Activated Autophagy Reduces The Apoptosis Of Human Nasal Mucosa Epithelial Cells Infected With Staphylococcus Aureus

BackgroundChronic rhinosinusitis(CRS)is a common nonspecific infectious disease of otolaryngology which course often exceeds 12 weeks,seriously affecting the quality of life of patients.Among triggers in its underlying pathogenic mechanisms,staphylococcus aureus(S.aureus)is regarded as a major one.Autophagy is an important catabolism process in vivo,which delivers cytoplasmic components to lysosomes for explanation and plays a crucial role in maintaining the balance between the synthesis,degradation and recycling of cell components.However,the mechanism of autophagy in sinusitis has not been clearly studied.Hence,in the present study,we evaluated the role of autophagy in controlling apoptotic cell death during S.aureus infection in human nasal mucosa epithelial cells(HNEpCs).ObjectiveThe purpose of this study was to discover the changes and interactions of autophagy and apoptosis in HNEpCs infected with S.aureus.Methods(1)To determine its logarithmic growth period under the present experimental conditions,a standard growth curve of s.aureus was drawn and the cells in logarithmic growth period would be used in subsequent experiments.(2)MTT assay:Firstly,we constructed a HNEpC model infected with S.aureus with different Manual Optical Inspector(MOI,1、25、50、100)for 6h、12h、18h、24h.3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,Thiazolyl Blue Tetrazolium Bromide(MTT)was added to make a reaction with cells.OD value at 570 nm was detected by enzyme labled instrument to determine the optimal MOI.In addition,we pretreated the cells with the rapamycin(100nmol/L,200nmol/L,1000nmol/L)and the bavolomycin(lnmol/L,10nmol/L,100nmol/L)for 2h and then repeat the above operation.The MTT assay was conducted to verify both the concentration of rapamycin and bavolomycin was safe for human nasal epithelial cells.(3)Western Blot:Fifteen microgram of each sample was analyzed using SDS PAGE.Protein quantification was carried out by the BCA method.Protein separation was performed by SDS-PAGE on 5%and 12%gels,and resolved proteins were transferred to membranes and incubated with calf serum for 120 min.Primary antibodies against cleaved caspase-3(1:800 dilution)beclin-1(1:1000 dilution),P62(1:1000 dilution),LC3II/LC3I(1:1000 dilution),and beta-actin(1:500 dilution)were added and incubated overnight at 4℃.The corresponding secondary antibodies(1:2000 dilution)were incubated for 2 h in a shaker at room temperature.Using the ECL chemiluminescence reaction,the protein expression levels of cleaved caspase-3,beclin-1,P62,LC3II/LC3I,and beta-actin in HNEpCs were analyzed using ImageJ software.Moreover,we pretreated the cells with the rapamycin(200nmol/L,)and the bavolomycin(10nmol/L)for 2h and then repeat the above operation.(4)Flow cytometry:Annexin V apoptosis detection kit FITC(Beyotime,Shanghai)was used according to the manufacturer’s instructions.Briefly,trypsin-digested cells were centrifuged at 300×g for 5 min,resuspended in 100 μl of 1×binding buffer containing 10 μl of PI and 5 μl of annexin V-FITC.After 15 minutes of reaction in the dark,each sample was supplemented with 400μl 1×binding buffer and analyzed with flow cytometry.Moreover,we pretreated the cells with the rapamycin(200nmol/L,)and the bavolomycin(10nmol/L)for 2h and then repeat the above operation.(5)Confocal Laser scanning Microscopy(CLSM):HNEpCs were plated in 6-well plates and allowed to reach 50%-70%confluence at the time of transfection.mRFP-GFP-LC3 adenoviral vectors were purchased from HanBioTechnology(Shanghai,China).The principle of the assay is based on different pH stability of green and red fluorescent proteins.The fluorescent signal of GFP could be quenched under the acidic condition(pH below 5)inside the lysosome,and the mRFP fluorescent signal has no significant change under the acidic condition.In green and red-merged images,autophagosomes are shown as yellow spots,while autolysosomes are shown as red spots.Autophagic flux is increased when both yellow and red spots are increased in cells,while autophagic flux is blocked when only yellow spots are increased without red spots alteration,or when both yellow and red spots are decreased in cells.Results(1)Under the conditions of this experiment,the 2-4.5 hours of s.aureus culture is logarithmic growth period,which is suitable for experiments.(2)25 is the optimal MOI of the HNEpCs model infected with S.aureus.(3)Western Blot、Flow cytometry、CLSM:The expression of cleaved caspase-3 and cell apoptosis increased in the model,accompanied by increased expression of the proteins Beclin-1 and LC3 and decreased expression of P62.After blocking autophagy degradation with BafA1,the expression of proteins LC3 and P62 was increased and expression of cleaved caspase-3 and apoptosis rates were significantly higher than the control group.On the contrary,after enhancing autophagy with RaPa,the expression levels of Beclin-1 and LC3 significantly up-regulated compared with the control group,while the expression of cleaved caspase-3 and the apoptosis rates were significantly down-regulated compared with the control group.ConclusionsThe study demonstrates that S.aureus induces apoptosis of human nasal mucosa epithelial cells through the caspase-3-dependent pathway.This kind of apoptotic cell death can be resisted by activated autophagy.These findings provide a new mechanistic insight into S.aureus-induced impairments of chronic rhinosinusitis.