The Role And Mechanism Of SPHK1 In Regulating Airway Epithelial Cells Autophagy In COPD Oxidative Stress Through TRAF2

Background:Chronic obstructive pulmonary disease(COPD)is a common and frequently occurring disease that seriously endangers human health,it is one of the third leading causes of death in the world.With the aging of the population,COPD will bring greater economic and social burdens.Smoking is the main pathogenic factor of COPD,but the pathogenesis is not clear.Cigarette smoke(CS)can stimulate airway epithelial cell’s oxidative stress reaction and damage bronchial epithelial cells.Autophagy is a cellular process that widely exists in eukaryotes and is necessary for normal homeostasis.Some studies have found that autophagy can affect the level of oxidative stress and participate in the occurrence and development of COPD,but the mechanism of autophagy in COPD is controversial.Through bioinformatics studies,we found that autophagy gene sphingosine kinase 1(SPHK1)is higher expressed in lung tissues of COPD patients than control,and some studies have confirmed that SPHK1 is involved in the pathogenesis of COPD,but the mechanism of SPHK1 affecting the occurrence and development of COPD is not clear.Through the analysis of protein interaction network,we found that SPEK1 interacts with TNF receptor associated factor 2(TRAF2),TRAF2 can ubiquitin modify the 63 lysine of autophagy protein Beclinl to promote autophagy.Therefore,we speculate that SPHK1 regulates autophagy of airway epithelial cells through TRAF2,which affects the level of oxidative stress and participates in the occurrence and development of COPD.The purpose of this study was to explore the effect of SPHK1 on autophagy and oxidative stress of bronchial epithelial cells stimulated by cigarette smoke extract(CSE)via knockdown and overexpression of SPHK1,and to explore the mechanism that SPHK1 regulating autophagy through TRAF2 by co-transfection SPHK1 and TRAF2 small interference and plasmid.Finally,emphysema mouse model and peripheral blood mononuclear cells(PBMC)of COPD patients were used to verify the expression of SPHK1 and the changes of autophagy and oxidative stress in vivo,which is helpful for us to clarify the regulation of SPHK1 on autophagy and the effect of oxidative stress in COPD,then find targeted therapeutic targets and provide new ideas for accurate treatment of COPD.Methods:1.Autophagy-related genes and sequencing data set GSE19407of lung tissue with COPD were obtained from HADb and Gene Expression Omnibus(GEO)database especially,and the differential expression of autophagy genes were analyzed by limma package in R software.2.BEAS-2B and 16HBE cells were treated with gradient concentration of CSE.Real-Time Quantitative Polymerase Chain Reaction(RT-qPCR)and Western Blotting were used to measure the expression of SPHK1 and autophagy protein.3.BEAS-2B and 16HBE cells were transfected by microtubule-associated protein 1 light chain 3(LC3B)autophagy double fluorescent lentivirus and stimulated by 3%CSE for 24 hours.The autophagy flow process was observed and the autophagy levels of normal bronchial epithelial cells and CSE stimulated cells were compared.At the same time,the number of autophagy bodies and autophagy lysosomes were observed by transmission electron microscope.4.Small interfering RNA(siRNA)and plasmid were used to knock down and overexpress SPHK1 molecules in CSE stimulated BEAS-2B and 16HBE cells respectively.The transfection efficiency and the expression of autophagy proteins were detected by RT-qPCR and Western Blotting,autophagy flow was observed under fluorescence microscope and autophagy lysosomes were observed under transmission electron microscope.SPHK1 molecule was also interfered in the stable strain cells of LC3B autophagy double fluorescent virus.The level of oxidative stress was detected by using reactive oxygen species(ROS),Superoxide dismutase(SOD)and Malondialdehyde(MDA)kits.In the recovery test,autophagy inhibitor Chloroquine(CQ)and autophagy inducer Rapamycin(Rapa)were added respectively and autophagy protein and oxidative stress were detected by Western Blotting and corresponding kits.5.Knockdown and overexpression SPHK1 molecules in BEAS-2B and 16HBE cells stimulated by CSE.RT-qPCR and Western Blotting were used to verify the efficiency and detect the level of TRAF2 mRNA and protein expression.Knock down and overexpress TRAF2,Western Blotting to verify efficiency and detect autophagy protein levels.Autophagy protein was detected by Western Blotting test in salvage experiment that co-transfection of TRAF2 and SPHK1 in BEAS-2B and 16HBE cells stimulated by CSE.6.The emphysema mouse model was established by CS exposure combined with intranasal instillation of Lipopolysaccharide(LPS).The lung tissue sections of mice were stained with hematoxylin-eosin(HE)and Immunohistochemistry(IHC),the level of oxidative stress was detected by corresponding kits,and the expression of SPHK1 and autophagy-related molecules were detected by Western Blotting and RT-qPCR.7.Peripheral blood of patients with COPD and control people were collected,PBMCs were extracted.The expression levels of SPHK1 and autophagy protein in human PBMC were detected by RT-qPCR and Western Blotting respectively.Results:1.Bioinformatics analysis showed that SPHK1 was higher expressed in lung tissues of COPD patients than control and with the increase of CSE concentration,the expression of SPHK1 increased and autophagy was impaired gradually.2.When BEAS-2B and 16HBE LC3B autophagy double fluorescence stable cells were stimulated by 3%CSE for 24 hours,fluorescence microscope observation result showed that autophagy flow decreased.and autophagosomes and autophagy lysosomes also were decreased in BEAS-2B and 16HBE cells were stimulated by 3%CSE under electron microscope.3.When BEAS-2B and 16HBE cells were stimulated by 3%CSE for 24 hours,SPHK1 molecule was knocked down,the expression of LC3BⅡ/Ⅰ and Beclinl were increased and the expression of P62 was decreased,autophagy flow was increased via fluorescence microscope,the number of autophagosomes and autophagy lysosomes also were increased under electron microscope,ROS and MDA levels were decreased,and SOD levels was increased compared with NC group.Those indicated that autophagy and oxidative stress of bronchial epithelial cells were increased in BEAS-2B and 16HBE cells stimulated by CSE and SPHK1 was knockdown.After overexpression of SPHK1 molecule,the expression of LC3B Ⅱ/Ⅰ and Beclin1 were decreased,the expression of P62 was increased,the autophagy flow was decreased and the number of autophagosomes and autophagy lysosomes also were decreased.the level of ROS and MDA increased,and the level of SOD decreased compared with NC group,those indicated that the level of autophagy was decreased and oxidative stress was increased in bronchial epithelial cells stimulated by CSE and overexpression the SPHK1.The positive and negative verification experiments showed that SPHK1 regulates the level of autophagy and oxidative stress in bronchial epithelial cells stimulated by CSE.4.3%CSE stimulated BEAS-2B and 16HBE cells for 24 hours and knocked down SPHK1.Then addition to autophagy inhibitor CQ,Western Blotting showed LC3B Ⅱ/Ⅰaccumulation,Beclinl was decreased,P62 increased,and ROS,MDA levels were increased,SOD levels was decreased.3%CSE stimulated BEAS-2B and 16HBE cells for 24 hours after overexpressed SPHK1,and adding autophagy activator Rapa,Western Blotting test showed that LC3B Ⅱ/Ⅰ and Beclinl were increased,P62 was decreased,ROS and MDA levels were decreased,SOD levels increased.The recovery experiments showed that SPHK1 affects the level of oxidative stress by regulating autophagy.5.When BEAS-2B and 16HBE cells were stimulated by 3%CSE for 24 hours,SPHK1 molecules were knocked down.RT-qPCR and Western Blotting showed the expression of TRAF2 was increased.Overexpression of SPHK1 showed the expression of TRAF2 was decreased,those suggested that SPHK1 regulated the expression of TRAF2.After knocking down TRAF2,RT-qPCR and Western Blotting showed that the level of autophagy was decreased.while the autophagy was increased after overexpression of TRAF2,suggested that TRAF2 regulates autophagy of bronchial epithelial cells stimulated by CSE.Co-transfection of SPHK1 siRNA and TRAF2 siRNA could reverse the increase of autophagy when SPHK1 was transfected alone and the decrease of autophagy when TRAF2 small interference was transfected alone.This phenomenon could also be observed when co-transfected with SPHK1 and TRAF2 plasmids.Those were suggested that SPHK1 affects autophagy by regulating TRAF2.6.The HE staining results of lung tissue sections of mouse exposed to cigarette smoke showed that the emphysema model was established successfully.The detection of IHC,Western Blotting and RT-qPCR showed that the expression of SPHK1 molecule was increased and the expression of autophagy-related molecules were decreased in the lung tissue of emphysema mouse.The level of MDA was increased and the level of SOD was decreased.Those suggested that the higher expression of SPHK1 in emphysema mouse model than control,and the level of autophagy was decreased,oxidative stress was increased.7.RT-qPCR and Western Blotting detection showed that the expression of SPHK1 in PBMCs of COPD patients were increased,LC3B Ⅱ/Ⅰ and Beclinl were decreased and autophagy was impaired.Those suggested that SPHK1 was highly expressed and autophagy was decreased in COPD patients.Conclusions:1.Autophagy-related gene SPHK1 was higher expression in lung tissues of patients with COPD than control.The higher expression of SPHK1 and the lower level of autophagy in bronchial epithelial cells stimulated by CSE than control.2.SPHK1 exacerbated autophagy damage was stimulated by CSE and enhanced oxidative stress level was mediated by autophagy.3.SPHK1 induced autophagy damage and increased the level of oxidative stress in bronchial epithelial cells stimulated by CSE via down-regulating TRAF2.4.The higher expression of SPHK1,the lower autophagy level and the higher oxidative stress level in PBMC with COPD and emphysema mouse than control.