弹性蛋白酶抑制剂对小鼠Lewis肺癌放射性肺损伤保护作用研究
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摘要
目的放疗是胸部肿瘤的重要治疗方法,但多数患者在放疗后会发生放射性肺损伤,其机制尚未明确。本研究旨在探讨中性粒细胞弹性蛋白酶抑制剂西维来司钠对小鼠Lewis肺癌放疗导致放射性肺损伤的保护作用,并评价西维来司钠对肿瘤放疗效果的影响。方法接种Lewis肺癌细胞(1×106)于雄性C57BL/6小鼠右侧腋下,8d后,随机分为对照组、单纯放射治疗组、放疗+50mg/kg西维来司钠组和放疗+100mg/kg西维来司钠组,10只/组。给予1次X射线20Gy照射肿瘤和小鼠全胸,腹腔注射西维来司钠1次/d,连续27d。放疗27d后,称取肿瘤质量;小鼠心脏穿刺取血,检测血清中弹性蛋白酶活性;取肺组织进行HE染色;取肺泡灌洗液,检测总蛋白浓度、有核细胞数和弹性蛋白酶活性;CD11b和Ly6G双参数标记,流式细胞仪检测肺组织中性粒细胞。结果放疗导致Lewis肺癌小鼠肺组织炎症改变。放疗+50mg/kg西维来司钠组和放疗+100 mg/kg西维来司钠组肺泡灌洗液中弹性蛋白酶相对活性分别为1.91±0.21和1.39±0.18,低于单纯放射治疗组(2.97±0.39),差异有统计学意义,F=31.844,P<0.001。放疗+50mg/kg西维来司钠组和放疗+100mg/kg西维来司钠组血清中弹性蛋白酶相对活性分别为1.93±0.15和1.31±0.19,低于单纯放疗治疗组(2.65±0.36),差异有统计学意义,F=25.580,P<0.001。病理显示放疗+50mg/kg西维来司钠组和放疗+100mg/kg西维来司钠组与单纯放射治疗组相比肺损伤减轻。放疗+50mg/kg西维来司钠组和放疗+100mg/kg西维来司钠组肺泡灌洗液中总蛋白浓度分别为(0.61±0.09)和(0.41±0.05)mg/mL,低于单纯放射治疗组(0.96±0.20)mg/mL,差异有统计学意义,F=18.465,P=0.001。放疗+50mg/kg西维来司钠组和放疗+100mg/kg西维来司钠组肺泡有核细胞数分别为(2.63±0.32)×105和(1.57±0.25)×105 mL-1,低于单纯放射治疗组(3.70±0.36)×105 mL-1,差异有统计学意义,F=53.210,P<0.001。放疗+50mg/kg西维来司钠组和放疗+100mg/kg西维来司钠组肺组织中性粒细胞分别为(4.50±0.51)%和(3.71±0.69)%,低于单纯放射治疗组(6.52±1.01)%,差异有统计学意义,F=14.589,P=0.001。单纯放射治疗组小鼠Lewis肺癌肿瘤质量为(2.3±0.6)g,低于对照组(7.9±1.0)g,F=206.162,P<0.001;放疗+50mg/kg西维来司钠组和放疗+100 mg/kg西维来司钠组肿瘤质量分别为(2.2±0.4)和(2.3±0.3)g,与单纯放射治疗组相比差异无统计学意义,P值分别为0.829和0.949。结论弹性蛋白酶抑制剂西维来司钠可减轻放射性肺损伤,不影响放疗对小鼠Lewis肺癌的治疗效果。
OBJECTIVE Radiotherapy is an effective treatment strategy for thoracic malignancies,but a significant proportion of patients experience radiation-induced lung injury(RILI)after therapeutic irradiation.The mechanisms regulating this event remain elusive.The aim of this study was to investigate the protective effect of neutrophil elastase inhibitor sivelestat on radiation-induced lung injury during the course of radiotherapy and evaluate whether sivelestat influences the outcome of radiotherapy in murine Lewis lung carcinoma model.METHODS Lewis lung carcinoma(LLC)cells(1×106)were inoculated into the right flank of the male mice.After 8 days,the mice were randomly divided into 4 treatment groups(10 mice/group):tumor model(control),radiotherapy,radiotherapy+sivelestat(50 mg/kg),radiotherapy+sivelestat(100 mg/kg).The mice were locally irradiated at the tumor and thorax with a single dose of 20 Gy.After irradiation,the mice were treated with sivelestat(50 mg/kg,100 mg/kg)via intraperitoneal injection once a day for 27 days.Twenty-seven days after thoracic irradiation,the tumors were collected and weighted.Blood was collected by cardiac puncture.Serum was collected and the activity of neutrophil elastase from serum was evaluated.The lungs were collected,fixed with formaldehyde,embedded in paraffin,cut,and stained with haematoxylin and eosin(HE).Total protein concentration and nucleated cells of bronchoalveolar lavage fluid(BALF)were determined.Lung-infiltrating neutrophils were analyzed by flow cytometry using CD11 band Ly6 Gdouble staining.RESULTS After irradiation,the LLC tumor-bearing mice showed pulmonary inflammation.The relative activity of neutrophil elastase in BALF of radiotherapy+50 mg/kg sivelestat group(1.91±0.21)and radiotherapy+100 mg/kg sivelestat group(1.39±0.18)were significantly lower than that of radiotherapy group(2.97±0.39)(F=31.844,P<0.001).The relative activity of neutrophil elastase in serum of radiotherapy+50 mg/kg sivelestat group(1.93±0.15)and radiotherapy+100 mg/kg sivelestat group(1.31±0.19)were significantly lower than that of radiotherapy group(2.65±0.36)(F=25.580,P<0.001).HE-stained histopathologic sections of lung tissue showed that sivelestat(50,100 mg/kg)protected the alveolar structure in tumor-bearing mice with radiotherapy.BALF protein of radiotherapy+50 mg/kg sivelestat group(0.61±0.09)mg/mL and radiotherapy+100 mg/kg sivelestat group(0.41±0.05)mg/ml were considerably lower than that of radiotherapy group(0.96±0.20)mg/ml(F=18.465,P=0.001).BALF nucleated cell counts of radiotherapy+50 mg/kg sivelestat group(2.63±0.32)×105 ml-1 and radiotherapy+100 mg/kg sivelestat group(1.57±0.25)×105 ml-1 were considerably lower than that of radiotherapy group(3.70±0.36)×105 ml-1(F=53.210,P<0.001).Neutrophil infiltration in lung tissues of radiotherapy+50 mg/kg sivelestat group(4.50±0.51)% and radiotherapy+100 mg/kg sivelestat group(3.71±0.69)%were significantly lower than that of mice with radiotherapy(6.52±1.01)%(F=14.589,P=0.001).The tumor weights were(7.9±1.0),(2.3±0.6),(2.2±0.4)and(2.3±0.3)g respectively in control mice,radiotherapy group,radiotherapy+50 mg/kg sivelestat group and radiotherapy+100 mg/kg sivelestat group.LLC tumor growth was inhibited after radiotherapy(F=206.162,P<0.001).Sivelestat treatment did not affect the tumor weights as compared to the radiotherapy group(P=0.829,P=0.949).CONCLUSION Neutrophil elastase inhibitor sivelestat ameliorates radiation-induced lung injury while preserving the therapeutic effect of radiotherapy in murine Lewis lung carcinoma model.
OBJECTIVE Radiotherapy is an effective treatment strategy for thoracic malignancies,but a significant proportion of patients experience radiation-induced lung injury(RILI)after therapeutic irradiation.The mechanisms regulating this event remain elusive.The aim of this study was to investigate the protective effect of neutrophil elastase inhibitor sivelestat on radiation-induced lung injury during the course of radiotherapy and evaluate whether sivelestat influences the outcome of radiotherapy in murine Lewis lung carcinoma model.METHODS Lewis lung carcinoma(LLC)cells(1×106)were inoculated into the right flank of the male mice.After 8 days,the mice were randomly divided into 4 treatment groups(10 mice/group):tumor model(control),radiotherapy,radiotherapy+sivelestat(50 mg/kg),radiotherapy+sivelestat(100 mg/kg).The mice were locally irradiated at the tumor and thorax with a single dose of 20 Gy.After irradiation,the mice were treated with sivelestat(50 mg/kg,100 mg/kg)via intraperitoneal injection once a day for 27 days.Twenty-seven days after thoracic irradiation,the tumors were collected and weighted.Blood was collected by cardiac puncture.Serum was collected and the activity of neutrophil elastase from serum was evaluated.The lungs were collected,fixed with formaldehyde,embedded in paraffin,cut,and stained with haematoxylin and eosin(HE).Total protein concentration and nucleated cells of bronchoalveolar lavage fluid(BALF)were determined.Lung-infiltrating neutrophils were analyzed by flow cytometry using CD11 band Ly6 Gdouble staining.RESULTS After irradiation,the LLC tumor-bearing mice showed pulmonary inflammation.The relative activity of neutrophil elastase in BALF of radiotherapy+50 mg/kg sivelestat group(1.91±0.21)and radiotherapy+100 mg/kg sivelestat group(1.39±0.18)were significantly lower than that of radiotherapy group(2.97±0.39)(F=31.844,P<0.001).The relative activity of neutrophil elastase in serum of radiotherapy+50 mg/kg sivelestat group(1.93±0.15)and radiotherapy+100 mg/kg sivelestat group(1.31±0.19)were significantly lower than that of radiotherapy group(2.65±0.36)(F=25.580,P<0.001).HE-stained histopathologic sections of lung tissue showed that sivelestat(50,100 mg/kg)protected the alveolar structure in tumor-bearing mice with radiotherapy.BALF protein of radiotherapy+50 mg/kg sivelestat group(0.61±0.09)mg/mL and radiotherapy+100 mg/kg sivelestat group(0.41±0.05)mg/ml were considerably lower than that of radiotherapy group(0.96±0.20)mg/ml(F=18.465,P=0.001).BALF nucleated cell counts of radiotherapy+50 mg/kg sivelestat group(2.63±0.32)×105 ml-1 and radiotherapy+100 mg/kg sivelestat group(1.57±0.25)×105 ml-1 were considerably lower than that of radiotherapy group(3.70±0.36)×105 ml-1(F=53.210,P<0.001).Neutrophil infiltration in lung tissues of radiotherapy+50 mg/kg sivelestat group(4.50±0.51)% and radiotherapy+100 mg/kg sivelestat group(3.71±0.69)%were significantly lower than that of mice with radiotherapy(6.52±1.01)%(F=14.589,P=0.001).The tumor weights were(7.9±1.0),(2.3±0.6),(2.2±0.4)and(2.3±0.3)g respectively in control mice,radiotherapy group,radiotherapy+50 mg/kg sivelestat group and radiotherapy+100 mg/kg sivelestat group.LLC tumor growth was inhibited after radiotherapy(F=206.162,P<0.001).Sivelestat treatment did not affect the tumor weights as compared to the radiotherapy group(P=0.829,P=0.949).CONCLUSION Neutrophil elastase inhibitor sivelestat ameliorates radiation-induced lung injury while preserving the therapeutic effect of radiotherapy in murine Lewis lung carcinoma model.
引文
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[12] Dau T,Sarker RS,Yildirim AO,et al.Autoprocessing of neutrophil elastase near its active site reduces the efficiency of natural and synthetic elastase inhibitors[J].Nat Commun,2015,6:6722.
[13] Talukdar S,Oh DY,Bandyopadhyay G,et al.Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase[J].Nat Med,2012,18(9):1407-1412.
[14] Polverino E,Rosales-Mayor E,Dale GE,et al.The role of neutrophil elastase inhibitors in lung diseases[J].Chest,2017,152(2):249-262.
[15] Boxio R,Wartelle J,Nawrocki-Raby B,et al.Neutrophil elastase cleaves epithelial cadherin in acutely injured lung epithelium[J].Respir Res,2016,17(1):129.
[16] Kido T,Muramatsu K,Yatera K,et al.Efficacy of early sivelestat administration on acute lung injury and acute respiratory distress syndrome[J].Respirology,2017,22(4):708-713.
[17] Wang T,Mathew B,Wu X,et al.Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury[J].Pulm Circ,2016,6(2):234-239.
[18] Kawabata K,Hagio T,Matsumoto S,et al.Delayed neutrophil elastase inhibition prevents subsequent progression of acute lung injury induced by endotoxin inhalation in hamsters[J].Am J Respir Crit Care Med,2000,161(6):2013-2018.
[19] Coffelt SB,Kersten K,Doornebal CW,et al.IL-17-producingγδT cells and neutrophils conspire to promote breast cancer metastasis[J].Nature,2015,522(7556):345-348.
[20]周海燕,高芳,田静,等.吡格列酮对放射性肺炎的预防作用及其机制研究[J].中华肿瘤防治杂志,2016,23(18):1213-1217.
[21] Wallrapp A,Riesenfeld SJ,Burkett PR,et al.The neuropeptide NMU amplifies ILC2-driven allergic lung inflammation[J].Nature,2017,549(7672):351-356.
[2]赵聪,刘兰平,汤良,等.双肺电子密度值和放疗模式与NSCLC放射性肺炎发生相关性[J].中华肿瘤防治杂志,2017,24(18):1305-1309.
[3] Meziani L,Mondini M,Petit B,et al.CSF1Rinhibition prevents radiation pulmonary fibrosis by depletion of interstitial macrophages[J].Eur Respir J,2018,51(3):1702120.
[4] Chen J,Zhang W,Zhang L,et al.Glycyrrhetinic acid alleviates radiation-induced lung injury in mice[J].J Radiat Res,2017,58(1):41-47.
[5] MacVittie TJ,Gibbs A,Farese AM,et al.AEOL 10150mitigates radiation-induced lung injury in the nonhuman primate:morbidity and mortality are administration schedule-dependent[J].Radiat Res,2017,187(3):298-318.
[6] Bickelhaupt S,Erbel C,Timke C,et al.Effects of CTGF blockade on attenuation and reversal of radiation-induced pulmonary fibrosis[J].J Natl Cancer Inst,2017,109(8):1-11.
[7] Yao Y,Zheng Z,Song Q.Mesenchymal stem cells:A double-edged sword in radiation-induced lung injury[J].Thorac Cancer,2018,9(2):208-217.
[8] Edvardsen H,Landmark-Hoyvik H,Reinertsen KV,et al.SNP in TXNRD2associated with radiation-induced fibrosis:a study of genetic variation in reactive oxygen species metabolism and signaling[J].Int J Radiat Oncol Biol Phys,2013,86(4):791-799.
[9] Sun Y,Du YJ,Zhao H,et al.Protective effects of ulinastatin and methylprednisolone against radiation-induced lung injury in mice[J].J Radiat Res,2016,57(5):505-511.
[10] Mishra A,Guo Y,Zhang L,et al.A critical role for P2X7receptor-induced VCAM-1shedding and neutrophil infiltration during acute lung injury[J].J Immunol,2016,197(7):2828-2837.
[11] Ortiz-Mu1oz G,Mallavia B,Bins A,et al.Aspirin-triggered 15-epi-lipoxin A4regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice[J].Blood,2014,124(17):2625-2634.
[12] Dau T,Sarker RS,Yildirim AO,et al.Autoprocessing of neutrophil elastase near its active site reduces the efficiency of natural and synthetic elastase inhibitors[J].Nat Commun,2015,6:6722.
[13] Talukdar S,Oh DY,Bandyopadhyay G,et al.Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase[J].Nat Med,2012,18(9):1407-1412.
[14] Polverino E,Rosales-Mayor E,Dale GE,et al.The role of neutrophil elastase inhibitors in lung diseases[J].Chest,2017,152(2):249-262.
[15] Boxio R,Wartelle J,Nawrocki-Raby B,et al.Neutrophil elastase cleaves epithelial cadherin in acutely injured lung epithelium[J].Respir Res,2016,17(1):129.
[16] Kido T,Muramatsu K,Yatera K,et al.Efficacy of early sivelestat administration on acute lung injury and acute respiratory distress syndrome[J].Respirology,2017,22(4):708-713.
[17] Wang T,Mathew B,Wu X,et al.Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury[J].Pulm Circ,2016,6(2):234-239.
[18] Kawabata K,Hagio T,Matsumoto S,et al.Delayed neutrophil elastase inhibition prevents subsequent progression of acute lung injury induced by endotoxin inhalation in hamsters[J].Am J Respir Crit Care Med,2000,161(6):2013-2018.
[19] Coffelt SB,Kersten K,Doornebal CW,et al.IL-17-producingγδT cells and neutrophils conspire to promote breast cancer metastasis[J].Nature,2015,522(7556):345-348.
[20]周海燕,高芳,田静,等.吡格列酮对放射性肺炎的预防作用及其机制研究[J].中华肿瘤防治杂志,2016,23(18):1213-1217.
[21] Wallrapp A,Riesenfeld SJ,Burkett PR,et al.The neuropeptide NMU amplifies ILC2-driven allergic lung inflammation[J].Nature,2017,549(7672):351-356.