Alantolactone suppresses inflammation, apoptosis and oxidative stress in cigarette smoke-induced human bronchial epithelial cells through activation of Nrf2/HO-1 and inhibition of the NF-κB pathways

Abstract

Background: It is well established that airway remodeling and inflammation are characteristics for chronic obstructive pulmonary disease (COPD). Moreover, cigarette smoke extract (CSE) promots inflammation, apoptosis and oxidative stress in COPD. And, there is evidence suggested that alantolactone (ALT), a sesquiterpene lactone isolated from Inula helenium, plays an adverse role in inflammation, apoptosis and oxidative stress. However, few studies have investigated the function and mechanism of ALT treatment on the COPD pathological process.

Methods: The levels of IL-1 β, TNF-α, IL-6 and IFN-γ were examined by ELISA. Cells' apoptosis and caspase-3 activity were detected by Cell Death Detection PLUS enzyme-linked immunosorbent assay and caspase-Glo 3/7 Assay, respectively. The content of malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by using MDA and SOD assay kits. Reactive oxygen species (ROS) generation was measured by DCFH-DA assay. Protein expression was assayed by Western blot.

Results: In the present study, we aimed to observe the protective effects of ALT against inflammation, apoptosis and oxidative stress in human bronchial epithelial Beas-2B and NHBE cells. Our results showed that different doses of CSE exposure induced Beas-2B and NHBE cell inflammatory cytokines IL-1 β, TNF-α, IL-6 and IFN-γ expression, cell apoptosis, caspase-3 activity and mediated oxidative stress markers MDA, ROS and SOD levels, while ALT treatment counteracted the effects of CSE. Further studies suggested that ALT attenuated NF-κB pathway activation. ALT also activated the Nrf2/HO-1 signal pathway through promoting Nrf2 nuclear aggregation and downstream HO-1 protein expression. HO-1 inhibitor tin protoporphyrin IX (SnPP IX) reversed the effects of ALT on Beas-2B and NHBE cell inflammation, apoptosis and oxidative stress.

Conclusions: The above results collectively suggested that ALT suppressed CSE-induced inflammation, apoptosis and oxidative stress by modulating the NF-ĸB and Nrf2/ HO-1 axis.

Keywords: Alantolactone; Apoptosis; Chronic obstructive pulmonary disease; Cigarette smoke extract; Inflammation; Oxidative stress.

Fig. 1

The chemical structure of alantolactone

Fig. 2

CSE promoted IL-1 β, TNF-α, IL-6 and IFN-γ production in Beas-2B and NHBE cells. a CSE increased Beas-2B cell inflammatory response; b CSE increased NHBE cell inflammatory response. Beas-2B and NHBE cells were treatment with 1, 2 and 5% CSE for 24 h. Data were expressed as mean ± S.D. from three independent experiments. ^*P values < 0.05 compared with the control group. CSE: cigarette smoke extract

Fig. 3

ALT inhibited CSE induced - IL-1 β, TNF-α, IL-6 and IFN-γ production in Beas-2B and NHBE cells. a ALT inhibited CSE induced inflammatory response in Beas-2B cells; b ALT inhibited CSE induced inflammatory response in NHBE cells. Beas-2B and NHBE cells were pre-treated with 1, 5 and 10 μM ALT for 2 h and then administrated with 5% CSE for 24 h. The commercially available ELISA kits were used to measure the level of IL-1 β, TNF-α, IL-6 and IFN-γ. Data were expressed as mean ± S.D. from three independent experiments. ^*P values < 0.05 compared with the control group, ^#P values < 0.05 compared with the CSE group. CSE: cigarette smoke extract; ALT: Alantolactone

Fig. 4

ALT inhibited CSE induced-LDH activity and apoptosis in Beas-2B a and NHBE b cells. LDH activity was measured using an LDH-cytotoxicity Kit; Cell Death Detection PLUS ELISA assay was used to detect cell apoptosis; Caspase-3 activity was measured by using Caspase-Glo 3/7 assay. Beas-2B and NHBE cells were pre-treated with 1, 5 and 10 μM ALT for 2 h and then administrated with 5% CSE for 24 h. Data were expressed as mean ± S.D. from three independent experiments. ^*P values < 0.05 compared with the control group, ^#P values < 0.05 compared with the CSE group. CSE: cigarette smoke extract; ALT: Alantolactone

Fig. 5

ALT inhibited CSE induced -oxidative stress in Beas-2B a and NHBE b cells. MDA assay kits was used to measure MDA content; SOD assay kits was used to measure SOD content; DCFH-DA method was used to measure ROS production. Beas-2B and NHBE cells were pre-treated with 1, 5 and 10 μM ALT for 2 h and then administrated with 5% CSE for 24 h. Data were expressed as mean ± S.D. from three independent experiments. ^*P values < 0.05 compared with the control group, ^#P values < 0.05 compared with the CSE group. CSE: cigarette smoke extract; ALT: Alantolactone

Fig. 6

ALT inhibited CSE induced - NF-κB pathways activation in Beas-2B and NHBE cells. Western blot was used to determine protein expression of p-p65 and total p65. Beas-2B and NHBE cells were pre-treated with 5 μM ALT for 2 h and then administrated with 5% CSE for 24 h. Data were expressed as mean ± S.D. from three independent experiments. ^*P values < 0.05 compared with the control group, ^#P values < 0.05 compared with the CSE group. CSE: cigarette smoke extract; ALT: Alantolactone

Fig. 7

ALT promoted Nrf2/HO-1 activation in CSE induced Beas-2B and NHBE cells. Western blot was used to determine protein expression of Nrf2 and HO-1. Beas-2B and NHBE cells were pre-treated with 5 μM ALT for 2 h and then administrated with 5% CSE for 24 h. Data were expressed as mean ± S.D. from three independent experiments. ^*P values < 0.05 compared with the control group, ^#P values < 0.05 compared with the CSE group. CSE: cigarette smoke extract; ALT: Alantolactone

Fig. 8

HO-1 inhibitor SnPP reversed the effects of ALT on CSE induced Beas-2B cells. a The commercially available ELISA kits were used to measure the level of TNF-α and IFN-γ; b Cell Death Detection PLUS ELISA assay was used to detect cell apoptosis; c DCFH-DA method was used to measure ROS production. Beas-2B cells were pre-treated with 5 μM ALT and SnPP (20 μM) for 2 h and then administrated with 5% CSE for 24 h. Data were expressed as mean ± S.D. from three independent experiments. ^*P values < 0.05 compared with the control group, ^#P values < 0.05 compared with the CSE group. CSE: cigarette smoke extract; ALT: Alantolactone

Fig. 9

A schematic diagram of function and mechnism of ALT in CSE-induced human bronchial epithelial cells