红霉素对烟草烟雾刺激下人巨噬细胞TNF-α释放的抑制作用及其机制

Inhibition of erythromycin on TNF-α release from human macrophages stimulated by cigarette smoke

目的探讨红霉素对烟草烟雾刺激下人巨噬细胞TNF-α释放的抑制作用及其机制。方法应用佛波酯(PMA)诱导人单核细胞系U937(以下称U937细胞)为巨噬细胞,用0.1%、1%、2.5%的烟草烟雾提取物(CSE)和0.1、1、10μg/mL的红霉素分别作用于巨噬细胞24、48、72 h,筛选对巨噬细胞增殖活性影响最小的红霉素、CSE作用浓度和作用时间。经筛选,选用1μg/mL红霉素溶液和1%CSE溶液、作用时间24 h进行后续实验。将U937细胞诱导分化为巨噬细胞,随机将细胞分为空白对照组、CSE组、CSE+红霉素组、TSA组。空白对照组不予处理;CSE组加入1%CSE溶液孵育24 h;红霉素+CSE组先加入1μg/mL红霉素溶液预孵育24 h,再加入1%CSE溶液孵育24 h;TSA组加入100 ng/mL TSA孵育24 h。收集各组培养液上清,采用ELISA法检测培养液上清TNF-α含量,采用Western blotting法检测各组HDAC1、NF-κB蛋白表达。结果空白对照组培养液上清TNF-α含量为(274.96±182.39)pg/mL,CSE组为(744.46±638.38)pg/mL,红霉素+CSE组为(646.57±603.53)pg/mL(P均<0.05)。与空白对照组比较,CSE组、红霉素+CSE组、TSA组HDAC1蛋白表达降低、NF-κB蛋白表达升高(P均<0.05);与CSE组比较,红霉素+CSE组HDAC1蛋白表达升高,NF-κB蛋白表达降低(P均<0.05);与红霉素+CSE组比较,TSA组HDAC1蛋白表达降低,NF-κB蛋白表达升高(P均<0.05)。结论红霉素能抑制烟草烟雾刺激下巨噬细胞TNF-α释放;其作用机制可能是通过恢复受烟草烟雾抑制的HDAC1蛋白表达,抑制NF-κB蛋白表达,使NF-κB依赖的TNF-α释放减少,继而减轻炎症反应。

Objective To investigate the inhibitory effect of erythromycin on the TNF-α release of human macrophages stimulated by cigarette smoke and its possible mechanism. Methods The human monocytic cell line U937 cells were differentiated into human macrophages by Phorbol esters（ PMA）. The cigarette smoke extract（ CSE）（ 0. 1,1,2. 5%） and erythromycin（ EM）（ 0. 1,1,10 μg/mL） were used to deal with the macrophages at different time points（ 24,48,and72 h）,respectively,and we chose the time and concentrations with the least effect on proliferation activity of macrophages.The 1 μg/mL CSE,1% EM and 24-hour treatment time were selected for the experiment. The differentiated U937 cells were randomly divided into four groups： the control group（ with no intervention）,CSE group（ 1% CSE stimulation for24 h）,CSE ＋ EM group（ pre-incubation with 1 μg/ml EM for 24 h before 1% CSE stimulation for 24 h）,and TSA group（ TSA stimulation for 24 h）. The TNF-α level in the culture supernatants was measured by ELISA; Western blotting was used to measure the expression of the histone deacetylase-1（ HDAC1） and NF-κB protein. Results The level of TNF-α in the culture supernatants of the control group,CSE group,and CSE ＋ EM group were（ 274. 96 ± 182. 39）,（ 744. 46 ±638. 38）,and（ 646. 57 ± 603. 53） pg/mL,respectively（ all P〈0. 05）. Compared with the control group,the expression of HDAC1 protein in the CSE group,EM ＋ CSE group,and TSA group decreased,and the expression of NF-κB protein increased（ all P〈0. 05）. Compared with the CSE group,the expression of HDAC1 protein increased,and the expression of NF-κB protein decreased in the EM ＋ CSE group（ both P〈0. 05）. Compared with the EM ＋ CSE group,the expression of HDAC1 protein in the TSA group decreased,and the expression of NF-κB protein increased（ both P〈0. 05）. Conclusions EM inhibits the release of TNF-α of human macrophages induced by cigarette smoke. The mechanism is that EM may restore the expression of HDAC1 and inhibit the expression of NF-κB,which decreases the release of TNF-α and thus alleviates the inflammatory response.