Effects Of Cigarette Smoke Extract On MTOR In Human Peripheral Blood Denfritic Cells

Tobacco hazards are the greatest preventable risk factor for human health Smoking can increase the incidence of respiratory diseases,cardiovascular diseases and cancers.According to the epidemiological survey,the number of deaths caused by smoking and second-hand smoke exposure worldwide reaches 6 million,while the number of deaths caused by smoking in China exceeds 1 million annually.Among the smokers,at least 25%of the persistent smokers develop chronic obstructive pulmonary disease(COPD),which brings heavy financial burden to society and familyPart 1 To investigate the expression and significance of p-mTOR in peripheral myeloid dendritic cells(mDCs)of smokersObjective:To investigate the expression and significance of p-mTOR in myeloid dendritic cells in peripheral blood of healthy smokersMethods 15 smokers with normal pulmonary function and 15 non-smokers as control group were collected.Flow cytometry was used to detect the phenotypic characteristics of mDCs in peripheral blood and the expression of p-mTOR in mDCs cells of the two groups;ELISA was used to detect the concentration of IL-6,IL-12 and IL-23 in serum of the two groups;correlation analysis was used to analyze the correlation between the expression of p-mTOR and CD86 in mDCs,p-mTOR in mDCs and IL-6,IL-12 and IL-23 in serum of smokers.Results(1)The expression of CD86 and p-mTOR in peripheral mDCs of smokers with normal pulmonary function was significantly higher than that of non-smokers.(2)Compared with non-smokers,the serum levels of IL-6,IL-12 and IL-23 in smokers with normal pulmonary function increased significantly(3)The correlation analysis showed that the serum levels of IL-6,IL-12 and IL-23 in smokers with normal pulmonary function were positively correlated with the expression of p-mTOR in mDCsConclusion Smoking can activate mDCs in peripheral blood,and mTOR phosphorylation may be one of the mechanisms of mDCs activationPart 2 Effects of Cigarette Smoke Extract(CSE)on the Activation of mTOR in Monocyte-derived Dendritic Cells(MoDCs)and Its Related MechanismsObjective To investigate the effect of cigarette smoke extract on the activation of mTOR in monocyte-derived dendritic cells and its related mechanism.Methods Human peripheral blood mononuclear cells were isolated and cultured for 6 days,then induced into MoDCs.CSE was used to stimulate the cells.Flow cytometry was used to detect the expression of co-stimulatory molecule and p-mTOR in the cells.Western Blot was used to detect the expression of p-mTOR,p-P70S6K and p-4EBP-1.ELISA was used to detect the concentration of IL-1?,IL-6,TNF-? and IL-12 in the supernatant of culture medium.The effect of rapamycin on the maturation of CSE-stimulated MoDCs was investigated.Results(1)CSE could significantly up-regulate the expression of p-mTOR,p-P70S6K and p-4EBP-1.CSE stimulated human MoDCs in vitro.(2)CSE stimulated human MoDCs in vitro.The results showed that CSE could robustly up-regulate the expression of costimulatory molecule CD86 in MoDCs,showing maturation and differentiation.(3)CSE can effectively stimulate the secretion of cytokines IL-1?,IL-6,IL-12 and TNF-a by MoDCs.(4)Rapamycin inhibits the expression of p-mTOR,p-P70S6K and p-4EBP-1 induced by CSE in MoDCs.(5)Rapamycin can inhibit CSE-induced expression of costimulatory molecule CD86 in MoDCs.(6)Rapamycin can inhibit the secretion of cytokines IL-1?,IL-6,IL-12,TNF-? and IL-12 by MoDCs.Conclusion Under cigarette smoke exposure,cigarette smoke can promote the maturation and differentiation of MoDCs through mTOR signaling pathway phosphorylation.