Effects Of Cigarette Smoke Exposure On Pyroptosis Of Mouse Lung And Mouse Bone Marrow-derived Dendritic Cells

PART I The effect of cigarette smoke exposure on the pyroptosis of lung cells in experimental mice.Objective: To observe the expression of pyroptosis associated proteins in emphysema mice.Methods: Ordinary C57BL/6 mice were divided into two groups according to a completely random method: one group was a smoking group,and the other group was an air control group.The mice in the smoke group received cigarette smoke exposure for 24 weeks,and the air group were kept in the same environment for 24 weeks.The experimental mice were sacrificed and lung tissue was collected.Some lung lobes were fixed in 4% formaldehyde,and pathological changes of lung tissue were observed by pathological method of hematoxylineosin staining,and compared with mean alveolar lining interval(MLI).Western blotting was used to detect the NLRP3,GSDMD-FL,Caspase-1 p10 and GSDMD-N-terminal protein expression in the total protein of the remaining lung tissues.Results:(1)Hematoxylin-eosin staining showed that in the air control mice,the alveolar structure was basically normal,the alveolar wall was not broken and fused,and there was no inflammatory cell infiltration.In the experimental group,the alveolar wall became thinner and ruptured,there was alveolar wall fusion and enlarged alveolar cavity,and there was obvious inflammatory cell infiltration with destruction of the tube wall.The alveolar lining interval of the cigarette smoke exposure group was significantly larger than that of the air exposure group(P<0.05).(2)The expression of total protein in lung tissue: Compared with the mice in the air exposure group,the NLRP3,Caspase-1 p10 and GSDMD-Nterminal protein expression in the lung tissue of the smoked group increased significantly,P<0.05(P=0.038,P=0.006,P=0.011).There was no significant difference in GSDMD-FL protein expression.Conclusion: In the model of mouse emphysema caused by cigarette smoke exposure,the expression of pyroptosis-related proteins in the lung tissue of mice is increased.Cigarette smoke exposure can activate the classic pyrolysis signaling pathway,induce cell pyrolysis,and play a role in the inflammatory mechanism of chronic obstructive pulmonary disease.PART Ⅱ Effect of Cigarette Smoke Extracts on Pyroptosis Pathway of Dendritic Cells from Mouse Bone Marrow.Objective: to observe the activation of pyroptosis pathway and the overexpression of il-1β in mouse bone-marrow derived dendritic cells stimulated by tobacco smoke extract.Methods: The experimental mice were sacrificed in accordance with animal ethics methods,and then mononuclear cells were extracted from bone marrow,and granulocyte-single colony stimulating factor(GM-CSF),interleukin-4(IL-4)and complete medium were added during the cultivation process Induce their differentiation into high-purity dendritic cells.The extracted cells were divided into blank control group and cigarette smoke extract stimulation group.Different concentrations of cigarette smoke extracts were used to stimulate dendritic cells.Western blotting was used to detect the related proteins expression in the pyrolysis pathway.Flow cytometry was used to detect the concentration of extracted dendritic cells and the IL-1β expression.Results: Significantly increased expression levels of NLRP3,caspase-1,GSDMD-N-terminal protein in bone marrow-derived dendritic cells in the stimulated group compared with the blank control group,and increased target protein expression with tobacco smoke extract concentration.Significantly increased(P<0.05).No significant difference in the GSDMD-FL protein expression.The purity of dendritic cells extracted by flow cytometry was 94.7%.The IL-1β expression in dendritic cells stimulated by tobacco smoke extract was significantly higher than that in the control group(P<0.05).Conclusion: Under the stimulation of cigarette tobacco extract,the expression of related proteins in the pyroptosis pathway of dendritic cells derived from mouse bone marrow increased.Cigarette tobacco extract can activate the Caspase-1 mediated pyroptosis signaling pathway and induce the pyroptosis of dendritic cells.The degree of induced cell pyroptosis is related to the extract concentration.And there was overexpression of IL-1β in the process of cell pyroptosis.Pyrocytosis may cause Th17 / Treg immune imbalance through the effector IL-1β and promote the occurrence and development of COPD.