摘要
为了研究PM2.5混悬液对博来霉素(BLM)诱导的肺纤维化小鼠的影响以及新对叶百部碱(NTS)的干预作用,本实验采用析因实验设计研究了PM2.5混悬液给药次数(每周1次或2次)和BLM剂量(1.5、3.0 U/kg)对小鼠肺纤维化模型的影响。小鼠于第0天经气管滴注1.5或3.0 U/kg的BLM;从第1天开始,小鼠每周1次或2次气管滴注5 mg/kg的PM2.5混悬液;从第8天开始,给药组小鼠每天1次灌胃给予30 mg/kg的NTS直至21 d;21 d后观察肺组织病理切片的苏木精-伊红(HE)染色和Masson染色结果,计算小鼠肺系数、肺组织羟脯胺酸(HYP)含量和基于病理切片的HE炎性评分和胶原容积分数(CVF)。结果显示,对于单独滴注PM2.5混悬液的小鼠,每周1次时只有HE炎性评分显著升高,每周2次时HYP和HE炎性评分增加但CVF无显著升高;而PM2.5与BLM联合滴注的各组小鼠的CVF均显著增加;新对叶百部碱能够显著改善联合滴注小鼠的肺组织病理改变、减少胶原沉积,降低小鼠的CVF和α-SMA蛋白的表达,具有抑制肺纤维化的作用。本研究结果表明,单独滴注PM2.5主要引起肺部炎症,即使多次给药引起肺纤维化的可能性也较小;但PM2.5与BLM联合滴注则小鼠肺组织胶原沉积显著增加,大大加重了BLM引起的肺纤维化,提示PM2.5对有呼吸系统疾病或基础性疾病的患者影响很大,而新对叶百部碱对联合滴注模型小鼠的肺纤维化具有明显的改善作用。
灰霾主要由SO2、氮氧化物和可吸入颗粒物组成。流行病学研究表明,可吸入颗粒物(particulate matter,PM)浓度的升高与人群疾病的发病率和病死率密切相
百部是润肺止咳的传统中药,对叶百部(Stemonatuberosa L.)是中药百部的主要基源之一,新对叶百部碱(neotuberostemonine,NTS)是对叶百部的主要的活性成分,具有止咳、改善肺损伤和肺纤维化的作
盐酸博来霉素(bleomycin,BLM,浙江海正辉瑞制药有限公司);尼达尼布(nintedanib,Nib,济南轩德化工有限公司);新对叶百部碱(neotuberostemonine,NTS)为本课题组从对叶百部(Stemonatuberosa L.)块根中分得,其纯度大于98%(HPLC归一化法
TH-150F型智能中流量TSP(total suspended particle,空气总悬浮微粒)采样器(南京长祺环保设备有限公司);DW-86L386式超低温冰箱(青岛海尔特种电器有限公司);3700型低温高速离心机(日本久保田株式会社);冷冻干燥机(日本东京理化器械株式会社);酶标仪、电泳及转膜系统(美国Bio-Rad公司)。
将TSP采集器置于室外(中国药科大学江宁校区)24 h,按照操作说明取下PM2.5收集滤片,剪碎,加双蒸水50 mL超声20 min,过滤。滤液于5 000 r/min离心20 min,弃上层,加双蒸水50 mL超声,重复上述步骤4次得到PM2.5混悬液,冷冻干燥即得。称取适量PM2.5粉末,充分分散于0.9%生理盐水中,制成所需浓度的PM2.5药液。现用现配,用前混匀。
小鼠禁食不禁水12 h,4%水合氯醛麻醉后固定,无创法气管滴入所需浓度的药液50 μL,将鼠板直立,旋转1 min,静置1 min,放回鼠笼休息。BLM剂量为1.5、3 U/kg,于第0天(D0)天滴注。PM2.5剂量为5 mg/kg(经预实验确定),分别滴注3次(D1、D8、D15)或6次(D1、D4、D8、D11、D15、D18),实验动物饲养至第21天。第22天处死小鼠,取肺组织,分别进行病理切片和相关指标的检测。
10%中性福尔马林溶液固定的肺组织制作常规石蜡切片(4 μm厚),苏木精-伊红(HE)或Masson染色。HE染色切片由专业研究人员于光学显微镜下阅片,根据肺泡隔是否增厚、肺内血管和气管周围是否有炎性细胞浸润、支气管上皮细胞是否变性脱落以及肺组织是否充血”进行炎性评分,每项的病变由基本正常到重度依次标记为0、1、2、3、4、5分,累加4项分数为炎性评分。Masson染色切片于光学显微镜下观察胶原纤维(蓝色)的分布情况,每张组织切片选取上、下、左、右和中间5个视野进行拍照,采用Image J软件选定并计算每张照片蓝色胶原部分面积和除空白外的总组织面积,计算胶原容积分数(CVF,collagen volume fraction),CVF(%)=蓝色胶原面积/总组织面积 × 100。
小鼠肺组织适量,加入RIPA裂解液,充分匀浆裂解后,于4 ℃、12 000 r/min离心10 min,取上清液,BCA法测定蛋白浓度,Westernblot方法检测α-SMA和TGF-β的蛋白表达水平。
为了探索BLM剂量与PM2.5给药次数对小鼠肺纤维化模型的影响,采用析因实验设计研究了BLM剂量(0、1.5、3.0 U/kg)和PM2.5混悬液(5 mg/kg,经预实验确定)给药频次(0、3、6次,即每周0次或1次或2次)对小鼠肺纤维化的影响。具体通过考察小鼠单给予PM2.5混悬液3次(P3)或6次(P6),或单给予BLM 1.5 U/kg(B1)或3U/kg(B2),或给予BLM 1.5U/kg+PM2.5混悬液3次(C13)或6次(C16),或给予BLM 3U/kg+PM2.5混悬液3次(C23)或6次(C26)的肺纤维化相关指标变化来进行分析。实验方案和结果如
BLM:Bleomycin; HYP: Hydroxyproline; CVF: Collagen volume fractionMice were given PM2.5 for 3 (P3) or 6 (P6) times,or given BLM in 1.5 (B1) or 3 (B2) U/kg,or given BLM in 1.5 U/kg and PM2.5 for 3 (C13) or 6 (C16) times,or given BLM in 3 U/kg and PM2.5 for 3 (C23) or 6 (C26) time

Figure 1 Effects of BLM dose and PM2.5 administration times on pulmonary fibrosis in mice
The mice grouping scheme and dosing plan are shown in Table 1. BLM were intratracheally given to mice on day 0 and PM2.5 on days 1, 8, 15 (3 times) or on days 1, 4, 8, 11, 15, 18 (6 times), and kept until day 21A: Lung injury was assessed by HE staining ( × 100); B: Collagen deposition was assessed by Masson's trichrome staining (blue,× 100)
析因实验设计结果的方差分析见
* The data was transformed logarithmically due to the inhomogeneity of variance; SS: Sum of squares of deviations from mean; MS: Mean square
综合上述实验结果,以BLM(1.5 U/kg)、PM2.5给药3次为模型M1,以BLM(1.5 U/kg)、PM2.5给药6次为模型M2,以美国FDA于2014批准的治疗肺纤维化药物尼达尼布(nintedanib,Nib
NTS: Neotuberostemonine; Nib: Nintedanib

Figure 2 Effect of NTS on body weight and lung coefficient in mice with pulmonary fibrosis ()
1.5 U/kg BLM was intratracheally given to mice on day 0,and 5 mg/kg PM2.5 was intratracheally given on days 1, 8, 15 (M1 model) or on days 1, 4, 8, 11, 15, 18 (M2 model). NTS and Nib (positive drug) were orally given to mice from day 8 to day 21A: Changes of body weight ; B: Lung coefficient

Figure 3 Effect of NTS on histopathological changes of lung tissue in mice with pulmonary fibrosis
A: HE staining lung section ( × 100); B: Masson staining lung section ( × 100,collagen stained in blue); C: inflammatory score (); D: collagen volume fraction (CVF % = collagen area / total tissue area × 100%, )

Figure 4 Effects of NTS on α-SMA and TGF-β levels in mice with pulmonary fibrosis
α-SMA and TGF-β levels were evaluated by Western blot ()
肺组织损伤后如果不能正常修复,就会引起成纤维细胞的异常大量增殖并转分化为肌成纤维细胞,后者分泌大量的细胞外基质(胶原蛋白)而导致肺纤维化发
我国2012年发布的《环境空气质量指数(AQI)技术规定(试行)》和《环境空气质量标准
预实验结果显示,BLM与PM2.5混合后滴注(第0天),小鼠纤维化程度明显小于非混合式的联合滴注(BLM第0天、PM2.5第1天);5、10 mg/kg的PM2.5与BLM联合给药均能明显加重BLM引起的肺纤维化,但高剂量时小鼠病死率高。因此本实验采用了5 mg/kg PM2.5、非混合式的联合给药方法,并采用析因实验设计进一步优化BLM剂量和PM2.5给药次数。结果显示,单独的PM2.5滴注3次只有HE评分较之假手术组明显升高,滴注6次时HYP和HE评分显著增加但胶原容积分数CVF无显著升高,表明PM2.5即使多次给药引起肺纤维化的可能性也较小。但也有研究显示,虽然PM2.5短期暴露(5.4 mg/kg、每3天1次、共10次)不会立即导致肺纤维化,但停止暴露后随着时间的延长可能会逐渐发展为肺纤维
关于PM2.5对肺纤维化的影响,临床流行病学调查研究的结果并不一
本研究结果显示,单纯的PM2.5对正常人的影响主要是引发肺部炎症,如果浓度低或持续时间短则能够自愈,不会发展成肺纤维化;但是对于有肺纤维化疾病(或其他呼吸道或肺部疾病)的人则影响较大,会极大地加速或加重原有疾病的发展。NTS对实验性肺纤维化具有较好的疗效。

References
Falcon-Rodriguez CI,Osornio-Vargas AR,Sada-Ovalle I,et al.Aeroparticles,composition,and lung diseases[J]. Front Immunol,2016,7:3. [百度学术]
Li T,Hu R,Chen Z,et al. Fine particulate matter (PM2.5): the culprit for chronic lung diseases in China[J]. Chronic Dis Transl Med,2018,4(3):176-186. [百度学术]
Yue CL,Liu HG. Research status of fine particulate matter (PM2.5) and its pathogenicity[J]. Chin J Clin Exp Pathol(临床与实验病理学杂志),2009,25(4):437-440. [百度学术]
Ou M,Wang CL,Xu WZ,et al. Research of PM2.5 on heart and lung injury[J].Med Recapitul(医学综述),2017,23(19):3817-3821. [百度学术]
Li KL,Lin YC. PM2.5 induced cardiac hypertrophy via CREB/GSK3b/SOS1 pathway and metabolomics alterations[J]. Oncotarget,2018,9(56):30748-30760. [百度学术]
Chen JJ,Ma WM,Yuan JL,et al. PM2.5 exposure aggravates left heart failure induced pulmonary hypertension[J]. Acta Cardiol,2019,74(3):238-244. [百度学术]
Richeldi L,Collard HR,Jones MG. Idiopathic pulmonary fibrosis[J]. Lancet,2017,389(10082):1941-1952. [百度学术]
Rozenberg D,Sitzer N,Porter S,et al. Idiopathic pulmonary fibrosis:a review of disease,pharmacological,and nonpharmacological strategies with a focus on symptoms,function,and health-related quality of life[J]. J Pain Symptom Manage,2020,59(6):1362-1378. [百度学术]
Duan Z,Wu CH. Exposure to PM2.5 enhances bleomycin-induced pulmonary fibrosis in rats[J]. Basic Clin Med(基础医学与临床),2015,35(6):781-785. [百度学术]
Li QL,Yang LW,Liu G. Effect of PM2.5 on bleomycin-induced pulmonary fibrosis in mice[J]. J Guangdong Med Univ(广东医科大学学报),2017,35(3):237-240. [百度学术]
Xu ZH,Li ZL,Liao ZY,et al. PM2.5 induced pulmonary fibrosis in vivo and in vitro[J]. Ecotox Environ Safe,2019,171:112-121. [百度学术]
Ye QY,Ma L,Ye XY,et al. Effect of Trolliusaltaicus flavonoids on the prevention and treatment of pulmonary fibrosis induced by PM2.5 in rats[J]. J Toxicol(毒理学杂志),2020,34(2):119-125. [百度学术]
Ministry of Ecology and Environment of the People's Republic of China. Technicalregulation on ambient air quality index (on trial) (HJ 633-2012)[EB/OL]. (2012-02-29)[2020-08-24].http://www.mee.gov.cn/ywgz/fgbz/bz/bzwb/jcffbz/201203/t20120302_224166.shtml. [百度学术]
Ministry of Ecology and Environment of the People's Republic of China. Ambient air quality standards (GB 3095-2012)[EB/OL]. (2012-02-29)[2020-08-24].http://www.mee.gov.cn/ywgz/fgbz/bz/bzwb/dqhjbh/dqhjzlbz/201203/t20120302_224165.htm. [百度学术]
Chung HS,Hon PM,Lin G,et al. Antitussive activity of stemona alkaloids from Stemona tuberosa[J]. Planta Med,2003,69(10):914-920. [百度学术]
Xu YT,Shaw PC,Jiang RW,et al. Antitussive and central respiratory depressant effects of Stemonatuberosa[J]. J Ethnopharmacol,2010,128(3):679-684. [百度学术]
Lee KY,Jeong EJ,Sung SH,et al. Stemona alkaloids isolated from Stemonatuberosa roots and their inhibitory activity on lipopolysaccharide-induced nitric oxide production[J]. Rec Nat Prod,2016,10(1):109-112. [百度学术]
Xiang J,Cheng S,Feng TL,et al. Neotuberostemonine attenuates bleomycin-induced pulmonary fibrosis by suppressing the recruitment and activation of macrophages[J]. Int Immunopharmacol,2016,36(6):158-164. [百度学术]
Gao Y,Wang J,Zhang CF,et al. Seven new alkaloids from the roots of Stemonatuberosa[J]. Tetrahedron,2014,70:967-974. [百度学术]
Martinez F J,Collard H R,Pardo A,et al. Idiopathic pulmonary fibrosis[J]. Nat Rev Dis Primers,2017,3:17074. [百度学术]
Liu Y,Luo ZL,Yan FL,et al. Effect of sanggenon C on pulmonary fibrosis induced by bleomycin in mice and potential mechanisms implicated[J]. Chin Tradit Herbal Drugs(中草药),2020,51(16):4244-4250. [百度学术]
Zhou XC,LiYX,Hong W,et al. Effect of Maculosin on fibrotic gene expression in lung fibroblasts[J]. J China Pharm Univ (中国药科大学学报),2014,45(4):491-495. [百度学术]
Liu W,Jiang SF,Tu L,et al. Mechanism of Jin Shui Bao capsule in ameliorating rat pneumoconiosis model[J].J China Pharm Univ(中国药科大学学报),2018,49(4):476-482. [百度学术]
Wang JX,Gu HD,Zhang ZN,et al. Contamination characterization and human exposure levels to polybrominated diphenyl ethers in indoor and outdoor air in industrial park of Suzhou city[J]. Environ Sci(环境科学),2018,39(4):1544-1551. [百度学术]
Sun BY,Shi YF,Li Y,et al. Short-term PM2.5 exposure induces sustained pulmonary fibrosis development during post-exposure period in rats[J]. J Hazard Mater,2020,385:121566. [百度学术]
Sese L,Nunes H,Cottin V,et al. Role of atmospheric pollution on the natural history of idiopathic pulmonary fibrosis[J]. Thorax,2018,73(2):145-150. [百度学术]
Johannson KA,Vittinghoff E,Morisset J,et al. Air pollution exposure is associated with lower lung function,but not changes in lung function,in patients with idiopathic pulmonary fibrosis[J]. Chest,2018,154(1):119-125. [百度学术]