Role Of Endoplasmic Reticulum Stress In Pyroptosis Of BEAS-2B Cells Induced By BPDE

Pyroptosis is the type of inflammatory programmed cell death.Endoplasmic reticulum stress（ERS）,which is a response to internal and external stimulation,has been reported to play an important role in pyroptosis.Benzo（a）pyrene（B（a）P）,as the main representative of polycyclic aromatic hydrocarbons（PAHs）,can promote inflammation and many chronic pulmonary diseases.However,the relationship between pyroptosis and ERS in B（a）P or BPDE-induced human bronchial epithelial cell injury has not been elucidated.ObjectiveThis study focused on the effects of ERS and pyroptosis in human bronchial epithelial cells（BEAS-2B）,and explored the relationship between ERS and pyroptosis,so as to provide a novel idea for the molecular mechanism of B（a）P cytotoxicityMethods1.The cell viability was detected and the appropriate dose was screened.After BEAS-2B cells were treated with BPDE（0、0.25、0.50、0.75、1.00、1.25、1.50、1.75 and 2.00μmol/L）for 24h,cell viability was detected by using CCK-8 method,and appropriate concentrations of BPDE were determined for subsequent experiments.2.ERS and pyroptosis were detected.Blank control group,dimethylsulfoxide（DMSO）solvent group and BPDE groups（0.50,0.75 and 1.00μmol/L）were set up.The morphology of endoplasmic reticulum was observed by transmission electron microscopy and the expression levels of ERS-related proteins（GRP78,PERK and p-PERK）were detected by Western blot to explore the effect of BPDE on ERS.The changes of cell membrane were observed by inverted microscopy and transmission electron microscopy respectively,the PI positive cell rate was detected by Hoechst33342/PI fluorescence staining,the expression levels of pyroptosis-related proteins（GSDMD,GSDMD-N,pro/cleaved-caspase 1,pro/cleaved-IL-1β）were detected by Western blot,and the expression of inflammatory cytokines IL-1βand IL-18 was detected by cellular immunohistochemistry to explore the effect of BPDE on pyroptosis.3.Intervention with ERS to detect cell pyroptosis.The classical inhibitor 4-PBA was used to inhibit ERS.Blank group,DMSO group,DMSO+4-PBA group,BPDE group and BPDE+4-PBA group were set up.The expression levels of proteins（GRP78,GSDMD,GSDMD-N,and cleaved-caspase 1）in each group were measured by Western blot.The rate of PI positive cell was detected by utilizing Hoechst33342/PI fluorescence staining.The expression of inflammatory cytokines（IL-1βand IL-18）was measured by immunohistochemistry.4.Statistical analysis.Experimental data were analyzed by Graph Pad Prism 8software and were expressed as mean±standard deviation（（?）±s）.One-way ANOVA were used for comparison between more than two groups.LSD method was utilized for multiple comparisons between groups.α=0.05 was regarded as the test level.Results1.Determination of BPDE doses and observation of cell morphologyCell viabilities were significantly inhibited after BPDE treatment（P<0.05）.The IC₅₀ of BPDE on BEAS-2B cells was calculated to be 1.147μmol/L.To ensure that the cell viability was over 50%,three concentrations（0.50,0.75 and 1.00μmol/L）of BPDE were chosen for subsequent experiments.Compared to control group,cellular swelling,indistinct cellular contours and poor growth condition were observed on BEAS-2B cells after BPDE exposure.2.Effects of BPDE on ERS of BEAS-2B cellsThe changes of ER morphology after BPDE stimulation were observed by TEM.The results showed that the ER was swollen and dilated.Western blot results showed that with the BPDE concentrations increasing,the expression levels of GRP78 and p-PERK were both significantly increased compared to DMSO group（P<0.05）.3.Effects of BPDE on pyroptosis of BEAS-2B cellsCellular swelling and indistinct cellular contours were observed in BPDE group by using inverted microscope.Cell membrane was damaged and incomplete in BPDE group under TEM.The rate of PI positive cell was improved with the increase of BPDE concentrations by using Hoechst 33342/PI fluorescence staining（P<0.05）.Compared with DMSO group,the expression levels of pyroptosis-related proteins（GSDMD-N,cleaved-caspase 1 and cleaved-IL-1β）were significantly increased after BPDE treatment（P<0.05）.The results of immunohistochemistry indicated that IL-1βand IL-18 proteins were expressed in the cytoplasm of BEAS-2B cells,and its expressions were enhanced after BPDE exposure（P<0.05）.4.Roles of ERS in pyroptosis induced by BPDE in BEAS-2B cells10mmol/L 4-phenylbutyrate（4-PBA）was selected as the suitable concentration for ERS inhibition.Compared to BPDE group,the rate of PI positive cell was declined,the protein expressions（GRP78,GSDMD-N and cleaved-caspase 1）were dropped,and the expressions of IL-1βand IL-18 in the cytoplasm were decreased in4-PBA+BPDE group（P<0.05）.ConclusionsERS and pyroptosis could be induced by BPDE in BEAS-2B cells,and ERS may promote the occurrence of pyroptosis induced by BPDE.