| 【Background】Bronchial asthma is a chronic airway inflammatory disease mainly mediated by T lymphocytes.There are about 358 million patients with asthma around the world.The prevalence is about 16.4%.Up to 400,000 people die of asthma every year.Smoking is very common among patients with asthma.Most non-smoking patients with asthma are exposed to second-hand smoke environment.Smoking is significantly correlated to asthma.Smokers have a nearly threefold increased risk of asthma.Smoking often aggravates airway lymphocyte inflammation in patients with asthma,and causes disorder of lymphocytes in secreting cytokines,aggravating the condition,making the symptoms more difficult to control and gradually insensitive to hormones.Accordingly,the asthma turns to severe asthma,bringing a heavy burden to patients.Therefore,exploring the mechanism of lymphocyte inflammation caused by smoking is of great significance to improve the diagnosis and treatment of smoking patients with asthma.Some studies have shown that SIRT1 is involved in the regulation of Th17/Treg imbalance in smoking patients with asthma,whose immune imbalance is mainly relevant to the expression of IL17 and IL10.Most of the existing experiments study from the perspectives of animal experiments and clinical trials.However,there is a lack of experiments that explore at the lymphocyte level.Therefore,this study intends to explore the expression and significance of SIRT1,IL-17 and IL-10 in lymphocytic inflammation under the action of cigarette extract by molecular biological technique at the cellular level.【Objective】To study the changes in the secretion of SIRT1,IL-17 and IL-10 by lymphocytes caused by cigarette smoke extract(CSE)and the significance at the cell experiment level with the lymphocytic inflammation model induced by CSE,so as to explore the possible mechanism by which smoking aggravates lymphocyte inflammation.【Methods】First,RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin streptomycin mixture was prepared for suspension culture of H9 human lymphocyte line.Then,a CSE extraction device was made by ourself,and the CSE was made by it.The cytotoxicity of CSE was detected by CCK8 method to explore its optimum concentration and timing for stimulating lymphocytes.In the experimental group,the cells were stimulated with complete medium containing the optimal concentration of cigarette smoke extract;In the blank control group,the cells were cultured with complete medium.The m RNA expression of SIRT1,IL-17 and IL-10 in each group was detected by RT-q PCR method after stimulating 24 hours.The protein expression of SIRT1 in each group was detected by Western Blot method after stimulating 48 hours.The protein expression of IL-17 and IL-10 in each group was detected by ELISA method after stimulating 48 hours.【Results】1.After the stimulation with CSE,the expression of SIRT1 m RNA and IL-10 m RNA in lymphocytes decreased(0.67±0.03;0.15±0.01 P<0.01),while the expression of IL-17 m RNA increased(3.40±0.31,P<0.01).2.After the stimulation with CSE,the expression of SIRT1 m RNA in lymphocytes decreased(0.25±0.12,P<0.01),the expression of IL-17 protein increased(3.43±0.65 pg/ml VS 1.17±0.42pg/ml,P<0.05),and the expression of IL-10 protein decreased significantly(0.49±0.17 pg/ml VS 1.58±0.42 pg/ml,P<0.05).3.Correlation analysis: Under the action of CSE,the expression of SIRT1 m RNA was positively correlated to that of IL-10 m RNA(r=0.999,P<0.05),and the expression of SIRT1 protein was positively correlated to that of IL-10 protein(r=0.989,P<0.05);The expression of SIRT1 m RNA was uncorrelated to that of IL-17 m RNA in lymphocytes(r=-0.959,P>0.05),and the expression of SIRT1 protein was negatively correlated to that of IL-17 protein(r=-0.921,P>0.05);The expression of IL-17 m RNA was uncorrelated to that of IL-10 m RNA(r=-0.971,P>0.05),and the expression of IL-17 protein was uncorrelated to that of IL-10 protein(r=-0.969,P>0.05).【Conclusion】1.Cigarette smoke extract causes inflammation of lymphocytes,and down-regulates the expression of SIRT1.2.Cigarette smoke extract causes inflammation of lymphocytes,increases IL-17 expression,down-regulates the expression of IL-10,and increases the proportion of IL-17/IL-10. |
