Apple Polyphenol Protects Against Cigarette Smoke-induced Acute Lung Injury And The Possible Signaling Pathway

Objective:Chronic obstructive pulmonary disease (COPD) is a complex chronic inflammatory disease involving oxidative stress as well as a wide variety of cells activation from smoking cigarette. There have been disappointingly only few therapeutic advances in the drug therapy of COPD. Plant polyphenols have been the topic of much research on their antioxidant activities, anti-inflammatory and immunomodulatroy effects. Furthermore, studies suggest that reactive oxygen species (ROS) mediate upregulation of TNF-alpha (TNF-α) expression by inhibiting MAPK pathways. In the present study, we ask whether the polyphenols from apple provides protection against cigarette smoke (CS)-induced acute lung injury, and investigate the possible signaling pathway of APP effects in CSE-induced NCI-H292cells.Methods:ICR mice were exposed to CS for four days with increasing exposure time for up to6h per day to elicit epithelial cells injury. One hour before smoke exposure, mice were treated with apple polyphenol (APP) by gavage;18h after the last CS exposure all examinations were performed. In vitro, cell viability was measured by using the MTT assay. Protein expressions of phospho-P38, ERK1/2, JNK were measured by Western Blot. Tumor necrosis factor-α (TNF-α) was measured by ELISA GSH levels as well as ROS levels were also measured in NCI-H292cells induced by CSE.Results:Eighteen hours after last CS-expose, mice were harvested for preparation of BALF, and the inflammatory cell counting and their difference were performed. We found total cell number in the BALF of vehicle group was greatly higher than in that of control group, neutrophils and macrophages, but not lymphocytes in the BALF of vehicle group was significantly higher than that in control group. Among these inflammatory cells, neutrophil numbers were predominately elevated in vehicle group as compared with macrophage and lymphocyte numbers. However, treatment with APP at30,100or300mg/kg, significantly reduced the numbers of total inflammatory cells as compared to the vehicle treatment, and the maximal effects were achieved at the dose of300mg/kg. APP at100mg/kg was as comparable as dexamethasone at1mg/kg and resveratrol at30mg/kg treatment. Furthermore, APP at30,100or300mg/kg significantly reduced the accumulation of neutrophil as well as macrophage in BALF as compared with the vehicle treatment, and the maximal inhibitory effects of APP on neutrophil and macrophage accumulation were achieved at the dose of300mg/kg. APP at100mg/kg was as comparable as dexamethasone at1mg/kg and resveratrol at30mg/kg treatment.To evaluate the histological changes after APP treatment in CS-treated mice, lung sections obtained18h after last CS-expose were subjected to H&E staining, in vehicle group, mice exhibited marked increases of inflammatory cell infiltration, predominately including macrophages, neutrophils and lymphocytes, especially, CS-expose instillation in vehicle group led to a marked infiltration of macrophages into the lung interstitium and alveolar spaces, alveolar wall thickening, intra-alveolar exudation and interstitial edema as compared with control group mice in which vehicle instillation caused no obviouse histological changes. However, in CS-exposed mice, APP treatment at the dose of300mg/kg markedly attenuated the inflammatory cell infiltration (especially macrophages), and improved alveolar wall thickening, intra-alveolar exudation and interstitial edema as compared with vehicle treatment in CS-exposed mice, which was comparable with those of dexamethasone treatment at dose of1mg/kg. To investigate the effects of APP on CS-induced pro-inflammatory factor mRNA expression, TNF-α and IL-1β mRNA expression levels in lungs were measured by RT-PCR. CXCL-1and MCP-1mRNA expression levels in lungs were measured by QRT-PCR. All TNF-α, IL-1β, CXCL-1and MCP-1mRNA expression levels were determined18h after last CS-expose. All pro-inflammatory factors were predominately elevated in vehicle group as compared with control. However, treatment with APP at30,100or300mg/kg significantly reduced the numbers of total inflammatory cells as compared to the vehicle treatment, and the maximal effects were achieved at the dose of300mg/kg. APP at30mg/kg was as comparable as resveratrol at30mg/kg or dexamethasone at1mg/kg treatment in IL-1β, TNF-α and CXCL-1mRNA expression. APP at100mg/kg was as comparable as resveratrol at30mg/kg or dexamethasone at1mg/kg treatment in MCP-1mRNA expression.To evaluate the effects of APP on oxidative stress, SOD activity in BALF, as well as MPO, GSH activity level and Nrf2mRNA and protein expression levels in lungs were determined.18h after last CS-expose, resulted in significant decreases of SOD activity in BALF and GSH level in lung and significant increases of lung MPO activity as well as Nrf2mRNA and protein expression levels in vehicle group as compared with control group. However, treatment with APP at the dose of30,100or300mg/kg significantly increased the SOD activity in CS-treated BALF and GSH level in CS-treated lung, and also significantly decreased the CS-induced increases of MPO activity as well as Nrf2mRNA and protein expression in lungs. APP at30mg/kg was as comparable as resveratrol at30mg/kg or dexamethasone at1mg/kg treatment in SOD activity, GSH level and Nrf2mRNA and protein levels. APP at100mg/kg was as comparable as resveratrol at30mg/kg or dexamethasone at1mg/kg treatment in MPO activity.Eighteen hours after exposure to CS for4consecutive days in mice, we examined protein expression of MMP-9/TIMP-1with immunohistochemical method, which found an increase in MMP-9as well as a degradation in TIMP-1. However, treatment with APP at the dose of300mg/kg significantly increased the CS-induced decrease of TIMP-1expression, also significantly decreased the CS-induced increases of MMP-9expression in lungs. APP at300mg/kg is as comparable as dexamethasone at1mg/kg treatment.In vitro, we found APP dose-dependently attenuated CSE-induced growth inhibition when cells pretreated with the0.1-10μg/ml concentrations of APP and exposed to2.5%CSE for24h. However, there was no statistically significant change when pre-treated with APP alone.Analyses of culture medium demonstrated that exposure of cells to various concentrations (0-5%) of CSE for24h concentration-dependently increased the protein level of TNF-a. Exposure to2.5%CSE for up to24h time-dependently increased the TNF-a protein level. However, there was no statistically significant change when pre-treated with APP alone. Cells pretreated with the0.1-10μg/ml concentrations of APP and exposed to2.5%CSE for24h showed the lowest TNF-a protein level was in the group with the concentration of APP at1μg/ml. Considering the efficacy of APP,1μg/ml of APP and2.5%CSE were chosen as standard concentrations for subsequent studies.NCI-H292cells were treated with various concentrations of CSE (0-7.5%) for24hours. The levels of GSH were significantly reduced by introduction of2.5%,5%, and7.5%CSE. Pre-incubation with APP (0.1-10μg/ml) significantly increased GSH level in2.5%CSE-introduced cells. Besides, APP (1μg/ml) inhibited the increase of intracellular ROS induced by CSE (2.5%) stimulation in cells.Related to the relationship between the effects of APP on ROS elimination and inhibition of TNF-a expression, we found either pre-treatment with DMTU (a ROS scavenger) at concentrations of2-20mM or APP (1μg/ml) significantly decreased TNF-a protein levels, suggesting that APP play a role in ROS-mediated TNF-a up-regulation.Pre-incubation of either SB203580(a P38inhibitor), SP600125(a JNK inhibitor), PD98059(an ERK1/2inhibitor) at10μM, or APP at1μg/ml significantly decreased2.5%CSE-induced increase of TNF-α protein level in cells. Besides, cells exposed to2.5%CSE increased the phosphorylation of MAPKs within20min. The CSE-induced increase in the phosphorylation of ERK1/2was prevented by pretreatment with both SB203580(10μM) and APP (1μg/ml). However, the phosphorylation of P38MAPK or JNK were respectively prevented by pretreatment with PD98059(10μM) or SP600125(10μM) but not APP (1μg/ml).Conclusion:In summary, the present study provided evidences for APP’s protective effects on inhibiting CS-exposed acute lung injury in mice and attenuating reactive oxygen species mediated TNF-a expression through the ERK1/2pathway in human airway epithelial cells.