气管狭窄肉芽组织中组蛋白去乙酰化酶2的表达及其与气管狭窄的关系
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摘要
目的研究组蛋白去乙酰化酶2(HDAC2)在良性气管狭窄动物模型中的表达,并探讨HDAC2在气管狭窄发生中的作用机制,为治疗良性气管狭窄寻求新靶标。方法将18只家兔完全随机分成空白对照组、模型组和红霉素组,每组各6只。模型组和红霉素组家兔行气管切开并用尼龙刷来回刷20余次损伤气管内壁制作良性气管狭窄的动物模型。术前7 d至术后9 d模型组给予生理盐水灌胃,红霉素组给予小剂量红霉素15 mg·kg~(–1)·d~(–1)灌胃,空白对照组不做任何处理。术后第10 d处死各组家兔,切取狭窄段气管,测量气管狭窄度,并取狭窄段肉芽组织提取RNA和蛋白质。用实时荧光定量PCR检测肉芽组织中HDAC2、白细胞介素-6(IL-6)、IL-8的mRNA相对表达量,用蛋白免疫印迹检测肉芽中HDAC2蛋白的相对表达量。结果模型组与空白对照组比较,气管狭窄明显[(84.60±1.14)%比(27.00±6.44)%],HDAC2 mRNA和蛋白表达下调(0.29±0.07比1.00±0.00,0.20±0.02比0.49±0.04),IL-6、IL-8 mRNA表达上调(4.22±0.67比1.00±0.00,162.72±23.23比1.00±0.00)。红霉素组与模型组比较,气管狭窄度减轻[(64.00±12.25)%比(84.60±1.14)%],HDAC2 mRNA和蛋白表达上调(0.42±0.14比0.29±0.07,0.43±0.01比0.20±0.02),IL-6、IL-8 mRNA表达下调(0.72±0.24比4.22±0.67,130.22±7.93比162.72±23.23),差异均具有统计学意义(均P<0.05)。气管狭窄度与HDAC2 mRNA相对表达量呈负相关(Pearson相关系数r=–0.96,P<0.05)。结论良性气管狭窄动物模型中HDAC2表达下调与气管狭窄的发生发展有关。小剂量红霉素具有治疗良性气管狭窄的作用,可能是小剂量红霉素上调HDAC2的表达,进而抑制气管损伤修复时的炎症失调有关。
Objective To investigate the expression of histone deacetylase 2(HDAC2) in animal model of benign tracheal stenosis, and explore the mechanism of HDAC2 in development of tracheal stenosis. Methods Eighteen rabbits were randomly divided into a blank control group, a model group, and an erythromycin group, with 6 rats in each group.The model group and the erythromycin group underwent tracheostomy, the inner wall of trachea was brushed back and forth with a nylon brush for more than 20 times to induce benign tracheal stenosis. From 7 days before surgery to 9 days after surgery, the model group received gavage with saline, the erythromycin group received gavage with low-dose erythromycin in dose of 15 mg·kg~(-1)·d~(-1), and the control group did not receive any treatment. On the 10 th day after operation, all the rabbits were sacrificed and the trachea was cut to measure the tracheal stenosis. RNA and protein were extracted from the granulation tissue in the stenosis and the relative m RNA expressions of HDAC2, interleukin(IL)-6 and IL-8 in the granulation tissue were detected by real-time fluorescence quantitative PCR. The relative expression of HDAC2 protein was detected by Western blot. Results Compared with the blank control group, the tracheal stenosis in the model group was more obvious [(84.60±1.14)% vs.(27.00±6.44)%], the mRNA and protein expressions of HDAC2 were decreased(0.29±0.07 vs. 1.00±0.00, 0.20±0.02 vs. 0.49±0.04), the m RNA expressions of IL-6 and IL-8 were up-regulated(4.22±0.67 vs. 1.00±0.00, 162.72±23.23 vs.1.00±0.00). Compared with the model group, tracheal stenosis in the erythromycin group was relieved [(64.00±12.25)% vs.(84.60±1.14)%], the mRNA and protein expressions of HDAC2 were increased(0.42±0.14 vs. 0.29±0.07, 0.43±0.01 vs. 0.20±0.02), the mRNA expressions of IL-6 and IL-8 were decreased(0.72±0.24 vs. 4.22±0.67, 130.22±7.93 vs. 162.72±23.23). All the differences were statistically significant(all P<0.05). The Pearson correlation coefficient between tracheal stenosis and HDAC2 mRNA relative expression was –0.96(P<0.05).Conclusions The down-regulation of HDAC2 expression in model of benign tracheal stenosis is related to the occurrence and development of tracheal stenosis. The low dose of erythromycin may be used to treat benign tracheal stenosis by up-regulating expression of HDAC2 and thus inhibiting the inflammatory disorder during tracheal injury repair.
Objective To investigate the expression of histone deacetylase 2(HDAC2) in animal model of benign tracheal stenosis, and explore the mechanism of HDAC2 in development of tracheal stenosis. Methods Eighteen rabbits were randomly divided into a blank control group, a model group, and an erythromycin group, with 6 rats in each group.The model group and the erythromycin group underwent tracheostomy, the inner wall of trachea was brushed back and forth with a nylon brush for more than 20 times to induce benign tracheal stenosis. From 7 days before surgery to 9 days after surgery, the model group received gavage with saline, the erythromycin group received gavage with low-dose erythromycin in dose of 15 mg·kg~(-1)·d~(-1), and the control group did not receive any treatment. On the 10 th day after operation, all the rabbits were sacrificed and the trachea was cut to measure the tracheal stenosis. RNA and protein were extracted from the granulation tissue in the stenosis and the relative m RNA expressions of HDAC2, interleukin(IL)-6 and IL-8 in the granulation tissue were detected by real-time fluorescence quantitative PCR. The relative expression of HDAC2 protein was detected by Western blot. Results Compared with the blank control group, the tracheal stenosis in the model group was more obvious [(84.60±1.14)% vs.(27.00±6.44)%], the mRNA and protein expressions of HDAC2 were decreased(0.29±0.07 vs. 1.00±0.00, 0.20±0.02 vs. 0.49±0.04), the m RNA expressions of IL-6 and IL-8 were up-regulated(4.22±0.67 vs. 1.00±0.00, 162.72±23.23 vs.1.00±0.00). Compared with the model group, tracheal stenosis in the erythromycin group was relieved [(64.00±12.25)% vs.(84.60±1.14)%], the mRNA and protein expressions of HDAC2 were increased(0.42±0.14 vs. 0.29±0.07, 0.43±0.01 vs. 0.20±0.02), the mRNA expressions of IL-6 and IL-8 were decreased(0.72±0.24 vs. 4.22±0.67, 130.22±7.93 vs. 162.72±23.23). All the differences were statistically significant(all P<0.05). The Pearson correlation coefficient between tracheal stenosis and HDAC2 mRNA relative expression was –0.96(P<0.05).Conclusions The down-regulation of HDAC2 expression in model of benign tracheal stenosis is related to the occurrence and development of tracheal stenosis. The low dose of erythromycin may be used to treat benign tracheal stenosis by up-regulating expression of HDAC2 and thus inhibiting the inflammatory disorder during tracheal injury repair.
引文
1Nouraei SA,Ma E,Patel A,et al.Estimating the population incidence of adult post-intubation laryngotracheal stenosis.Clin Otolaryngol,2007,32(5):411-412.
2 吴旋,苏振忠,胡丽茎,等.560例气管切开行机械通气患者并发气管狭窄的危险因素分析.中华耳鼻咽喉头颈外科杂志,2007,42(11):839-842.
3李莉华,徐明鹏,甘罗曼,等.小剂量红霉素对气管损伤后肉芽组织增生的影响.中华医学杂志,2017,97(10):777-781.
4 李梅华,钟小宁,文明智,等.红霉素对香烟烟雾刺激的人巨噬细胞组蛋白去乙酰化酶2表达降低的抑制作用.国际呼吸杂志,2014,34(18):1383-1387.
5Nakagishi Y,Morimoto Y,Fujita M,et al.Rabbit model of airway stenosis induced by scraping of the tracheal mucosa.Laryngoscope2005,115(6):1087-1092.
6 Mazhar K,Gunawardana M,Webster P,et al.Bacterial biofilms and increased bacterial counts are associated with airway stenosis Otolaryngol Head Neck Surg,2014,150(5):834-840.
7宋岩,杨静,季文樾.大耳白兔气管切开术后炎症及机械损伤导致气管狭窄的实验研究.中国医科大学学报,2009,38(1):50-51,59
8 李莉华,黎雨,甘罗曼,等.静脉用抗菌素对良性气管狭窄呼吸内镜介入治疗术后长期疗效的影响.国际呼吸杂志,2017,37(15)1154-1160.
9López-Boado YS,Rubin BK.Macrolides as immunomodulatory medications for the therapy of chronic lung diseases.Curr Opin Pharmacol,2008,8(3):286-291.
10 Seemungal TA,Wilkinson TM,Hurst JR,et al.Long-term erythromycin therapy is associated with decreased chronic obstructive pulmonary disease exacerbations.Am J Respir Crit Care Med,2008,178(11):1139-1147.
11Blasi F,Mantero M,Aliberti S.Antibiotics as immunomodulant agents in COPD.Curr Opin Pharmacol,2012,12(3):293-299.
12Friedlander AL,Albert RK.Chronic macrolide therapy in inflammatory airways diseases.Chest,2010,138(5):1202-1212.
13 Yatsunami J,Tsuruta N,Hara N,et al.Inhibition of tumor angiogenesis by roxithromycin,a 14-membered ring macrolide antibiotic.Cancer Letters,1998,131(2):137-143.
14He ZY,Ou LM,Zhang JQ,et al.Effect of 6 months of erythromycin treatment on inflammatory cells in induced sputum and exacerbations in chronic obstructive pulmonary disease.Respiration,2010,80(6):845-852.
15 牛卉,梅和坤,胡雪栋,等.红霉素长期小剂量超说明书用药的文献计量分析.中国临床药理学杂志,2015,31(3):223-226.
16Yao H,Rahman I.Current concepts on oxidative/carbonyl stress,inflammation and epigenetics in pathogenesis of chronic obstructive pulmonary disease.Toxicol Appl Pharmacol,2011,254(2):72-85.
17 Adenuga D,Yao H,March TH,et al.Histone deacetylase 2 is phosphorylated,ubiquitinated,and degraded by cigarette smoke.Am J Respir Cell Mol Biol,2009,40(4):464-473.
18Bhavsar P,Ahmad T,Adcock IM.The role of histone deacetylases in asthma and allergic diseases.J Allergy Clin Immunol,2008,121(3):580-584.
19 Ito K,Hanazawa T,Tomita K,et al.Oxidative stress reduces histone deacetylase 2 activity and enhances IL-8 gene expression:role of tyrosine nitration.Biochem Biophys Res Commun,2004,315(1):240-245.
20Marwick JA,Kirkham PA,Stevenson CS,et al.Cigarette smoke alters chromatin remodeling and induces proinflammatory genes in rat lungs.Am J Respir Cell Mol Biol,2004,31(6):633-642.
21 Guven M,Turan F,Eyibilen A,et al.A comparison of the efficacy of 5-fluorouracil/triamcinolone,carnitine and dexamethasone therapy on wound healing in tracheal injury:potential for preventing tracheal stenosis?.Eur Arch Otorhinolaryngol,2012,269(1):201-206.
2 吴旋,苏振忠,胡丽茎,等.560例气管切开行机械通气患者并发气管狭窄的危险因素分析.中华耳鼻咽喉头颈外科杂志,2007,42(11):839-842.
3李莉华,徐明鹏,甘罗曼,等.小剂量红霉素对气管损伤后肉芽组织增生的影响.中华医学杂志,2017,97(10):777-781.
4 李梅华,钟小宁,文明智,等.红霉素对香烟烟雾刺激的人巨噬细胞组蛋白去乙酰化酶2表达降低的抑制作用.国际呼吸杂志,2014,34(18):1383-1387.
5Nakagishi Y,Morimoto Y,Fujita M,et al.Rabbit model of airway stenosis induced by scraping of the tracheal mucosa.Laryngoscope2005,115(6):1087-1092.
6 Mazhar K,Gunawardana M,Webster P,et al.Bacterial biofilms and increased bacterial counts are associated with airway stenosis Otolaryngol Head Neck Surg,2014,150(5):834-840.
7宋岩,杨静,季文樾.大耳白兔气管切开术后炎症及机械损伤导致气管狭窄的实验研究.中国医科大学学报,2009,38(1):50-51,59
8 李莉华,黎雨,甘罗曼,等.静脉用抗菌素对良性气管狭窄呼吸内镜介入治疗术后长期疗效的影响.国际呼吸杂志,2017,37(15)1154-1160.
9López-Boado YS,Rubin BK.Macrolides as immunomodulatory medications for the therapy of chronic lung diseases.Curr Opin Pharmacol,2008,8(3):286-291.
10 Seemungal TA,Wilkinson TM,Hurst JR,et al.Long-term erythromycin therapy is associated with decreased chronic obstructive pulmonary disease exacerbations.Am J Respir Crit Care Med,2008,178(11):1139-1147.
11Blasi F,Mantero M,Aliberti S.Antibiotics as immunomodulant agents in COPD.Curr Opin Pharmacol,2012,12(3):293-299.
12Friedlander AL,Albert RK.Chronic macrolide therapy in inflammatory airways diseases.Chest,2010,138(5):1202-1212.
13 Yatsunami J,Tsuruta N,Hara N,et al.Inhibition of tumor angiogenesis by roxithromycin,a 14-membered ring macrolide antibiotic.Cancer Letters,1998,131(2):137-143.
14He ZY,Ou LM,Zhang JQ,et al.Effect of 6 months of erythromycin treatment on inflammatory cells in induced sputum and exacerbations in chronic obstructive pulmonary disease.Respiration,2010,80(6):845-852.
15 牛卉,梅和坤,胡雪栋,等.红霉素长期小剂量超说明书用药的文献计量分析.中国临床药理学杂志,2015,31(3):223-226.
16Yao H,Rahman I.Current concepts on oxidative/carbonyl stress,inflammation and epigenetics in pathogenesis of chronic obstructive pulmonary disease.Toxicol Appl Pharmacol,2011,254(2):72-85.
17 Adenuga D,Yao H,March TH,et al.Histone deacetylase 2 is phosphorylated,ubiquitinated,and degraded by cigarette smoke.Am J Respir Cell Mol Biol,2009,40(4):464-473.
18Bhavsar P,Ahmad T,Adcock IM.The role of histone deacetylases in asthma and allergic diseases.J Allergy Clin Immunol,2008,121(3):580-584.
19 Ito K,Hanazawa T,Tomita K,et al.Oxidative stress reduces histone deacetylase 2 activity and enhances IL-8 gene expression:role of tyrosine nitration.Biochem Biophys Res Commun,2004,315(1):240-245.
20Marwick JA,Kirkham PA,Stevenson CS,et al.Cigarette smoke alters chromatin remodeling and induces proinflammatory genes in rat lungs.Am J Respir Cell Mol Biol,2004,31(6):633-642.
21 Guven M,Turan F,Eyibilen A,et al.A comparison of the efficacy of 5-fluorouracil/triamcinolone,carnitine and dexamethasone therapy on wound healing in tracheal injury:potential for preventing tracheal stenosis?.Eur Arch Otorhinolaryngol,2012,269(1):201-206.