Study On The Mechanism Of A549 Cell Pyroptosis Caused By Oxygen Glucose Deprivation/Recovery

Section1:The effect of oxygen glucose deprivation/recovery on A549 cell pyroptosisPurpose: Acute lung injury(ALI)caused by cardiopulmonary bypass(CPB)is the most important complication of large-scale cardiac surgery,with high mortality and poor prognosis.Ischemia-reperfusion is an important cause of acute lung injury in CPB.pyroptosis is a type of programmed death dependent on inflammatory cysteine protease(Caspase).The inflammatory response is a significant feature of pyroptosis.This process is similar to the accumulation of high levels of inflammatory factors in the lung tissue of cardiopulmonary bypass..Studies have shown that the simulated cardiopulmonary bypass ischemia-reperfusion process can induce lung tissue cell pyroptosis and lead to lung injury.Therefore,whether the cell pyroptosis occurs after lung tissue ischemia-reperfusion during cardiopulmonary bypass needs to be further studied.This study passed Establish an Oxygen-glucose Deprivation/Recovery(OGD/R)model of A549 alveolar epithelial cells,detect pyrosis-related indicators,and observe the effect of OGD/R on the levels of A549 cell pyrosis-related indicators.Explore the mechanism of cardiopulmonary bypass(CPB)related lung injury to provide reference.Methods:(1)Culture A549 cells,perform OGD/R(6h/6h)modeling,and use real-time quantitative PCR(quantity real-time reverse transcription PCR,RT-q PCR)method and Western-blot method to detect the pyroptosis-related gene Caspase1,ASC,NLRP3,GSDMD m RNA transcription and protein expression levels;(2)A549 cells were pretreated with Caspase1 specific antagonist VX-765 for 3 hours and then OGD/R(6h/6h)modeling was performed to detect Caspase1 and pyroptosis Effect of GSDMD,the expression level of Caspase1 pyroptosis product IL-1βand IL-18 in culture supernatant and cell survival rate.Results:(1)Compared with the control group,Caspase1 activity in A549 cells increased after OGD/R,and the m RNA and protein expressions of pyroptosis-related genes Caspase1,ASC,NLRP3,GSDMD were up-regulated;(2)Caspase1 specific antagonist was used After pretreatment with VX-765 for 3 hours,the expression of pyroptosis-related proteins Caspase1 and GSDMD protein was significantly lower than that in the OGD/R group,and the levels of pyroptosis marker products cytokines IL-1β and IL-18 in the cell culture supernatant were higher than those in the OGD/R group.The R group also decreased significantly,and the cell survival rate was significantly restored compared with the OGD/R group.With the pyroptosis-related inhibitor VX-765,the pyroptosis-related protein and the corresponding products of pyroptosis markers in A549 cells were reduced and reduced.OGD/R damage in A549 cells.Conclusion: Oxygen and glucose deprivation/restoration can induce A549 alveolar epithelial cells to develop pyroptosis,and inhibiting the occurrence of pyroptosis has a protective effect on the oxygen and glucose deprivation/restoration cell model.Section2:The role of oxidative stress in OGD/R-induced pyroptosis of alveolar epithelial cells of A549 cellsPurpose: Our previous experiments suggest that oxygen glucose deprivation/recovery can induce pyroptosis of A549 alveolar epithelial cells,and inhibiting the occurrence of pyroptosis has a protective effect on the oxygen glucose deprivation/recovery cell model.But under OGD/R conditions,the specific mechanism of A549 cell pyroptosis needs to be further studied.Studies have shown that ROS,an important indicator of oxidative stress,mediates the activation of NLRP3 inflammasomes in brain,heart,kidney and testicular ischemia/reperfusion injury.In this study,whether A549 cell pyroptosis caused by OGD/R is also The activation of ROS by oxidative stress is uncertain.In this part of the study,the A549 alveolar epithelial cell oxygen glucose deprivation/recovery model was established to detect the effect of OGD/R on the oxidative stress in A549cells;to observe the antioxidant acetylcysteine ??(N-Acetyl-L-cysteine,NAC)The effect on the level of pyroptosis and oxidative stress of A549 alveolar epithelial cells will further provide a reference for exploring the mechanism of cardiopulmonary bypass(CPB)related lung injury.Methods: The experimental groups were divided into(1)Control group: A549 cells were treated without any treatment;(2)OGD/R group:A549 cells were treated with OGD for 6 hours and recovered for 6 hours(3)OGD/R+NAC group: anti-The A549 cells were pretreated with NAC(10mmol/L)for 1 hour and then OGD/R was performed.MTT was used to detect the viability of A549 cells,LDH was used to detect the degree of cell membrane damage;flow cytometry was used to detect the intracellular ROS content of A549 cells;MDA,SOD,GSH kits were used to detect oxidative stress changes in A549 cells;RT-q PCR and Western-blot were used to detect A549 The m RNA transcription and protein expression levels of Caspase1,ASC,NLRP3,GSDMD related to cell pyroptosis,and the expression levels of IL-1β and IL-18 in the cell supernatant were detected by an ELISA kit.Results: Compared with the Control group,A549 cell viability decreased and cell membrane damage in OGD/R group,oxidative stress products ROS and MDA levels increased,anti-oxidative stress products SOD and GSH levels decreased,and pyroptosis-related genes Caspase1,ASC,The m RNA and protein expression levels of NLRP3 and GSDMD are increased,and the expression levels of cytokines IL-1 β and IL-18 are significantly increased.This indicates that in the OGD/R model,A549 cells are seriously damaged,and the expression of cell oxidative stress is increased.The expression level of death-related m RNA and protein increased;compared with the OGD/R group,the cell survival rate of the OGD/R+NAC group was improved,the cell membrane damage was reduced,the expression of ROS and MDA in the cell was reduced,and the expression of SOD and GSH was increased.The m RNA and protein expression levels of apoptosis-related genes Caspase1,ASC,NLRP3,GSDMD and the expression levels of cytokines IL-1β and IL-18 were significantly down-regulated,suggesting that the use of antioxidant NAC can effectively alleviate the damage of A549 cells by oxygen and glucose deprivation and effectively inhibit A549 cells.Oxidative stress reduces the expression of pyroptosis-related m RNA,protein and pyroptosis effector factors.Conclusion: In the OGD/R model,the viability of A549 cells decreases and oxidative stress damages.NAC reduces cell oxidative stress damage and reduces the products of A549 cell pyroptosis.Oxygen glucose deprivation/recovery can induce A549 alveolar epithelial cells to undergo pyroptosis and oxidative stress damage.NAC effectively protects the cell oxygen glucose deprivation/recovery model by inhibiting cell oxidative stress and pyroptosis.