Cigarette smoking causes persistent lung inflammation that is mainly regulated by redox-sensitive pathways. We have previously reported that cigarette smoke (CS) activates reactive oxygen species- (ROS-) sensitive mitogen-activated protein kinases (MAPKs)/nuclear factor-κB (NF-κB) signaling leading to induction of lung inflammation. Paeonol, the main phenolic compound present in the Chinese herb Paeonia suffruticosa, has antioxidant and anti-inflammatory properties. However, whether paeonol has similar beneficial effects against CS-induced lung inflammation remains unclear. Using a murine model, we showed that chronic CS exposure for 4 weeks caused pulmonary inflammatory infiltration, increased lung vascular permeability, elevated lung levels of chemokines, cytokines, and 4-hydroxynonenal (an oxidative stress biomarker), and induced lung inflammation; all of these CS-induced events were suppressed by chronic treatment with paeonol. Using human bronchial epithelial cells (HBECs), we demonstrated that cigarette smoke extract (CSE) sequentially increased extracellular and intracellular levels of ROS, activated the MAPKs/NF-κB signaling, and induced interleukin-8 (IL-8); all these CSE-induced events were inhibited by paeonol pretreatment. Our findings suggest a novel role for paeonol in alleviating the oxidative stress and lung inflammation induced by chronic CS exposure in vivo and in suppressing CSE-induced IL-8 in vitro via its antioxidant function and an inhibition of the MAPKs/NF-κB signaling.
The mechanism underlying the inflammatory role of TRPA1 in lung epithelial cells (LECs) remains unclear. Here, we show that cigarette smoke extract (CSE) sequentially induced several events in LECs. The Ca2+ influx was prevented by decreasing extracellular reactive oxygen species (ROS) with the scavenger N-acetyl-cysteine, removing extracellular Ca2+ with the chelator EGTA, or treating with the TRPA1 antagonist HC030031. NADPH oxidase activation was abolished by its inhibitor apocynin, EGTA, or HC030031. The increased intracellular ROS was halted by apocynin, N-acetyl-cysteine, or HC030031. The activation of the MAPKs/NF-κB signaling was suppressed by EGTA, N-acetyl-cysteine, or HC030031. IL-8 induction was inhibited by HC030031 or TRPA1 siRNA. Additionally, chronic cigarette smoke (CS) exposure in wild-type mice induced TRPA1 expression in LECs and lung tissues. In CS-exposure trpa1
−/− mice, the increased BALF level of ROS was similar to that of CS-exposure wild-type mice; yet lung inflammation was lessened. Thus, in LECs, CSE may initially increase extracellular ROS, which activate TRPA1 leading to an increase in Ca2+ influx. The increased intracellular Ca2+ contributes to activation of NADPH oxidase, resulting in increased intracellular ROS, which activate the MAPKs/NF-κB signaling leading to IL-8 induction. This mechanism may possibly be at work in mice chronically exposed to CS.
Clinical studies suggest that smokers with chronic obstructive pulmonary disease who use menthol cigarettes may display more severe lung inflammation than those who smoke non-menthol cigarette. However, the mechanisms for this difference remain unclear. Menthol is a ligand of transient receptor potential melastatin-8 (TRPM8), a Ca2+-permeant channel sensitive to reactive oxygen species (ROS). We previously reported that exposure of human bronchial epithelial cells (HBECs) to non-menthol cigarette smoke extract (Non-M-CSE) triggers a cascade of inflammatory signaling leading to IL-8 induction. In this study, we used this in vitro model to compare the inflammatory effects of menthol cigarette smoke extract (M-CSE) and Non-M-CSE and delineate the mechanisms underlying the differences in their impacts. Compared with Non-M-CSE, M-CSE initially increased a similar level of extracellular ROS, suggesting the equivalent oxidant potency. However, M-CSE subsequently produced more remarkable elevations in intracellular Ca2+, activation of the mitogen-activated protein kinases (MAPKs)/nuclear factor-κB (NF-κB) signaling, and IL-8 induction. The extracellular ROS responses to both CSE types were totally inhibited by N-acetyl-cysteine (NAC; a ROS scavenger). The intracellular Ca2+ responses to both CSE types were also totally prevented by NAC, AMTB (a TRPM8 antagonist), or EGTA (an extracellular Ca2+ chelator). The activation of the MAPK/NF-κB signaling and induction of IL-8 to both CSE types were suppressed to similar levels by NAC, AMTB, or EGTA. These results suggest that, in addition to ROS generated by both CSE types, the menthol in M-CSE may act as another stimulus to further activate TRPM8 and induce the observed responses. We also found that menthol combined with Non-M-CSE induced greater responses of intracellular Ca2+ and IL-8 compared with Non-M-CSE alone. Moreover, we confirmed the essential role of TRPM8 in these responses to Non-M-CSE or M-CSE and the difference in these responses between the both CSE types using HBECs with TRPM8 knockdown and TRPM8 knockout, and using HEK293 cells transfected with hTRPM8. Thus, compared with exposure to Non-M-CSE, exposure to M-CSE induced greater TRPM8-mediated inflammatory responses in HBECs. These augmented effects may be due to a double-hit on lung epithelial TRPM8 by ROS generated from CSE and the menthol in M-CSE.
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