Gliotoxin Induced Bronchial Epithelial Cell Apoptosis And Its Influence On Expression Of Apoptosis Related Genes And Proteins

Objective Using an in vitro model of human bronchial epithelial cell（HBE）infected by Aspergillus fumigatus（AF），we hope to find some effects and potentialmechanisms of gliotoxin（GT） when AF infect HBE by observing the concentrationin cell culture supernatant、genes and protein related to apoptosis of HBE and cellapoptosis rate.Methods1）Fungal culture：Inoculate AF into PDA plate，put the plate into30℃incubator for4days，activation of two consecutive times，use PBS to suspend sporeand count；2) HBE culture：Inoculate HBE into10cm petri dishes as the density of106/ml，each dish with5ml cell suspension，add moderate fresh medium containing10%FBS and culture in the CO2incubator for48h；3) When the cell density is morethan80%, according to the ratio of1：1000（fungus spore count: the number of HBE）to join the fungal spore suspension，incubate under the condition of37℃,5%CO2for3h, PBS washing，abandon the supernatant, add a certain amount of RPMI1640complete，and put into cell incubator for further culture；4）results：terminate theco-culture experiments at12h,24h,36h respectively, collect cell culture supernatantand HBE and detect GT concentration, expressing of genes and protein related toHBE apoptosis and apoptosis rate by the technologies of liquid chromatography-massspectrometry (LC-MS), real-time PCR, Western Blot and flow cytometry (AnnexinV-FITC staining).Results1.The results of the concentration of GT detected by LC-MS revealed：comparingwith the control group（0h point-in-time，AF in state of spores)，the GT concentrationof the supernatant of AF293and AF B5233WT co-culture group increased over time（P<0.05）；The supernatant of AF B5233Δ Glip co-culture group did not detect GT.The GT concentration of the supernatant of AF293separate culture group and AF293HBE co-culture group increased over time (P <0.05) and at36h point-in-time the concentration of AF293HBE co-culture was higher than the AF293separateculture group，having statistical significance (P <0.05).2. The results of HBE genes related apoptosis analyzed by real-time PCR revealed：comparing with the control group（0h point-in-time，AF in state of spores)，Bcl-2expressing level of cells of each co-culture group was gradually reduced (P <0.05)，Bak expression level was gradually increased (P <0.05)；Compared AF B5233Δ Glipco-culture group with AF293and AF B5233WT co-culture group respectively，at36hpoint-in-time the expression level of the apoptosis genes Bcl-2and Bak all hadstatistically significant (P <0.005).3. The results of HBE genes related apoptosis analyzed by Western-blot revealed：comparing with the control group（0h point-in-time，AF in state of spores)，Bcl-2protein synthesis level of cells of each co-culture group was gradually reduced (P <0.05)，Bak protein synthesis level was gradually increased (P <0.05)；Comparing withAF B5233Δ Glip co-culture group with AF293and AF B5233WT co-culture grouprespectively，at36h point-in-time the protein synthesis of the apoptosis Bcl-2and Bakall had statistically significant (P <0.005).4. The results of HBE apoptosis rate detected by flow cytometry revealed：Comparingwith the control group（0h point-in-time，AF in state of spores)，the cells apoptosisrate of each co-culture group was gradually increased (P <0.05)；Comparing AFB5233Δ Glip co-culture group with AF293and AF B5233WT co-culture grouprespectively， the cells apoptosis rate had statistically significant at36h point-in-time(P <0.005).Conclusion1. GT may have an important toxic effect in the process of AF infecting HBE.2. GT can induce HBE death, and Bcl-2、Bak may be the important sites where GTplays its role in the process of AF infecting HBE.3. AF not only secrete one virulence factor such as GT in the process of AF infectingHBE.4. HBE may stimulate AF to secrete more GT.