Anti-malarial drug,artesunate,as a novel therapeutic drug target for airway wall remodeling in asthma

OBJECTIVE Airway wall remodeling(AWR),which refers to structural changes in the airway,is a key characteristic of asthma.Airway smooth muscle(ASM)cell hypertrophy and hyperplasia contributes to AWR.Glucocorticoids,which are used as first line therapy for the treatment of asthma,reduce ASM proliferation but the magnitude of their anti-proliferative actions is dependent on the mitogen used.Moreover,glucocorticoid therapy is accompanied by many side effects.Artesunate,a semi-synthetic artemisinin derivative,has been widely used to treat malaria with minimal toxicity.Artesunate has been shown to attenuate allergic airway inflammation in mice.However,its role in treating AWR in asthma is not known.In this study,we hypothesize that artesunate has anti-proliferative actions on ASM cells,potentially reversing AWR.METHODS and RESULTS Quiescent primary human ASM cells were pre-treated(1h)with artesunate(3,10,30μmol·L-1)before being stimulated with either FBS(10%)or thrombin(0.3U·mL-1).Following 48 h stimulation with mitogen,cells were counted using a haemocytometer.Dexamethasone(Dex,100nmol·L-1)was used as a positive control.Artesunate concentration-dependently reduced cell number and the magnitude of inhibition appeared to be non-mitogen dependent.Moreover,we examined the effect of artesunate on two important signalling proteins involved in cell proliferation,ERK1/2phosphorylation and cyclin D1 protein levels.Artesunate reduced cyclin D1 protein levels significantly following 20 h stimulation with either thrombin or FBS but had no effect on ERK1/2 phosphorylation following 6h stimulation.Importantly,artesunate(30μmol·L-1),but not Dex,inhibited the phosphorylation of Akt,which is upstream of cyclin D1.Next,we show that the inhibitory effect of artesunate,but not Dex,on ASM cell number is retained at least 24h post-treatment following stimulation with FBS.In an acute murine model of allergic asthma,artesunate treatment decreased sm-α-actin positive cells and cyclin D1 protein abundance in the ovalbumin sensitized and challenged mice.CONCLUSION We have shown that artesunate regulates the PI3K/Akt pathway to inhibit the proliferation of primary human cultured ASM cells.This is an alternative mode of action,in comparison to glucocorticoids such as Dex.The anti-proliferative effect of artesunate was further validated in vivo.Thus,our study provides a basis for the future development of artesunate as a novel anti-AWR agent that targets ASM hyperplasia via the PI3K/Akt pathway.Moreover,artesunate may be used as an add-on therapy for asthmatic patients.

Vitamin E isoform γ-tocotrienol alleviates cigarette smoke-induced chronic obstructive pulmonary disease

OBJECTIVE Cigarette smoke-induced chronic obstructive pulmonary disease(COPD)is a leading cause of death,where inflammation and oxidative stress are involved in the pathogenesis.Vitamin E isoformγ-tocotrienol possesses both anti-oxidative and anti-inflammatory properties.We hypothesized thatγ-tocotrienol may have protective effects against COPD.METHODS BALB/c mice were exposedto cigarette smoke daily for 2 weeks with oralγ-tocotrienol treatment in the second week.Bronchoalveolar lavage(BAL)fluid was assessed for total and differential cell counts,oxidative damage biomarkers,and cytokine levels.Lung tissues were examined for the expression of antioxidants and pro-inflammatory biomarkers.In order to measure changes in lung functions in COPD,another set of mice was exposed to cigarette smoke for 2 months with oralγ-tocotrienol treatment in the last 2 weeks.RESULTSγ-Tocotrienol dose-dependently abated cigarette smoke-induced elevation of BAL fluid total and neutrophil cell counts,cytokine and chemokine(IL-1β,IL-6,IL-17,LIX,G-CSF,KC,RANTES and VEGF)levels,as well as oxidative/nitrosative damage biomarker(advanced oxidation of protein products,8-isoprostane,8-hydroxy-2-deoxyguanosine and 3-nitrotyrosine)levels.γ-Tocotrienol promoted total lung antioxidant capacity and endogenous antioxidant activities of superoxide dismutase,catalase and glutathione peroxidase.More importantly,γ-tocotrienol markedly restored work of breathing and lung functions(total lung capacity,static compliance and FEV100/FVC)in chronic experimental COPD.Furthermore,γ-tocotrienol demonstrated better anti-oxidative,anti-inflammatory,and restoration of lung functions in COPD than prednisolone.CONCLUSION We have shown for the first time the efficacy of vitamin E isomerγ-tocotrienol in protection against cigarette smoke-induced COPD by direct neutralization of free radicals,abating oxidative damage,and restoring antioxidants activities,coupled with anti-inflammatory actions in the inflamed airways.

Andrographolide restores steroid sensitivity to block LPS/IFNγ-induced IL-27 and airway hyper-responsiveness in mice

OBJECTIVE Steroid-resistant airway hyper-responsiveness(AHR)has been proposed to be related to the activation of innate host defense pathways such as those induced by LPS,IFN-γ,and LPS/IFN-γ-stimulated essential mediator IL-27.We investigated whether andrographolide,apreviously demonstrated anti-inflammatory bioactive molecule extracted from the plant Andrographis paniculata,could restore steroid sensitivity to block LPS/IFN-γ-induced IL-27 production and AHR viaits anti-oxidative property.METHODS Mouse macrophage cell line Raw264.7,mouse primary pulmonary monocyte/macrophage,and BALB/c mouse were treated with LPS/IFN-γ,in the presence and absence of increasing doses of dexamethasone and/or andrographolide.mRNA and protein levels of IL-27 in vitro and in vivo were examined,and mouse AHR was assessed.RESULTS Dexamethasone alone failed to inhibit LPS/IFN-γ-induced IL-27 and AHR in mice.Andrographolide significantly facilitated the suppressive effect of dexamethasone on LPS/IFN-γ-induced IL-27 level in macrophage cell line and primary monocyte/macrophage,mouse bronchoalveolar lavage fluid and lung tissue,and furthermore on the incurring AHR.LPS/IFN-γdid not impede nuclear translocation of glucocorticoid receptors but diminishthe protein level of histone deacetylase-2(HDAC2),an essential epigenetic enzymeresponsible for steroid anti-inflammatory action.Andrographolide at low doses(5μmol·L-1 in vitro;1mg·kg-1,ip in vivo)restored nuclear HDAC2 protein levels both in cells and in mouse lungs,possibly via suppression of PI3K/Akt signaling pathway and up-regulation of the anti-oxidative transcription factor Nrf-2level.CONCLUSION Our data suggest that andrographolide may resensitize steroid action on blocking LPS/IFN-γ-induced IL-27 and resultant AHR by restoring HDAC2 level.

Non-typeable haemophilus influenzae-induced exacerbation on a cigarette smoke lung injury model:Protective effect of andrographolide

OBJECTIVE To develop a 2-week cigarette smoke(CS)acute lung injury model exacerbated by haemophilus influenzae(NTHi)and study the protective effect of andrographolide in this COPD model.METHODS Female BALB/c mice,6-8-week-old,were exposed to 4% 3R4 FCS delivered using aperistaltic pump daily for 2 weeks to induce an acute lung injury model.After 2 weeks of smoking,mice were inoculated intratracheally with NTHi to induce exacerbation on the model.Mice were sacrificed 48 h after last bacteria challenge and lung samples were collected for various analyses.RESULTS After developing a 2-week CS acute lung injury model exacerbated by NTHi,the CS+NTHi group was shown to have a higher inflammatory response,higher bacterial clearance,an upregulation of MMP12 mRNA levels and decrease in TIMP1 mRNA levels in the lungs.Administration of Andrographolide suppressed BALF lung cellular infiltrates,TNF-α,CXCL1/KC,IL-1βand 8-OHdG protein levels,together with increased HO-1 and GR mRNA levels and decreased MMP-8 and MMP-9 mRNA levels.Andrographolide was able to ameliorate lung histopathology as observed with H&E staining and inflammation scoring.Andrographolide was also shown to reduce Keap-1 level in lungs without affecting DJ-1 level.CONCLUSION This study demonstrates the protective effect of andrographolide in a novel 2-week CS acute lung injury model exacerbated by NTHi and presents it as a potential therapeutic for COPD.