OBJECTIVE To investigate the genome protective effects of anti-malaria drug,artesunate in an experimental allergic asthma model.METHODS Mice were sensitized on day 0 and 7 and challenged on day 14 with 100μg house du...OBJECTIVE To investigate the genome protective effects of anti-malaria drug,artesunate in an experimental allergic asthma model.METHODS Mice were sensitized on day 0 and 7 and challenged on day 14 with 100μg house dust mite(HDM)via intratracheal administration.Artesunate(30mg·kg-1)was administered intra-peritoneally on day 6,7,8,13,14 and 15.Samples were collected on day 1,3 and 5 post last HDM-challenge for analysis of air way inflammation and DNA damage.Lung sections were immunofluorescence(IF)-stained for DNA double strand breaks(DSBs)markers,γH2AX and 53BP1.Levels of DNA repair proteins Ku70 and Rad51,which are involved in non-homologous end joining(NHEJ)and homologous recombination(HR)DNA DSB repair pathways respectively,were measured.To quantify cell death in asthmatic lung,TUNEL staining was performed.Comet assay,a single cell gel electrophoresis was employed to detect DNA damage induced by HDM in BEAS-2Bhuman bronchial epithelial cell line,in vitro.RESULTS Artesunate treatment significantly reduces immune cells infiltration in BAL fluid of asthmatic mice,collected on day 3 and 5 post-challenge.Importantly,artesuante is able to protect bronchial epithelium from DNA DSBs induced by asthma,as detected by the reduced level of γH2AX and 53BP1 foci formation in the nucleus.This genome protective effect is evident even on day 1 post-challenge,when immune cells infiltration remained high.This indicates that artesunate confers protection on bronchial epithelium in the presence of inflammation.Additionally,artesunate is also able to reduce cell death in asthmatic lung revealed by TUNEL assay and cleaved caspase 3 level.Interestingly,the levels of DNA repair proteins in artesuante-treated asthmatic mice are unchanged as compared to HDM-only mice,suggesting that artesunate treatment does not augment the level of DNA repair proteins.When human bronchial epithelial BEAS-2 Bcells were exposed to HDMin vitro,we observed an increase in the levels of DNA damage.Artesunate(60μmol·L-1)co-incubated with HDM is not able to prevent direct DNA damage induced by the allergen.Together,these studies suggest that the genome protective effect of artesunate in vivo may be attributed to physiological effects(such as its anti-inflammatory effects)rather than serving to directly prevent DNA damage.CONCULSION This study highlights a novel role for artesunate in protecting bronchial epithelial cells from asthma-induced DNA damage.展开更多
基金The project supported by a NMRC grant NMRC/CBRG/0027/2012from the National Medical Research Council of Singapore,with additional support from the Singapore-MIT Alliance for Research and Technology
文摘OBJECTIVE To investigate the genome protective effects of anti-malaria drug,artesunate in an experimental allergic asthma model.METHODS Mice were sensitized on day 0 and 7 and challenged on day 14 with 100μg house dust mite(HDM)via intratracheal administration.Artesunate(30mg·kg-1)was administered intra-peritoneally on day 6,7,8,13,14 and 15.Samples were collected on day 1,3 and 5 post last HDM-challenge for analysis of air way inflammation and DNA damage.Lung sections were immunofluorescence(IF)-stained for DNA double strand breaks(DSBs)markers,γH2AX and 53BP1.Levels of DNA repair proteins Ku70 and Rad51,which are involved in non-homologous end joining(NHEJ)and homologous recombination(HR)DNA DSB repair pathways respectively,were measured.To quantify cell death in asthmatic lung,TUNEL staining was performed.Comet assay,a single cell gel electrophoresis was employed to detect DNA damage induced by HDM in BEAS-2Bhuman bronchial epithelial cell line,in vitro.RESULTS Artesunate treatment significantly reduces immune cells infiltration in BAL fluid of asthmatic mice,collected on day 3 and 5 post-challenge.Importantly,artesuante is able to protect bronchial epithelium from DNA DSBs induced by asthma,as detected by the reduced level of γH2AX and 53BP1 foci formation in the nucleus.This genome protective effect is evident even on day 1 post-challenge,when immune cells infiltration remained high.This indicates that artesunate confers protection on bronchial epithelium in the presence of inflammation.Additionally,artesunate is also able to reduce cell death in asthmatic lung revealed by TUNEL assay and cleaved caspase 3 level.Interestingly,the levels of DNA repair proteins in artesuante-treated asthmatic mice are unchanged as compared to HDM-only mice,suggesting that artesunate treatment does not augment the level of DNA repair proteins.When human bronchial epithelial BEAS-2 Bcells were exposed to HDMin vitro,we observed an increase in the levels of DNA damage.Artesunate(60μmol·L-1)co-incubated with HDM is not able to prevent direct DNA damage induced by the allergen.Together,these studies suggest that the genome protective effect of artesunate in vivo may be attributed to physiological effects(such as its anti-inflammatory effects)rather than serving to directly prevent DNA damage.CONCULSION This study highlights a novel role for artesunate in protecting bronchial epithelial cells from asthma-induced DNA damage.
