摘要
羧甲司坦(CMC)是治疗慢性阻塞性肺病的常用药,长期服用对胃肠道产生严重刺激。L-精氨酸是一氧化氮(NO)合酶(NOS)的底物,在体内可转化为对心血管及胃肠道等有益的NO。L-精氨酸属碱性氨基酸,能与某些含羧酸基团的化合物成盐以改善原药的水溶性,并可能由于促进NO的释放带来活性的提高或毒副作用的缓解。因此,本文设计、合成了CMC的L-精氨酸盐(CMCA),并测试其理化性质以及在香烟烟雾诱导的人支气管上皮细胞损伤模型中清除活性氧(ROS)、抗细胞凋亡和NO释放的能力。结果表明,CMCA能有效捕获ROS,释放NO,并抑制细胞凋亡,效果优于CMC或L-精氨酸,提示该化合物值得深入研究和开发。
慢性阻塞性肺疾病(chronic obstructive pulmonary diseases,COPD)是一种常见的慢性气道疾病,具有高发病率、致残率和致死率的特
羧甲司坦(carboxymethylcysteine,CMC)是临床常用的黏痰溶解药。临床研究发现,应用CMC可明显减少COPD患者病情急性加重并延长发作间
L-精氨酸(L-arginine,L-Arg)是一种半必需氨基酸,可被内源性NOS代谢为NO和瓜氨酸,在维持气道张力和功能方面起着重要作用。值得一提的是,只有L-精氨酸才能作为NOS的底物释放NO,而D-精氨酸不具有产生NO的能
基于上述背景,本研究设计、合成了一种CMC的L-精氨酸盐化合物(CMCA),并测试了CMCA的理化性质及其在人支气管上皮细胞(16-HBE)中抗氧化、抗凋亡和NO释放的作用。
羧甲司坦(成都西亚化工股份有限公司,Lot:20200710);L-精氨酸[梯希爱(上海)化成工业发展有限公司,Lot:22RPO];商用卷烟(万宝路红标);16-HBE细胞系、DCFH-DA活性氧检测试剂盒、Annexin V-APC/7-AAD细胞凋亡检测试剂盒、DAF-FM DA一氧化氮检测试剂盒(江苏凯基生物技术股份有限公司);6孔培养板(美国Corning Costar公司);AV-500核磁共振仪(德国Bruker公司);MAT95XP型高分辨质谱仪[赛默飞世尔科技(中国)有限公司];X-4精密显微熔点测定仪(北京福凯仪器有限公司);pH计[梅特勒-托利多(中国)有限公司];FACS Calibur流式细胞仪(美国Becton-Dickinson公司)。
CMCA的合成途径如路线1所示,将L-精氨酸(1.74 g,10 mmol)溶于蒸馏水5 mL中,逐量加入CMC(1.79 g,10 mmol)始终保持反应液为澄清状,于室温25 ℃下搅拌2 h,向反应液中滴加无水乙醇50 mL,开始时滴速稍快,至有白色晶体析出时减慢滴速,滴毕,搅拌0.5 h,抽滤,用少量乙醇洗涤滤饼,40 ℃真空干燥,得白色CMCA结晶3.20 g,收率约90.7%。mp 203.8 ~ 205.2 ℃;UV(H2O) λmax 211.5(lg ε 3.717) nm;IR(KBr,ν):918.6,1 223.9,1 380.0,1 690.7,3 336.9,3 438.0 c

Scheme 1 Synthetic route of carboxymethylcysteine L-arginate (CMCA)
香烟烟雾溶液的制备方法如文献所
本研究采用16-HBE细胞系建立CSE模型,参照文献方
DCFH-DA是一种被广泛用于检测ROS的荧光探针。用PBS洗涤细胞1次(离心200 r/min,5 min)收集并调整细胞浓度为每毫升1 × 1
将对数生长期的16-HBE细胞接种到6孔板中,次日,待细胞贴壁后,根据组别设置相应的含药培养基,同时设立阴性对照组。药物作用18 h后,用0.25%胰酶(不含EDTA)消化收集细胞,用PBS洗涤细胞两次(离心200 r/min,5 min)调整细胞浓度为每毫升5 ×1
DAF-FM DA是一种可用于检测细胞内低浓度NO的荧光探针。将细胞消化、计数、配制成浓度为每毫升5 × 1
将CMC与CMCA配制成为1 mg/mL的水溶液,使用pH计测试二者pH,结果如
CMC: Carboxymethylcysteine; CMCA: Carboxymethylcysteine L-arginate
首先,本研究测试了CMCA清除CSE诱导16-HBE细胞内ROS的能力,以证明其抗氧化作用。如

Figure 1 Effect of CMCA on cigarette smoke extract (CSE)-induced reactive oxygen species (ROS) generation in 16-HBE cells ()
###P < 0.001 vs control group;
CMCA抑制CSE引起的16-HBE细胞凋亡情况见图

Figure 2 Effect of CMCA on CSE-induced 16-HBE cells apoptosis ()
###P < 0.001 vs control group;
为了验证NO在16-HBE细胞中发挥的作用,进一步研究了16-HBE细胞内NO的含量情况(

Figure 3 Effect of CMCA on CSE-induced NO concentration in 16-HBE cells ()
###P < 0.001 vs control group;
香烟烟雾中含有醛类、尼古丁类、氰化物、氧自由基、焦油等大量有害成分,可直接对气道和肺组织造成损伤,产生并释放TNF-α、IL-8及IL-6等炎症介质,促进气道炎症,是导致COPD最主要的原因。CSE从香烟烟雾中提取并溶于细胞培养基中,被广泛用于体外研究
实验结果表明,CMCA显著改善了CMC本身的强酸性,从而可能减轻对消化道的刺激作用。此外,在等物质的量浓度下CMCA较CMC或L-精氨酸有着更好的清除ROS和抗细胞凋亡的能力,并且呈现浓度依赖性关系,提示在体外CSE诱导的细胞损伤模型中CMCA较CMC有着更好的治疗效果。
随后使用DAF-FM DA荧光探针检测细胞内NO的含量,考察CMCA细胞保护作用是否依赖于NO。研究发现,模型组较对照组NO含量显著下降,可能是由于10% CSE的加入,使细胞内自身的NO与CSE刺激产生的ROS进一步反应生成NO2、N2O3和ONO
总之,本研究合成的CMCA较CMC具有更优的理化性质,在体外CSE诱导的细胞损伤模型中较CMC有着更好的清除ROS和抗细胞凋亡的作用。此外,CMCA还可能降低应用CMC带来的胃肠道不良反应,改善患者的依从性,较CMC具有更广泛的应用前景,值得深入研究。
References
GBD 2015 Disease and Injury Incidence and Prevalence Collaborators. Global,regional,and national incidence,prevalence,and years lived with disability for 310 diseases and injuries,1990-2015:a systematic analysis for the Global Burden of Disease Study 2015[J]. Lancet,2016,388(10053):1545-1602. [百度学术]
GBD 2015 Mortality and Causes of Death Collaborators. Global,regional,and national life expectancy,all-cause mortality,and cause-specific mortality for 249 causes of death,1980-2015:a systematic analysis for the Global Burden of Disease Study 2015[J]. Lancet,2016,388(10053):1459-1544. [百度学术]
Collaborators GBD2CRD. Global,regional,and national deaths,prevalence,disability-adjusted life years,and years lived with disability for chronic obstructive pulmonary disease and asthma,1990-2015:a systematic analysis for the Global Burden of Disease Study 2015[J]. Lancet Respir Med,2017,5(9):691-706. [百度学术]
Adeloye D,Chua S,Lee C,et al. Global and regional estimates of COPD prevalence:systematic review and meta-analysis[J]. J Glob Health,2015,5(2):020415. [百度学术]
Chronic Obstructive Pulmonary Disease Group of Chinese Thoracic Society,Chronic Obstructive Pulmonary Disease Committee of Chinese Association of Chest Physician. Guidelines for the diagnosis and management of chronic obstructive pulmonary disease(revised version 2021)[J]. Chin J Tuberc Respir Dis(中华结核和呼吸杂志),2021,44(3):170-205. [百度学术]
ChenYH. Interpretation of global strategy for the diagnosis,management,and prevention of chronic obstructive pulmonary disease 2021 report[J]. Chin J Front Med Sci[中国医学前沿杂志(电子版)],2021,13(1):16-37. [百度学术]
Yan P,Ye LB,Chen WQ. Progress in therapeutic targets and development of drugs against chronic obstructive pulmonary disease[J]. J China Pharm Univ(中国药科大学学报),2021,52(2):144-155. [百度学术]
Zhou YM,Chen RC. Risk factors and intervention for chronic obstructive pulmonary disease in China[J]. Respirology,2013,18(Suppl 3):4-9. [百度学术]
van der Toorn M,Slebos DJ,de Bruin HG,et al. Critical role of aldehydes in cigarette smoke-induced acute airway inflammation[J]. Respir Res,2013,14(1):45. [百度学术]
Zuo L,He F,Sergakis GG,et al. Interrelated role of cigarette smoking,oxidative stress,and immune response in COPD and corresponding treatments[J]. Am J Physiol Lung Cell Mol Physiol,2014,307(3):L205-L218. [百度学术]
Ishibashi Y,Takayama G,Inouye Y,et al. Carbocisteine normalizes the viscous property of mucus through regulation of fucosylated and sialylated sugar chain on airway mucins[J]. Eur J Pharmacol,2010,641(2/3):226-228. [百度学术]
Zheng JP,Kang J,Huang SG,et al. Effect of carbocisteine on acute exacerbation of chronic obstructive pulmonary disease(PEACE Study):a randomised placebo-controlled study[J]. Lancet,2008,371(9629):2013-2018. [百度学术]
Rahman I,MacNee W. Antioxidant pharmacological therapies for COPD[J]. Curr Opin Pharmacol,2012,12(3):256-265. [百度学术]
Yasuda H,Yamaya M,Sasaki T,et al. Carbocisteine inhibits rhinovirus infection in human tracheal epithelial cells[J]. Eur Respir J,2006,28(1):51-58. [百度学术]
Asada M,Yoshida M,Hatachi Y,et al. L-carbocisteine inhibits respiratory syncytial virus infection in human tracheal epithelial cells[J]. Respir Physiol Neurobiol,2012,180(1):112-118. [百度学术]
Wang W,Guan WJ,Huang RQ,et al. Carbocisteine attenuates TNF-α-induced inflammation in human alveolar epithelial cells in vitro through suppressing NF-κB and ERK1/2 MAPK signaling pathways[J]. Acta Pharmacol Sin,2016,37(5):629-636. [百度学术]
Palmer RM,Ashton DS,Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine[J]. Nature,1988,333(6174):664-666. [百度学术]
Pernow J,Wang QD. The role of the L-arginine/nitric oxide pathway in myocardial ischaemic and reperfusion injury[J]. Acta Physiol Scand,1999,167(2):151-159. [百度学术]
Aydin M,Altintas N,Cem Mutlu L,et al. Asymmetric dimethylarginine contributes to airway nitric oxide deficiency in patients with COPD[J]. Clin Respir J,2017,11(3):318-327. [百度学术]
Grasemann H,Al-Saleh S,Scott JA,et al. Asymmetric dimethylarginine contributes to airway nitric oxide deficiency in patients with cystic fibrosis[J]. Am J Respir Crit Care Med,2011,183(10):1363-1368. [百度学术]
Scott JA,Grasemann H. Asymmetric dimethylarginine:a disease marker for asthma[J]?Chest,2013,144(2):367-368. [百度学术]
Wallace JL. Nitric oxide in the gastrointestinal tract:opportunities for drug development[J]. Br J Pharmacol,2019,176(2):147-154. [百度学术]
Kato S,Kitamura M,Korolkiewicz RP,et al. Role of nitric oxide in regulation of gastric acid secretion in rats:effects of NO donors and NO synthase inhibitor[J]. Br J Pharmacol,1998,123(5):839-846. [百度学术]
Ichikawa T,Ishihara K,Saigenji K,et al. Lafutidine-induced stimulation of mucin biosynthesis mediated by nitric oxide is limited to the surface mucous cells of rat gastric oxyntic mucosa[J]. Life Sci,1998,62(16):PL259-PL264. [百度学术]
Elliott SN,Wallace JL. Nitric oxide:a regulator of mucosal defense and injury[J]. J Gastroenterol,1998,33(6):792-803. [百度学术]
Kurose I,Kubes P,Wolf R,et al. Inhibition of nitric oxide production. Mechanisms of vascular albumin leakage[J]. Circ Res,1993,73(1):164-171. [百度学术]
Elliott SN,McKnight W,Cirino G,et al. A nitric oxide-releasing nonsteroidal anti-inflammatory drug accelerates gastric ulcer healing in rats[J]. Gastroenterology,1995,109(2):524-530. [百度学术]
Wallace JL,Del SP,Cirino G,et al. Nitric oxide-releasing NSAIDs:GI-safe antithrombotics[J]. Drugs Investig Drugs J,1999,2(4):321-326. [百度学术]
Arginine acetylsalicylate[J]. Chin Pharm J(中国药学杂志),1982,12:49-50. [百度学术]
Muscará MN,Lovren F,McKnight W,et al. Vasorelaxant effects of a nitric oxide-releasing aspirin derivative in normotensive and hypertensive rats[J]. Br J Pharmacol,2001,133(8):1314-1322. [百度学术]
Geusens P. Naproxcinod,a new cyclooxygenase-inhibiting nitric oxide donator(CINOD)[J]. Expert Opin Biol Ther,2009,9(5):649-657. [百度学术]
Feng XC,Ji H,Zhang YH,et al. Effect of ZLR-8 on the healing of gastric ulcer and NO releasing in vitro[J]. J China Pharm Univ(中国药科大学学报),2004,35(4):357-360. [百度学术]
Wang WD,Zhang YH,Ji H,et al. Synthesis and antiinflammatory activities of NO releasing-flobufen derivatives[J]. Chin J Med Chem(中国药物化学杂志),2004,14(1):1-8. [百度学术]
Su Y,Han W,Giraldo C,et al. Effect of cigarette smoke extract on nitric oxide synthase in pulmonary artery endothelial cells[J]. Am J Respir Cell Mol Biol,1998,19(5):819-825. [百度学术]
Pace E,Ferraro M,Siena L,et al. Cigarette smoke increases Toll-like receptor 4 and modifies lipopolysaccharide-mediated responses in airway epithelial cells[J]. Immunology,2008,124(3):401-411. [百度学术]
Vayssier-Taussat M,Camilli T,Aron Y,et al. Effects of tobacco smoke and benzo[a]Pyrene on human endothelial cell and monocyte stress responses[J]. Am J Physiol Heart Circ Physiol,2001,280(3):H1293-H1300. [百度学术]
Carnevali S,Petruzzelli S,Longoni B,et al. Cigarette smoke extract induces oxidative stress and apoptosis in human lung fibroblasts[J]. Am J Physiol Lung Cell Mol Physiol,2003,284(6):L955-L963. [百度学术]
van der Toorn M,Smit-de Vries MP,Slebos DJ,et al. Cigarette smoke irreversibly modifies glutathione in airway epithelial cells[J]. Am J Physiol Lung Cell Mol Physiol,2007,293(5):L1156-L1162. [百度学术]
Puchelle E,Zahm JM,Tournier JM,et al. Airway epithelial repair,regeneration,and remodeling after injury in chronic obstructive pulmonary disease[J]. Proc Am Thorac Soc,2006,3(8):726-733. [百度学术]