Medicinal Foods, YT and RH Combination, Suppress Cigarette Smoke-Induced Inflammation and Oxidative Stress by Inhibiting NF- κ B/ERK Signaling Pathways

Abstract

Background: Cigarette smoke is a risk factor for Chronic Obstructive Pulmonary Disease (COPD). Given the lack of COPD curative treatment, dietary management for COPD patients has become important. This study investigated whether the medicinal foods (YT and RH) could suppress cigarette smoke exposure-induced inflammation and oxidative stress.

Methods: Chronic pulmonary inflammation in male C57 mice was induced by a 4-week exposure to cigarette smoke (CS). The medicinal foods YT and RH were orally administered 1 week prior to CS exposure. The protective effects were assessed by measuring the pulmonary function and histopathological evaluations. Inflammatory cell numbers and cytokines levels in BALF and blood serum were analyzed by enzyme-linked immunosorbent assay (ELISA). Malondialdehyde (MDA) and superoxide dismutase (SOD) levels of the lung were analyzed. Furthermore, the levels of phosphorylated ERK and NF-κB in both the mice lungs and RAW264.7 cells were also detected.

Results: YT and RH combination (YT + RH) significantly improved pulmonary function and suppressed the inflammation, including cell number and cytokines in BALF relative to the CS group; histological examination revealed protective effects of YT + RH in the lungs of mice exposed to CS. Moreover, the MDA level in the lung of the YT + RH group of mice was lower, the SOD activity was higher, and in vitro treatment of YT and RH combination attenuated reactive oxygen species (ROS) expression in mouse macrophage RAW264.7 cells stimulated with cigarette smoke (CSE). YT + RH combination significantly reduced the expression of pNF-κB and pERK in the lung tissues and macrophage stimulated with CSE.

Conclusions: YT and RH combination attenuates cigarette smoke-induced inflammation and oxidative stress through inhibition of the NF-κB/ERK signaling pathway.

Figure 1

YT + RH improves pulmonary function and emphysema in CS-exposed mice. (a) Mice were exposed to CS for 4 weeks and pretreated. (b, c) The pulmonary function of mice was measured. (d) Pathological changes in the lungs of mice (H&E stained). Scale bar = 50 μm, n = 6 in each group. The values are presented as the mean ± SD. ^#P＜0.05 and ^###P ＜ 0.001 versus the Blank group;  ^∗P ＜ 0.05 versus the CS group. YT + RH reduces CS-induced inflammation in mice.

Figure 2

YT + RH reduces CS-induced inflammation in mice. (a) The inflammation cells differentials in BALF were stained with diff-quick staining. (b–d) The total counts of cells, macrophages, and neutrophils from the BALF were counted. (e–j) Inflammatory cytokines were detected by ELISA. n = 7 in the Blank group, CS group, YT group, and YT + RH group, n = 6 in RH group, and n = 5 in SCMC group. The values are presented as the mean ± SD. ^##P＜0.01 and ^###P＜0.001 versus the Blank group;  ^∗P ＜ 0.05,  ^∗∗P ＜ 0.01, and  ^∗∗∗P ＜ 0.001 versus the CS group.

Figure 3

YT + RH attenuates CS-induced oxidative stress in mice. (a, b) SOD activity and MDA levels in the lungs of mice MDA and SOD activity of lungs were measured. (c) HO1 expression of the lung was measured by WB, n = 5 in each group. ^##P ＜ 0.01 and ^###P ＜ 0.001 versus the Blank group;  ^∗P ＜ 0.05, ^∗∗P ＜ 0.01, and  ^∗∗∗P ＜ 0.001 versus the CS group.

Figure 4

YT + RH attenuates CSE-induced oxidative stress in macrophages. (a) HO1 expression of the lung was measured by WB. (b) ROS production in RAW267.4 cell was analyzed by fluorescence microscopy. The values are presented as the mean ± SD of five individual experiments. ^##P＜0.01 and ^###P＜0.001 versus the Blank group;  ^∗P＜0.05, ^∗∗P＜0.01, and  ^∗∗∗P＜0.001 versus the CS group.

Figure 5

YT + RH treatment inhibits CS-induced activation of pERK1/2 and pNF-κB in vivo. (a) The expression of pNF-κB in the lungs was detected by immunohistochemical experiments. (b) The expression of phosphorylation ERK of the lung was detected by WB. The values are presented as mean ± SD of five individual experiments. ^##P ＜ 0.01 and ^###P ＜ 0.001 versus the Blank group;  ^∗P ＜ 0.05, ^∗∗P ＜ 0.01, and  ^∗∗∗P ＜ 0.001 versus the CS group.

Figure 6

YT + RH treatment inhibits CS-induced activation of pNF-κB in vitro. (a) The phosphorylation NF-κB expression of cell was detected by WB. (b) The phosphorylation NF-κB expression of cell was detected by IF. The values are presented as the mean ± SD of five individual experiments. ^#P ＜ 0.05 versus the Blank group;  ^∗P ＜ 0.05 versus the CS group.

Figure 7

YT + RH treatment inhibits CS-induced phosphorylation of ERK in vitro. (a) The phosphorylation ERK expression of cell was detected by WB. (b) The phosphorylated ERK expression of cell was detected by IF. The values are presented as the mean ± SD of five individual experiments. ^#P ＜ 0.05 versus the Blank group;  ^∗P ＜ 0.05 versus the CS group.