF-κB expression in CSC stimulated H292 cells. (a) representative figure for Nrf2, (b) representative figure for NF-κB, (c) quantitative analysis of Nrf2 expression, (d) quantitative analysis of NF-κB. The treatment of LTE increased Nrf2 translocation into the nucleus but decreased NF-κB translocation into the nucleus in CSC stimulated H292 cells. This experiment was performed in triplicate. Scale bar = 50 μm. Data shown as the mean ± SD. ^#, ^##, vs. Control, p < 0.05 and <0.01, respectively, *, vs. CSC-treated cells, p < 0.05.

Figure 7

Effects of rhamnetin 3-ramnoside and quercitrin on ROS production. (a) representative figure of DCFDA stained HeLa cells, (b) quantitative analysis of ROS production in rhamnetin 3-rhamnoside-treated cells, (c) quantitative analysis of ROS production in quercitrin-treated cells. rhamnetin 3-ramnoside and quercitrin-treated cells exhibited marked reduction in ROS production induced by H₂O₂ treatment. This experiment was performed in triplicate. Scale bar = 50 μm. Data shown as the mean ± SD. ^##, vs. Control, p < 0.01, **, vs. H₂O₂-treated cells, p < 0.01.

Figure 8

Effects of LTE on pathophysiological factors in CSC+LPS exposed mice. (a) Total cells in BALF, (b) neutrophils in BALF, (c) macrophages in BALF, (d) IL-1β levels in BALF, (e) IL-6 levels in BALF, and (f) TNF-α levels in BALF. ^##, vs. NC, p < 0.01, *, **, vs. CSC+LPS, p < 0.05 and <0.01, respectively.

Figure 9

Effects of LTE on histological alteration in CSC+LPS exposed mice. (a) representative figure for H&E, PAS and ICH. (b) inflammatory index, (c) mucus production index, (d) Nrf2 expression value, (e) NF-κB expression value. Pulmonary inflammation and mucus production of lung tissue were determined using hematoxylin and eosin and PAS staining, respectively. The expression of Nrf2 and NF-κB on lung tissue was determined using immunohistochemistry. Scale bar = 100 μm. ^##, vs. NC, p < 0.01, *, **, vs. CSC+LPS, p < 0.05 and <0.01, respectively.