| Background and objectiveChronic obstructive pulmonary disease(COPD)is characterized by airflow limitation which is usually progressive,not fully reversible and associated with an abnormal inflammatory response of the lung to noxious particles or gases.Cigarette smoke(CS),the major risk factor for the development of COPD,contains a variety of noxious components which induce pulmonary inflammation.Besides inflammation,the imbalances of oxidant/antioxidant and protease/antiprotease are intimately associated with the progression and exacerbation of COPD.The root of Glycyrrhiza uralensis(licorice)is a widely-used herb in traditional Chinese medicine.This herb is sweet in taste and neutral in nature,and is distributed to the Spleen,Stomach,Heart and Lung Channels.The action and indications of licorice contains invigorating the spleen and replenishing Qi,dispelling phlegm and relieving cough,clearing away heat and removing toxin,relieving spasm and pain,and regulating drug properties.Licorice contains several kinds of bioactive ingredient,such as glycyrrhizin,glycyrrhetinic acid,flavonoids,alkaloid and amino acid.Licochalcone A(LA),a major component of flavonoid from licorice,has shown various anti-inflammatory,anti-bacterium,antioxidant and anti-tumor biochemical activities.However,the effect of LA on cigarette smoke-mediated acute lung injury has not been studied.Therefore,in this study,we initially investigated the protective roles of LA against CS-induced acute lung injury model in mice and cigarette smoke extract(CSE)-induced cell injury in human lung epithelial cells(BEAS-2B).The possible signaling pathways of LA's effects were also studied.Methods1.In vivoCigarette smoke-induced acute lung injury in mice:C57BL/6 mice were divided into six groups and assigned to different treatments.Treatment groups were administered orally with LA(10,20,40 mg/kg)by gavage.As a positive control drug,dexamethasone(Dex,1 mg/kg)was administered as well as LA.Control and model animals received solvent as placebo.Mice were pretreated with LA,Dex or solvent for two days before the first CS exposure.1 hour after administration of LA.Dex or vehicle,mice were exposed to CS or room air for four consecutive days.The bronchoalveolar lavage fluid(BALF)was conducted 18 hours after the last CS exposure.Total cell counts,differential cell counts and the protein expression levels of keratinocyte-derived chemokine(KC),tumor necrosis factor alpha(TNF-?)and interleukin 1?(IL-1?)in the BALF were determined.The partial left upper lobe was fixed and sectioned.The paraffin sections were prepared and stained with H&E and immunohistochemical methods.Lung homogenates were prepared for analysis of oxidative stress and the isolation of mRNA.The myeloperoxidase(MPO),superoxide dismutase(SOD)activities and glutathione(GSH)levels were quantified using appropriate kits.The mRNA expression levels of KC,TNF-?,IL-1? and matrix metalloproteinases(MMP)-9 were measured by quantitative real-time PCR.2.In vitroCigarette smoke extract(CSE)-induced cell injury in the BEAS-2B cells:Human lung epithelial cells(BEAS-2B)were exposed to different concentrations of CSE,which induce cell injury.The cells were treated with different concentrations of LA(0.1-10?M/L)and cell viability was measured by using MTT assay.BEAS-2B cells were incubated with LA(1?2.5?5 ?M/L)followed by treatments with CSE.The releases of interleukin 8(IL-8)and MMP-9 were assessed by qRT-PCR.To elucidate the underlying mechanism,we studied the signaling pathways.The phosphorylation of mitogen-activated protein kinases(MAPKs,including Erkl/2,p38 and JNK)and nuclear factor-?B(NF-?B)p65 protein were analyzed by Western blotting.Results1.In vivo1.1 Effects of LA on inflammatory cell accumulation in lungs of cigarette smoke-exposed miceIn the BALF,CS exposure resulted in higher numbers of total inflammatory cells,neutrophils,macrophages,and lymphocytes compared to air exposure(P<0.001).Pretreatment with LA significantly decreased the total inflammatory cells and neutrophils in a dose-dependent manner(P<0.001?0.05).LA at a concentrations of 40 mg/kg significantly reduced the accumulation of lymphocytes(P<0.001)and macrophages(P<0.05)in lungs.1.2 The role of LA in regulation of inflammatory cells infiltration into lungs mediated by CSLung sections stained with H&E displayed increases in the influx of neutrophils and macrophages into the lungs of CS-exposed animals compared to the air exposed control animals.LA,compared to CS exposure alone,markedly inhibited infiltration of inflammatory cells into lungs.1.3 Effects of LA on the mRNA levels of inflammatory mediators in lungs of cigarette smoke-exposed miceCigarette smoke induced releases of KC,TNF-?,IL-1? and MMP-9(P<0.001).CS-induced mRNA levels of KC and TNF-? were significantly inhibited by LA in dose-dependent manner(P<0.01?0.05),which were accompanied by decreases in IL-1? and MMP-9 mRNA level by LA at the highest tested concentration(P<0.05).1.4 Effects of LA on the protein levels of inflammatory mediators in the bronchoalveolar lavage fluids of cigarette smoke-exposed miceCigarette smoke induced releases of KC,TNF-? and IL-1?(P<0.001?0.01).CS-induced protein levels of KC and TNF-a were significantly inhibited by LA in dose-dependent manner(P<0.01?0.05),which was accompanied by a decrease in IL-1? level by LA at 20 and 40 mg/kg(P<0.05).1.5 Effects of LA on MPO,SOD activity and GSH level in the lung tissues of cigarette smoke-exposed miceIn the CS-exposed mice,the MPO activity in lungs was higher(P<0.01),meanwhile the SOD activity(P<0.05)and GSH level(P<0.01)were significantly decreased compared with the air-exposed animals.CS-induced activity of MPO was significantly inhibited,which were accompanied by increases in SOD and GSH levels by LA.1.6 Effect of LA on the protein expression of MMP-9 in the lungs of mice exposed to CSThe paraffin sections were prepared and stained with immunohistochemical method for examining protein expression of MMP-9.LA significantly decreased the expression of MMP-9 in response to cigarette smoke(P<0.05).2.In vitro2.1 Effect of LA on BEAS-2B cell viabilityThe cells were exposed to different concentrations of LA(0.1-10 ?M/L).Then the cell viability was measured by MTT assay.LA had no toxicity to BEAS-2B cells(P>0.05).2.2 Effects of LA on the mRNA levels of inflammatory mediators induced by CSE in BEAS-2B cellsBEAS-2B cells were treated with different concentrations of CSE(0?2.5%)for 48 hours.Then another group of BEAS-2B cells were exposed to 2.5%CSE for different times(0-48 hours).We found a greatest elevation of IL-8 and MMP-9 when cells were treated with 2.5%CSE for 48 hours.Cells pretreated(1h)with different concentrations of LA were exposed to air or 2.5%CSE for 48h.Cigarette smoke extract induced releases of IL-8 and MMP-9(P<0.001).CSE-induced mRNA levels of IL-8 and MMP-9 were significantly inhibited by LA at 2.5 and 5 ?M/L(P<0.01-0.05).2.3 Effects of LA on phosphorylation of p38,JNK and Erkl/2The phosphorylation of p38,JNK,Erkl/2 were detected by Western blotting.Treatment with CSE led to the activations of p38,JNK and Erkl/2.The CSE-induced phosphorylation of Erkl/2 was prevented by pretreatment with LA(P<0.01).However,LA pre-treatment did not affect the p-JNK and p-p3 8 expression.2.4 Effect of LA on NF-?B activationThe activation of nucleus NF-?B p65 was detected by Western blotting.Treatment with 2.5%CSE led to the activation of NF-?B(P<0.01).LA significantly decreased CSE-induced nucleus NF-?B p65 protein expression(P<0.05).ConclusionWe observe that LA has protective effects on CS-exposed acute lung injury in mice via preventing CS-induced pulmonary inflammation,oxidative stress and protease rise.The exploration of the mechanisms of CSE-induced injury in human lung epithelial cells suggests that LA exerts protective effects through suppression of NF-?B p65 activation and Erk1/2 MAPK signaling.Consequently,LA may be a potential therapy for acute lung injury. |
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