The Expression Of IGFBPs In Alveolar Epithelial Cells Type Ⅱ Stimulated By Cigarette Smoke Extract And The Possible Interaction With P21 In The Process

BackgroundSmoking is the main cause of chronic obstructive pulmonary disease(COPD).Studies have shown that it can induce cell senescence,apoptosis and autophagy,but the mechanism is not clear.Current studies suggest that cigarette smoke can cause oxidative stress in alveolar epithelial cells,and excessive reactive oxygen species(ROS)can also lead to DNA damage and cell cycle arrest,eventually leading to apoptosis,but the specific signaling pathways and mechanisms have not yet been elucidated.Insulin-like growth factor-binding proteins(IGFBPs)are widely expressed in most tissues.They can bind to insulin-like growth factor(IGF)and regulate it.Studies have shown that IGFBPs have a regulatory effect independent of IGF,and they are widely involved in a series of physiological and pathological processes in the human body.It includes basal metabolism,immune regulation,neurological diseases and cancers,and is also associated with cell proliferation,survival,differentiation,migration,invasion,senescence and autophagy.The p21 and p53 genes are a class of tumor suppressor genes that are widely recognized in the traditional sense.They limit the growth of cancer cells and promote cell senescence and apoptosis by regulating cell cycle arrest.In recent studies,sulforaphane(SFN)is a kind of natural antioxidant product extracted from vegetable plants,which can play its antioxidant role by activating Nrf2-related pathway.However,there is no research to prove whether it has a corresponding protective effect in COPD.At present,the research of IGFBPs is more extensive,but it is rarely associated with COPD.In the previous experiments of our laboratory,it was found that the expression of IGFBP-3 in cells treated with cigarette smoke increased and there was apoptosis and cell cycle arrest.Therefore,it is necessary to comprehensively explore the role of IGFBPs in COPD.ObjectiveThis experiment intends to explore whether there is a change in the level of IGFBPs in A549 cells and RLE-6TN cells after cigarette smoke extract(CSE)treatment and the correlation between p21 protein and gene levels related to cell cycle regulation.The protein-protein interaction between IGFBPs and p21 was investigated.The purpose of this study was to explore whether SFN could exert antioxidant protective effect through Nrf2 related signaling pathway.Method(1)The human type Ⅱ alveolar epithelial cells A549 and rat alveolar epithelial cells RLE-6TN were treated with cigarette smoke extract(CSE)and compared with the untreated control group.Real-time fluorescence quantitative PCR was used to determine changes in gene expression of IGFBP-1,IGFBP-2,IGFBP-3,IGFBP-4,IGFBP-5,IGFBP-6,IGFBP-7,p21,p53,Nrf2,HO-1,Fas and Fas L.(2)The changes of intracellular corresponding proteins were determined by western blotting assay and the changes of proteins in the supernatant of cell culture were determined by ELISA kit.Caspase-8 and 9 activity changes were detected with the caspase activity detection kit of Beyotime Company.(3)Design and use siRNA sequences to reduce the expression of IGFBP-1,and determine the state of cell apoptosis by flow cytometry.(4)The co-immunoprecipitation of IGFBP-1,2,3 and p21 proteins was performed to observe whether there was any interaction between the proteins.(5)Attempted to construct the pEGFP-N1-IGFBP1 expression plasmid.Results(1)The mRNA expression levels of IGFBP-1,IGFBP-2,IGFBP-3,p21,HO-1,Fas and Fas L were increased in A549 cells treated by CSE(p < 0.05),while m RNA expression levels of IGFBP-5 were decreased(p < 0.05).The protein expressions of IGFBP-1,IGFBP-2,IGFBP-3,p21 and HO-1 were increased,and the content of IGFBP-1 protein in supernatant was increased(p < 0.05).m RNA expression levels of IGFBP-2,IGFBP-3,IGFBP-5,HO-1,Fas and Fas L were increased in RLE-6TN cells(p < 0.05),while m RNA expression levels of p21 were decreased(p < 0.05).(2)There was no significant change in the activity of caspase-8 in A549 cells treated by CSE,and the activity of caspase-9 was decreased(p < 0.05).(3)After siRNA interference,the expression of IGFBP-1 protein in A549 cells was decreased(p < 0.05),but flow cytometry showed that knockdown of IGFBP-1 had no significant effect on apoptosis induced by CSE.(4)The co-localization relationship between IGFBP-2 and p21 proteins was found in A549 cells.Conclusion(1)IGFBP-1,IGFBP-2,and IGFBP-3 are upregulated at both nucleic acid and protein levels in human alveolar type Ⅱ epithelial cells treated with CSE;(2)There exists the interaction between IGFBP-2 and p21 protein in the CSE injury model of A549 cells,indicating that IGFBP-2 may inhibit cell cycle by regulating p21;(3)The Nrf2-HO-1 signaling pathway is involved in CSE-induced cell damage and may play a protective role through anti-oxidant mechanisms.