Preliminary Study On Recombinant CC16 Protein Inhibiting Inflammatory Response Induced By Cigarette Smoke

Objective:The number of morbidity and mortality of chronic obstructive pulmonary disease(COPD)has been increasing year by year in recent years,but the current clinical treatment drugs commonly used bronchodilators and glucocorticoids to treat its airway obstruction and systemic inflammatory response,with obvious adverse effects,while CC16 protein is the most abundant protective endogenous protein in bronchus and lung,if CC16 protein is purified in vitro as a therapeutic drug,it may reduce the adverse effects in treatment.adverse effects and provide new ideas for clinical treatment of COPD.In this experiment,human recombinant CC16 protein was prepared in vitro by genetic engineering,and its effect on cigarette smoke-induced inflammatory response and possible mechanisms were investigated by verifying it at in vitro and in vivo levels.Methods:1.p GEX-6P-1-hCC16 recombinant plasmid was constructed,GST-hCC16 protein was induced to be expressed and purified,and pure hCC16 protein was obtained by enzymatic cleavage.2.Cigarette smoke extract(CSE)was prepared and the CSE and rhCC16 protein action concentration was screened using the CCK-8 method.The m RNA levels of CC16,IFN-γ,TNF-α,IL-1β,IL-4 and IL-5 were detected in each group of cells by q PCR.The protein expression levels of c-Jun,p-c-Jun,c-Fos,p-c-Fos and the nuclear entry levels of cJun and c-Fos in each group of cells were detected using Weastern blotting.3.A passive smoking method was used to establish a COPD model in mice exposed to cigarette smoke,and an rhCC16 intervention group was set up.After modelling,lung function was measured using a lung function meter,bronchoalveolar lavage fluid(BALF)for cell counting,ELISA for CC16,IFN-γ,TNF-α,IL-1β,IL-4,IL-5 protein levels in BALF,immunohistochemical staining for lung CC16 protein expression levels,histopathological analysis of morphological changes in mouse lungs,q PCR for IFN-γ,TNF-α,IL-1β,IL-4,IL-5 m RNA levels in lung tissues,Weastern blotting for c-Jun,p-c-Jun,c-Fos,p-c-Fos expression levels in lung tissues.Results:1.Obtain rhCC16 protein with > 95% purity and biological activity.2.Compared to the control group,5% CSE intervention cells caused an increase in IFN-γ,TNF-α and IL-1β(P<0.05),a decrease in IL-4 and IL-5(P<0.05),an increase in both pc-Jun/c-Jun and p-c-Fos/c-Fos(P<0.05)and promoted their nuclear translocation;all indices were alleviated in the rhCC16 intervention group.3.Compared with the control group,the lung function of COPD model mice decreased(P<0.05);inflammatory cell count and inflammatory factor protein level in BALF increased(P<0.05)and CC16 protein level decreased;CC16 immunohistochemistry results showed that the COPD model group was weakly positive;The m RNA levels of inflammatory factors in lung tissues were elevated(P<0.05);collapsed alveolar walls with multiple fractures,irregularly enlarged and fused alveoli,and different degrees of inflammatory cell infiltration in the interstitium of the lung were seen in the COPD model group;the mean alveolar lining interval increased,the mean alveolar number decreased,and the alveolar destruction index increased(P<0.05);p-c-Jun/c-Jun and p-c-Fos/c-Fos were both increased(P<0.05);all indices were alleviated in the rhCC16 intervention group.Conclusion:1.RhCC16 protein was successfully induced to be expressed and purified.2.RhCC16 protein can inhibit the inflammatory response produced by CSE-induced HBEC cells.3.RhCC16 protein can inhibit the inflammatory response in COPD model mice.4.Inhibition of CSE-induced inflammatory response by rhCC16 is dependent on inhibition of AP-1 signaling pathway activation.