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香烟对大鼠肺白介素—8、谷胱甘肽及组蛋白去乙酰化酶2的影响

2014-09-24罗鹏等

中国医学创新 2014年22期
关键词:香烟戒烟烟雾

罗鹏等

【摘要】 目的:探讨香烟暴露及戒烟对大鼠肺白介素-8、谷胱甘肽(GSH)及组蛋白去乙酰化酶2(HDAC2)的影响。方法:健康雄性SD大鼠40只随机分对照组、吸烟组(1月、2月组)、戒烟组(1月、2月组)。每组8只,吸烟组及戒烟组大鼠每天给予烟熏两次,吸烟组烟熏1月、2月取材;戒烟组烟熏2月后再继续饲养1月和2月后取材,分别检测支气管肺泡灌洗液(BALF)中白介素-8和GSH、肺组织HDAC2的表达。结果:比较吸烟组、戒烟组和对照组BALF中白介素-8含量升高、GSH浓度下降,戒烟后GSH渐升高,但仍不能达到正常;吸烟组肺组织HDAC2含量逐渐降低,戒烟后HDAC2又逐渐升高,但与对照组比较,仍有下降。结论:香烟暴露可诱导气道氧化应激反应,导致GSH减少,HDAC2减少,戒烟可有所改善。

【关键词】 香烟; 白介素-8; 谷胱甘肽; 组蛋白去乙酰化酶2

【Abstract】 Objective: To evaluate the impacting of IL-8, GSH and HDAC2 in the lung of cigarette exposure and cessation rats. Method: 40 SD rats were randomly divided into control group, smoking group (January, February group), smoking cessation group (January, February group), smoking group were sacrificed at 1 month and 2 month, smoking cessation group was sacrificed when smoking cessation for 1 month and 2 months. To collect the cells in BALF and lung tissue, and detect the IL-8,GSH and HDAC2 in rats lung tissue. Result: Compared with control group, the levels of IL-8 in smoking group and smoking cessation group increased (P<0.01), the levels of GSH and HDAC2 in smoking group and smoking cessation group decreased (P<0.01), but smoking groups level was lower than that of smoking-cessation groups. Conclusion: It shows that cigarette exposure can induce airway oxidative stress. It can decrease after quitting, but still can't back to the normal.

【Key words】 Smoke; IL-8; GSH; HDAC2

First-authors address: Xili Peoples Hospital in Nanshan District of Shenzhen City, Shenzhen 518055, China

doi:10.3969/j.issn.1674-4985.2014.22.006

吸烟是导致慢性阻塞性肺疾病(Chronic obstructive pulmonary disease, COPD)的重要因素[1]。慢性气道炎症是COPD的典型特征,香烟烟雾通过激活吸烟者和COPD患者肺部的氧化应激反应触发了气道的慢性炎症[2]。本文利用烟熏大鼠造成大鼠COPD模型,分析吸烟及戒烟后大鼠支气管肺泡灌洗液中谷胱甘肽(Glutathione, GSH)及肺组织中组蛋白去乙酰化酶2(histone deacetylase 2, HDAC2)的变化,进一步探讨吸烟导致COPD的分子机制。

1 材料与方法

1.1 材料 GSH ELIASA试剂盒(美国RD公司),兔抗鼠HDAC2抗体及免疫组化SP试剂盒购自北京博奥森生物技术有限公司。

1.2 实验分组 健康清洁级雄性大鼠40只(安徽医科大学实验动物中心提供),随机分五组,对照组、吸烟1月组、吸烟2月组、戒烟1月组和戒烟2月组。

1.3 模型制备 红三环牌过滤嘴香烟:焦油含量11 mg/支,烟气一氧化碳量15 mg/支,烟碱量0.8 mg/支,由安徽中烟工业公司滁州卷烟厂出品。各实验组均在自制吸烟箱(80 cm×70 cm×50 cm)每天两次,每次10支香烟持续烟熏半小时。戒烟组在烟熏两个月后停止并继续饲养1月及2月。

1.4 取材 大鼠腹腔注射10%的水合氯醛(3 mL/kg)麻醉,开胸分离肺后结扎左主支气管,右肺注入10 mL生理盐水后抽取肺泡灌洗液,重复3次,回收液离心后留上清液于-70 ℃冰冻备检。左肺组织送病理和免疫组化检测。

1.5 指标检测 IL-8、GSH和HDAC2严格按照ELIASA试剂盒操作。

1.6 统计学处理 使用SPSS 13.0软件,不同组间差异性采用单因素方差分析,两变量间的相关性分析用Pearson相关,以P<0.05为差异有统计学意义。

2 结果

2.1 肺组织病理改变 实验组大鼠双肺有不同程度气肿及淤血。光镜下:对照组大鼠肺泡结构正常连续;吸烟1月组肺泡结构受损,可见支气管黏膜上皮纤毛倒伏、粘连,肺泡内见淋巴细胞和浆细胞浸润;吸烟2月组损害加重,肺泡腔扩大融合,弹力纤维层破坏,肺泡数量减少;戒烟组仍可见肺泡结构受损、肺泡融合,淋巴细胞和浆细胞浸润较吸烟组减少;戒烟2月组较1月组病理改变轻。endprint

2.2 支气管肺泡灌洗液(BALF)IL-8的变化 对照组(39.85±8.83)pg/mL、吸烟1月组

(50.29±5.60)pg/mL、吸烟2月组(56.29±4.81)pg/mL,

戒烟1月组(54.31±6.43)pg/mL、戒烟2月组(51.35±7.47)pg/mL。吸烟组与戒烟组各组间比较,差异无统计学意义(P=0.300)。对照组与吸烟组和戒烟组比较差异均有统计学意义(P<0.05)。见表1。

2.3 大鼠BALF中GSH的变化 对照组BALF中GSH含量(133.42±10.33)mg/L、吸烟1月组(125.42±9.54)mg/L、吸烟2月组(116.96±6.85)mg/L、戒烟1月组(120.51±8.58)mg/L、戒烟2月组(130.94±6.96)mg/L。对照组与吸烟组比较,差异有统计学意义(P<0.05);对照组与戒烟组比较,差异无统计学意义(P>0.05);吸烟组与戒烟组比较,差异无统计学意义(P>0.05)。见表1。

2.4 大鼠肺组织HDAC2的变化 HDAC2的累积光密度值(IOD):对照组(0.87±0.03),吸烟1月组(0.78±0.02),吸烟2月组(0.73±0.01),戒烟1月组(0.75±0.01),戒烟2月组(0.76±0.04)。对照组与吸烟组比较,差异有统计学意义(P<0.05),其余两两组间比较,差异无统计学意义(P>0.05)。见表1。

2.5 BALF 中IL-8、GSH与肺组织HDAC2蛋白相关性分析采用Pearson相关分析,大鼠支气管肺泡灌洗液IL-8与GSH呈负相关(r=-0.63,P<0.05),大鼠肺组织HDAC2蛋白表达与支气管肺泡灌洗液IL-8水平呈负相关(r=-0.59,P<0.05),与GSH水平呈正相关(r=0.43,P<0.05)。

3 讨论

慢性阻塞性肺部疾病(COPD)是一种常见的慢性炎症性疾病,以进行性发展的气流受限不完全可逆为特征,与肺部对有害气体或有害颗粒的异常炎症反应有关[3]。吸烟可导致COPD,在COPD和吸烟者的肺部均可以观察到由于氧化应激反应增强触发的炎症反应[4]。目前研究认为,香烟烟雾通过激活巨噬细胞、中性粒细胞、T淋巴细胞释放蛋白酶和活性氧等引起细胞损伤和气道炎症[4]。

IL-8是一种多功能细胞因子,通过激活、趋化中性粒细胞并在气道内聚集,导致细胞变形反应和脱颗粒反应、呼吸爆发反应后释放溶酶体和形成超氧化物,参与COPD的炎症反应[5]。研究显示与健康吸烟者和不吸烟者相比,COPD患者诱导痰中IL-8含量显著升高,测定肺内外的IL-8可反应COPD患者的病情[6-7]。香烟烟雾诱导IL-8的释放可能是通过氧化还原介导的信号转导通路,特别是NF-κB的激活[8-9]。笔者的数据表明吸烟组较健康对照组肺泡灌洗液IL-8含量升高,比较差异有统计学意义(P<0.05),且随着吸烟时间延长,IL-8水平升高更为显著。

每一口香烟烟雾中含有大约1017种氧化剂/氧自由基和4700种化合物,包括活性醛和醌类。活性醛和醌类还可以进一步生成羟自由基。大量的氧化剂和自由基激活氧化应激反应。GSH是重要的抗氧化剂,对于保持气道上皮细胞完整性和抵御肺部损伤与炎症等方面有重要作用,现常将GSH作为评价氧化反应的一个指标[10]。笔者的研究发现吸烟后GSH含量下降,提示香烟烟雾降低GSH水平,并且GSH水平下降和IL-8水平升高具有显著相关性(r=-0.63,P<0.05),提示GSH下降与肺泡巨噬细胞释放IL-8有关,与相关研究一致[11-12]。Hirohiko[13]研究证明戒烟2周就可以改善长期吸烟者的氧化抗氧化失衡状态,可以检测到GSH升高;Tavilani等研究显示长期吸烟者在戒烟1年后体内氧化抗氧化指标可恢复正常,但重新吸烟后GSH等指标又再次上升[14-15]。笔者的研究也发现戒烟后GSH逐渐回升,与既往研究结果相似。

组蛋白去乙酰化酶家族包括11种异构体,划分为三类,Ⅰ类HDACS(HDAC1,2,3,8和11)几乎只存在于细胞核内,Ⅱ类HDACS(HDAC4,5,6,7,9和10)能够响应于某些细胞的细胞核和细胞质之间的信号穿梭,第Ⅲ类功能尚未完全破译。Ⅰ类HDACS已被证明在调节细胞增殖和炎症反应中发挥重要作用[16]。HDAC2主要参与炎症反应,是Ⅰ类HDAC。最近的报道HDAC2是糖皮质激素介导的抗炎活性所必需的酶,糖皮质激素通过诱导组蛋白去乙酰化酶蛋白和基因表达增加细胞HDAC2的活性[17]。笔者的研究发现香烟烟雾介导的IL-8的释放与HDAC活性下降有关。有研究通过支气管镜活检COPD患者和吸烟者的肺组织送检,结果显示肺泡巨噬细胞HDAC活性有显著下降,并且HDAC活性下降幅度与疾病严重程度相关[18-19]。笔者的研究数据显示肺组织HDAC2活性下降与早前的报道一致[20],随着吸烟时间的延长,HDAC2活性降低越明显,戒烟可使之有所恢复。

总之,本研究为香烟烟雾诱导基因转录和增强巨噬细胞炎症反应的分子机制提供了新的重要资料。香烟烟雾导致HDAC活性和HDAC2水平下降,HDAC活性下降与细胞内GSH的消耗有关。抗氧化剂可能通过升高细胞内GSH,恢复HDAC活性/水平和废除香烟烟雾介导的IL-8的释放来逆转氧化应激反应,因此有可能作为一种有用的治疗方法来调节COPD患者细胞内的核信号转导途径和高水平的氧化应激反应。此外,香烟烟雾介导的炎症反应是由细胞的氧化还原状态来调节的。

参考文献

[1] van Dijk W D, Heijdra Y, Lenders J W, et al. Cigarette smoke retention and bronchodilation in patients with COPD. A controlled randomized trial[J]. Respir Med,2013,107(1):112-119.endprint

[2] Yao H, Sundar I K, Ahmad T, et al. SIRT1 protects against cigarette smoke-induced lung oxidative stress via a FOXO3-dependent mechanism[J]. Am J Physiol Lung Cell Mol Physiol,2014,306(9):816-828.

[3]陈文明,黄树红,王桂英,等.COPD患者诱导痰中IL-6、TNF-α水平及其与气流受限的关系[J].中国医学创新,2012,9(6):12-13.

[4] Arja C, Surapaneni K M, Raya P, et al. Oxidative stress and antioxidant enzyme activity in South Indian male smokers with chronic obstructive pulmonary disease[J]. Respirology,2013,18(7):1069-1075.

[5] 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[J]. Biochem Biophys Res Commun,2004,315(1):240-245.

[6] Montuschi P, Collins J V, Ciabattoni G, et al. Exhaled 8-isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers[J]. Am J Respir Crit CareMed,2000,162(3Pt1):1175-1177.

[7] Angelis N, Porpodis K, Zarogoulidis P, et al. Airway inflammation in chronic obstructive pulmonary disease[J]. J Thorac Dis,2014,6(Suppl 1):167-172.

[8] Rahman I, Gilmour P S, Jimenez L A, et al. Oxidative stress and TNF-α induce histone acetylation and NF-κB/AP-1 activation in alveolar epithelial cells: potential mechanism in gene transcription in lung inflammation[J]. Mol Cell Biochem,2002,234-235(1-2):239-248.

[9] Li M, Zhong X, He Z, et al. Effect of erythromycin on cigarette-induced histone deacetylase protein expression and nuclear factor-κB activity in human macrophages in vitro[J]. Int Immunopharmacol,2012,12(4):643-650.

[10]闫莉,刘春霞,魏欣,等.谷氨酰胺对慢性阻塞性肺病患者营养免疫调节和抗氧化治疗的作用[J].中国老年学杂志,2010,30(16):2277-2279.

[11] Yang S R, Chida A S, Bauter M R, et al. Cigarette smoke induces proinflammatory cytokine release by activation of NF-kappa B and posttranslational modifications of histone deacetylase in macrophages[J]. Am J Physiol Lung Cell Mol Physiol,2006,291(1):46-57.

[12] Tsai J J, Liao E C, Hsu J Y, et al. The differences of eosinophil-and neutrophil-related inflammation in elderly allergic and non-allergic chronic obstructive pulmonary disease[J]. J Asthma,2010,47(9):1040-1044.

[13] Hirohiko Morita M D, Hisao Ikeda M D, PhD, Nobuya Haramaki M D, et al. Only two-week smoking Cessation improves platelet aggregability and intraplatelet redox imbalance of long-term smokers[J]. Journal of the American College of Cardiology,2005,45(4):589-594.

[14] Zhou J F, Yan X F, Guo F Z, et al. Effects of cigarette smoking and smoking cessation on plasma constituents and enzyme activities related to oxidative stress[J]. Biomed Environ Sci,2000,13(1):44-55.endprint

[15] Tavilani H, Nadi E, Karimi J, et al. Oxidative stress in COPD patients, smokers, and non-smokers[J]. Respir Care,2012,57(12):2090-2094.

[16] De Ruijter A J, van Gennip A H, Caron H N, et al. Histone deacetylases (HDACs): characterization of the classical HDAC family[J]. Biochem J,2003,370(Pt 3):737-749.

[17] Pace E, Ferraro M, Di Vincenzo S, et al. Comparative cytoprotective effects of carbocysteine and fluticasone propionate in cigarette smoke extract-stimulated bronchial epithelial cells[J]. Cell Stress Chaperones,2013,18(6):733-743.

[18] Ito K, Ito M, Elliott W M, et al. Decreased histone deacetylase activity in chronic obstructive pulmonary disease[J]. N Engl J Med,2005,352(19):1967-1976.

[19] Li D Z, Ruan S W, Chen Z B, et al. Effect of Quanzhenyiqitang on apoptosis of alveolar macrophages and expression of histone deacetylase 2 in rats with chronic obstructive pulmonary disease[J]. Exp Ther Med,2014,7(5):1327-1331.

[20] Cosio B G, Tsaprouni L, Ito K, et al. Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages[J]. J Exp Med,2004,200(5):689-695.

(收稿日期:2014-06-30) (本文编辑:王宇)endprint

[15] Tavilani H, Nadi E, Karimi J, et al. Oxidative stress in COPD patients, smokers, and non-smokers[J]. Respir Care,2012,57(12):2090-2094.

[16] De Ruijter A J, van Gennip A H, Caron H N, et al. Histone deacetylases (HDACs): characterization of the classical HDAC family[J]. Biochem J,2003,370(Pt 3):737-749.

[17] Pace E, Ferraro M, Di Vincenzo S, et al. Comparative cytoprotective effects of carbocysteine and fluticasone propionate in cigarette smoke extract-stimulated bronchial epithelial cells[J]. Cell Stress Chaperones,2013,18(6):733-743.

[18] Ito K, Ito M, Elliott W M, et al. Decreased histone deacetylase activity in chronic obstructive pulmonary disease[J]. N Engl J Med,2005,352(19):1967-1976.

[19] Li D Z, Ruan S W, Chen Z B, et al. Effect of Quanzhenyiqitang on apoptosis of alveolar macrophages and expression of histone deacetylase 2 in rats with chronic obstructive pulmonary disease[J]. Exp Ther Med,2014,7(5):1327-1331.

[20] Cosio B G, Tsaprouni L, Ito K, et al. Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages[J]. J Exp Med,2004,200(5):689-695.

(收稿日期:2014-06-30) (本文编辑:王宇)endprint

[15] Tavilani H, Nadi E, Karimi J, et al. Oxidative stress in COPD patients, smokers, and non-smokers[J]. Respir Care,2012,57(12):2090-2094.

[16] De Ruijter A J, van Gennip A H, Caron H N, et al. Histone deacetylases (HDACs): characterization of the classical HDAC family[J]. Biochem J,2003,370(Pt 3):737-749.

[17] Pace E, Ferraro M, Di Vincenzo S, et al. Comparative cytoprotective effects of carbocysteine and fluticasone propionate in cigarette smoke extract-stimulated bronchial epithelial cells[J]. Cell Stress Chaperones,2013,18(6):733-743.

[18] Ito K, Ito M, Elliott W M, et al. Decreased histone deacetylase activity in chronic obstructive pulmonary disease[J]. N Engl J Med,2005,352(19):1967-1976.

[19] Li D Z, Ruan S W, Chen Z B, et al. Effect of Quanzhenyiqitang on apoptosis of alveolar macrophages and expression of histone deacetylase 2 in rats with chronic obstructive pulmonary disease[J]. Exp Ther Med,2014,7(5):1327-1331.

[20] Cosio B G, Tsaprouni L, Ito K, et al. Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages[J]. J Exp Med,2004,200(5):689-695.

(收稿日期:2014-06-30) (本文编辑:王宇)endprint

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