Recombinant CC16 Protein Inhibits Cigarette Smoke-Induced Lung Epithelial Cell Senescence In A Preliminary Study

Objective:Chronic obstructive pulmonary disease（COPD）is a chronic disease with high mortality rate worldwide,and the number of morbidity and mortality is increasing year by year,but there is no safe and effective drug to reduce COPD mortality,and there is a close relationship between COPD and aging.CC16 is a strong anti-inflammatory protein secreted by Club cells,and its role in inhibiting inflammation in COPD has been widely studied,but its role in anti-cellular senescence has not been described.Methods:1.Cigarette smoke extract（CSE）was prepared and CCK-8 assay was performed to detect the cytotoxicity of CSE and rhCC16 protein on HBECs.Cellular senescence was prepared using 5%CSE for 72h and the COPD mouse model was constructed using 6-month passive smoking method,the protective role of rhCC16 was assayed.2.Western blot assay was performed to detect the levels of P16,P21,P38,p-P38,ERK1/2,p-ERK1/2,JNK and p-JNK in the control group,CSE group,rhCC16 group,P38 inhibitor group,ERK1/2 inhibitor group,JNK inhibitor group,α4β1 receptor inhibitor（BIO-1211）group,and DMSO group.3.The mRNA expression of P16,P21,IL-6,IL-8,IL-1α,IL-1β,MMP-1,MMP-3,CXCL-1,CXCL-2 in cells of control group,CSE group and rhCC16 group was measured by RT-qPCR.4.SA-β-Gal staining kit was used to detect β-galactosidase activity of cells in control group,CSE group and rhCC16 group and the positive rates were analyzed.5.Immunofluorescence staining assay was used to detect the number of positive cells in senescence-associated heterochromatic foci（SAHF）and the level of intracellular reactive oxygen species（DCFH-DA）in each group.6.COPD mouse model was constructed by passive smoking method.The lung function of mice in control group,COPD group,COPD+rhCC16 group and PBS group was determined using small animal lung function test,and the morphological changes of mice’s lungs were observed.7.Total RNA from mouse lung tissues was extracted and the mRNA expression of P16,P21,IL-6,IL-8,IL-1α,IL-1βand CXCL-1 in lung tissues of control group,COPD group,COPD+rhCC16 group and PBS group was detected by RT-qPCR.8.Immunohistochemical staining was performed to detect the levels of P16,P21,SA-β-Gal,p-P38,p-ERK1/2 proteins in lung tissues of control group,COPD group,COPD+rhCC16 group and PBS group.Results:1.Cellular senescence was successfully induced by CSE.Compared with the control group,the CSE group showed slightly increased cell volume with a flattened shape and a twitching phenomenon,which was consistent with the morphological changes of senescent cells;the levels of senescence markers P16 and P21 were substantially increased in protein and mRNA（P<0.05）.Senescence-associated secretory phenotypes（SASP）,IL-6,IL-8,IL-1α,IL-1β,MMP-1,MMP-3,CXCL-1,CXCL-2,were significantly raised in mRNA（P<0.05）.β-galactosidase staining cells was remarkable increased（P<0.05）.The positive rate of senescence-associated heterochromatic foci（SAHF）staining cells was specially enhanced.（P<0.05）.2.The COPD mouse model was successfully constructed using passive smoking.Compared with the control group,mice in the COPD model group had sparse,dull yellow and lusterless hair,some mice had hair loss,were irritable,liked to pile up when smoking,huddled,sweated easily,and breathed rapidly,which were consistent with the characteristics of the COPD.FEV_0.3/FVC ratio in COPD mice was decreased（P<0.05）,airway resistance was increased（P<0.05）,and lung compliance was declined（P<0.05）.Lung tissue showed dark red due to prolonged inhalation of cigarette smoke,and foci of congestion with deep red petechiae were seen on the lung surface;alveolar wall ruptured and collapsed,irregular enlargement of alveoli,formation of large alveoli with inflammatory cell infiltration,partial loss of airway mucosal epithelium,cilia atrophy,luminal narrowing,and thickening of the duct wall.The expression of P16 and P21mRNA were greatly elevated（P<0.05）.SASP factors,IL-6,IL-8,IL-1α,IL-1β,and CXCL-1 were significantly augmented（P<0.05）.The levels of P16,P21,and SA-β-Gal in lung tissues of COPD mice were raised.3.rhCC16 protein had an inhibitory effect on cigarette smoke-induced senescence in vitro and in vivo.The morphology changes of cellular senescence was restored in rhCC16group compared with those in the CSE group.The levels of P16 and P21 in proteins and mRNA were hugely decreased（P<0.05）.IL-6,IL-8,IL-1α,IL-1β,MMP-1,MMP-3 were significantly descended（P<0.05）.β-galacto glycosidase staining cells were enormously declined（P<0.05）.Senescence-associated heterochromatic foci（SAHF）staining cells were highly declined（P<0.05）.ROS levels were significantly reduced.（P<0.05）.Compared with the COPD group,FEV_0.3/FVC ratio increased in rhCC16 roup（P<0.05）,airway resistance decreased（P<0.05）,and lung compliance increased（P<0.05）.Lung tissue congestion foci were reduced.Alveolar structure was restored;the expression of P16 and P21 mRNA were significantly abated（P<0.05）.The expression of IL-6,IL-8,IL-1α,IL-1β,CXCL-1 mRNA was thoroughly decreased（P<0.05）.The positive cells of P16,P21,and SA-β-Gal staining were reduced.4.In vitro experiments,p-P38 and p-ERK1/2 in the CSE group was augmented compared to the control group（P<0.05）.Compared with the CSE group,rhCC16 protein effectively inhibited the phosphorylation of P38 and ERK1/2（P<0.05）.The MAPK pathway inhibitor decreased the phosphorylation of P38 and ERK1/2,also reversed the anti-senescence of rhCC16.The phosphorylation of P38 and ERK1/2 in lung tissues was increased in the COPD mice compared with those in control group（P<0.05）.rhCC16protein effectively inhibited the phosphorylation of P38 and ERK1/2 in lung tissues.5.rhCC16 protein bound to integrinα4β1 exerted anti-senescence effects.The levels of P16,P21,p-P38,and p-ERK1/2 were elevated in the BIO-1211 group compared to the rhCC16 group.Conclusion:rhCC16 protein binds toα4β1 to enter cells and inhibit cigarette smoke-induced cellular senescence via the P38 MAPK/ERK1/2 signaling pathway.